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Sample records for alpha-1 foundation dna

  1. Alpha-1-antitrypsin augmentation therapy in deficient individuals enrolled in the Alpha-1 Foundation DNA and Tissue Bank

    Directory of Open Access Journals (Sweden)

    Adriano R Tonelli

    2009-12-01

    Full Text Available Adriano R Tonelli1, Farshid Rouhani1, Ning Li2, Pam Schreck1, Mark L Brantly11Alpha-1 Research Program, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, 2Department of Epidemiology and Biostatistics, University of Florida, Gainesville, Florida, USAIntroduction: Intravenous augmentation therapy with purified intravenous alpha-1 antitrypsin replaces the deficient protein and is the only currently approved treatment for alpha-1 antitrypsin deficiency (AATD related lung disease. While augmentation therapy has been available for more than 20 years, there are a limited number of studies evaluating the effect of augmentation on lung function.Material and methods: We examined the decline in forced expiratory volume in one second (FEV1 in patients enrolled in the Alpha-1 Foundation DNA and Tissue Bank in relation to the use or not of alpha-1 antitrypsin augmentation therapy. For the purpose of our analysis we included 164 patients with AATD and PI ZZ genotype.Results: Mean age of the patients was 60 years, 52% were females, 94% were white and 78% ex-smokers. The mean FEV1 at baseline was 1.7 L and the mean FEV1 % of predicted was 51.3%. The mean follow-up time was 41.7 months. A total of 124 (76% patients received augmentation therapy (augmented group while 40 patients (24% did not received it (non-augmented group. When adjusted by age at baseline, sex, smoking status, baseline FEV1 % of predicted, the mean overall change in FEV1 was 47.6 mL/year, favoring the augmented group (?FEV1 10.6 ± 21.4 mL/year in comparison with the non-augmented group (?FEV1 −36.96 ± 12.1 mL/year (P = 0.05. Beneficial ?FEV1 were observed in ex-smokers and the group with initial FEV1 % of predicted of <50%. No differences were observed in mortality.Conclusions: In conclusion, augmentation therapy improves lung function in subjects with AATD when adjusted by age, gender, smoking status and baseline FEV1 % of predicted. The beneficial

  2. A review of the alpha-1 foundation: its formation, impact, and critical success factors.

    Science.gov (United States)

    Walsh, John W; Snider, Gordon L; Stoller, James K

    2006-05-01

    Patient-advocacy organizations have proliferated because they can be an effective method to advance research and clinical care for those with the index condition, and can produce substantial benefits for the affected community, especially when the condition is uncommon. To clarify critical success factors in organizing a patient-advocacy organization and to provide a blueprint for others, including the respiratory-care advocacy community, this report examines features of one highly successful organization, the Alpha-1 Foundation, which is committed to helping those with the genetic condition alpha-1 antitrypsin deficiency. Features of the Alpha-1 Foundation that underlie its success include: consistently creating partnerships with key stakeholders, including the scientific and clinical communities, government, and pharmaceutical manufacturers; bringing passion to the cause (eg, by assuring that organizational leadership is provided by individuals affected by alpha-1 antitrypsin deficiency); and developing strategic business partnerships, as with a company that administers alpha-1 antitrypsin treatment (so-called intravenous augmentation therapy) and employs individuals with alpha-1 antitrypsin deficiency. Funds allocated by the company help to underwrite the foundation's research-funding commitment. The foundation also recruits and retains talent, including alpha-1 patients, to leadership roles (eg, on the board of directors) and has a voluntary group of committed scientists and clinicians. We believe that attention to these factors can help assure the success of patient-advocacy groups.

  3. PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

    Science.gov (United States)

    Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

    2009-07-01

    Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

  4. cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

    Science.gov (United States)

    Hwang, Jae-Ho; Yokoyama, Yoshihiro; Mizuta, Shoshi; Yoshinaka, Reiji

    2006-05-01

    A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.

  5. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

    OpenAIRE

    Kristie, T M; Roizman, B

    1986-01-01

    Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

  6. Diagnosis of alpha-1-antitrypsin deficiency by DNA analysis of children with liver disease

    Directory of Open Access Journals (Sweden)

    De TOMMASO Adriana Maria Alves

    2001-01-01

    Full Text Available Background - Alpha-1-antitrypsin deficiency is a genetic disorder which is transmitted in a co-dominant, autosomal form. Alpha-1-antitrypsin deficiency affects mainly the lungs and the liver leading, in the latter case, to neonatal cholestasis, chronic hepatitis or cirrhosis. A precise diagnosis of Alpha-1-antitrypsin deficiency may be obtained by biochemical or molecular analysis. Objective - The purpose of this study was to use DNA analysis to examine the presence of an alpha-1-antitrypsin deficiency in 12 children suspected of having this deficiency and who showed laboratory and clinical characteristics of the disease. Patients and Methods - Twelve patients, aged 3 months to 19 years, who had serum alpha-1-antitrypsin levels lower than normal and/or had hepatic disease of undefined etiology were studied. The mutant alleles S and Z of the alpha-1-antitrypsin gene were investigated in the 12 children. Alpha-1-antitrypsin gene organization was analyzed by amplification of genoma through the polymerase chain reaction and digestion with the restriction enzymes Xmnl (S allele and Taq 1 (Z allele. Results - Seven of the 12 patients had chronic liver disease of undefined etiology and the other five patients had low serum levels of alpha-1-antitrypsin as well as a diagnosis of neonatal cholestasis and/or chronic liver disease of undefined etiology. Five of the 12 patients were homozygous for the Z allele (ZZ and two had the S allele with another allele (*S different from Z. Conclusion - These results show that alpha-1-antitrypsin deficiency is relatively frequent in children with chronic hepatic disease of undefined etiology and/or low alpha-1-antitrypsin levels (41.6%. A correct diagnosis is important for effective clinical follow-up and for genetic counseling.

  7. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  8. Increased humoral immunity by DNA vaccination using an alpha-tocopherol-based adjuvant

    DEFF Research Database (Denmark)

    Karlsson, Ingrid; Borggren, Marie; Nielsen, Jens

    2017-01-01

    approaches. We tested whether the emulsion-based and alpha-tocopherol containing adjuvant Diluvac Forte® has the ability to enhance the immunogenicity of a naked DNA vaccine (i.e., plasmid DNA). As a model vaccine, we used plasmids encoding both a surface-exposed viral glycoprotein (hemagglutinin......) and an internal non-glycosylated nucleoprotein in the Th1/Th2 balanced CB6F1 mouse model. The naked DNA (50 µg) was premixed at a 1:1 volume/volume ratio with Diluvac Forte®, an emulsion containing different concentrations of alpha-tocopherol, the emulsion alone or endotoxin-free phosphate-buffered saline (PBS......). The animals received two intracutaneous immunizations spaced 3 weeks apart. When combined with Diluvac Forte® or the emulsion containing alpha-tocopherol, the DNA vaccine induced a more potent and balanced immunoglobulin G (IgG)1 and IgG2c response, and both IgG subclass responses were significantly enhanced...

  9. Forensics and mitochondrial DNA: applications, debates, and foundations.

    Science.gov (United States)

    Budowle, Bruce; Allard, Marc W; Wilson, Mark R; Chakraborty, Ranajit

    2003-01-01

    Debate on the validity and reliability of scientific methods often arises in the courtroom. When the government (i.e., the prosecution) is the proponent of evidence, the defense is obliged to challenge its admissibility. Regardless, those who seek to use DNA typing methodologies to analyze forensic biological evidence have a responsibility to understand the technology and its applications so a proper foundation(s) for its use can be laid. Mitochondrial DNA (mtDNA), an extranuclear genome, has certain features that make it desirable for forensics, namely, high copy number, lack of recombination, and matrilineal inheritance. mtDNA typing has become routine in forensic biology and is used to analyze old bones, teeth, hair shafts, and other biological samples where nuclear DNA content is low. To evaluate results obtained by sequencing the two hypervariable regions of the control region of the human mtDNA genome, one must consider the genetically related issues of nomenclature, reference population databases, heteroplasmy, paternal leakage, recombination, and, of course, interpretation of results. We describe the approaches, the impact some issues may have on interpretation of mtDNA analyses, and some issues raised in the courtroom.

  10. Human pro. cap alpha. 1)(I) collagen: cDNA sequence for the C-propeptide domain

    Energy Technology Data Exchange (ETDEWEB)

    Maekelae, J K; Raassina, M; Virta, A; Vuorio, E

    1988-01-11

    The authors have previously constructed a cDNA clone pHCAL1, covering most of the C-terminal propeptide domain of human pro..cap alpha..1(I) collagen mRNA,by inserting a 678 bp EcoRI-XhoI fragment of cDNA into pBR322. Since the XhoI/SalI ligation prevented removal of the insert, they used the same strategy to obtain a similar clone in pUC8. RNA was isolated from fetal calvarial bones. The cDNA was digested with EcoRI and XhoI and fractionated on a 1 % agarose gel. Fragments of 650-700 bp were cloned in pUC8 at the polylinker site, which now permits easy removal of the insert. The new clone was named pHCAL1U since the RNA was isolated from another individual. The approach outlined is useful for studies on individual variation which is important to recognize when searching for disease-related mutations in type I collagen.

  11. Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

    Science.gov (United States)

    Schulmeister, Ulrike; Hochwallner, Heidrun; Swoboda, Ines; Focke-Tejkl, Margarete; Geller, Beate; Nystrand, Mats; Härlin, Annika; Thalhamer, Josef; Scheiblhofer, Sandra; Keller, Walter; Niggemann, Bodo; Quirce, Santiago; Ebner, Christoph; Mari, Adriano; Pauli, Gabrielle; Herz, Udo; Valenta, Rudolf; Spitzauer, Susanne

    2009-06-01

    Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

  12. Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with alpha-acetates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA).

    Science.gov (United States)

    Cheng, Guang; Wang, Mingyao; Upadhyaya, Pramod; Villalta, Peter W; Hecht, Stephen S

    2008-03-01

    The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.

  13. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  14. The alpha-spectrin gene is on chromosome 1 in mouse and man.

    Science.gov (United States)

    Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J

    1985-06-01

    By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.

  15. Alpha Particles and X Rays Interact in Inducing DNA Damage in U2OS Cells.

    Science.gov (United States)

    Sollazzo, Alice; Brzozowska, Beata; Cheng, Lei; Lundholm, Lovisa; Haghdoost, Siamak; Scherthan, Harry; Wojcik, Andrzej

    2017-10-01

    Survivors of the atomic bombings of Hiroshima and Nagasaki are monitored for health effects within the Life Span Study (LSS). The LSS results represent the most important source of data about cancer effects from ionizing radiation exposure, which forms the foundation for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The A-bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons. However, until now the possible interaction of the two radiation types of inducing biological effects has not been taken into consideration. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after pure gamma-ray irradiation (such as in the Fukushima prefecture) leads to an overestimation of risk. To analyze the possible interaction of radiation types, a mixed-beam exposure facility was constructed where cells can be exposed to sparsely ionizing X rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein (GFP). The induction and repair of DNA damage, which are known to be related to cancer induction, were analyzed. The results suggest that alpha particles and X rays interact, leading to cellular and possibly cancer effects, which cannot be accurately predicted based on assuming simple additivity of the individual mixed-beam components.

  16. Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

    International Nuclear Information System (INIS)

    Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W.

    1989-01-01

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with [ 3 H] ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major [ 3 H]ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release [ 3 H]ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues

  17. Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Markus Böhm

    2016-05-01

    Full Text Available Alpha-melanocyte-stimulating hormone (alpha-MSH increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation.

  18. Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density

    DEFF Research Database (Denmark)

    Barres, Romain; Osler, Megan E; Yan, Jie

    2009-01-01

    -CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis factor-alpha (TNF-alpha) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non......-CpG methylation of PGC-1alpha and decreased mtDNA and PGC-1alpha mRNA. We provide evidence for PGC-1alpha hypermethylation, concomitant with reduced mitochondrial content in type 2 diabetic patients, and link DNMT3B to the acute fatty-acid-induced non-CpG methylation of PGC-1alpha promoter....

  19. RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

    Science.gov (United States)

    Yu, W; Sanders, B G; Kline, K

    1997-01-01

    RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

  20. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status

    Energy Technology Data Exchange (ETDEWEB)

    Lyckesvärd, Madeleine Nordén, E-mail: madeleine.lyckesvard@oncology.gu.se [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden); Delle, Ulla; Kahu, Helena [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden); Lindegren, Sture [Department of Radiation Physics, Sahlgrenska Academy, University of Gothenburg (Sweden); Jensen, Holger [The PET and Cyclotron Unit Copenhagen University Hospital, Rigshospitalet (Denmark); Bäck, Tom [Department of Radiation Physics, Sahlgrenska Academy, University of Gothenburg (Sweden); Swanpalmer, John [Department of Medical Physics and Biomedical Engineering, Sahlgrenska University Hospital, Gothenburg (Sweden); Elmroth, Kecke [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden)

    2014-07-15

    Highlights: • We study DNA damage response to low-LET photons and high-LET alpha particles. • Cycling primary thyrocytes are more sensitive to radiation than stationary cells. • Influence of radiation quality varies due to cell cycle status of normal cells. • High-LET radiation gives rise to a sustained DNA damage response. - Abstract: Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ({sup 211}At), concentrated in the thyroid by the same mechanism as {sup 131}I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ({sup 60}Co) and alpha particles from {sup 211}At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24 h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to {sup 211}At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1 Gy {sup 211}At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative

  1. Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

    OpenAIRE

    Liew, C C; Jandreski, M A

    1986-01-01

    A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequen...

  2. Detection of alpha particles using DNA/Al Schottky junctions

    Energy Technology Data Exchange (ETDEWEB)

    Al-Ta' ii, Hassan Maktuff Jaber, E-mail: hassankirkukly@gmail.com, E-mail: vengadeshp@um.edu.my [Low Dimensional Materials Research Centre (LDMRC), 50603 Kuala Lumpur (Malaysia); Department of Physics, Faculty of Science, University of Al-Muthana, Al-Muthana 66001 (Iraq); Periasamy, Vengadesh, E-mail: hassankirkukly@gmail.com, E-mail: vengadeshp@um.edu.my [Low Dimensional Materials Research Centre (LDMRC), 50603 Kuala Lumpur (Malaysia); Amin, Yusoff Mohd [Department of Physics, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2015-09-21

    Deoxyribonucleic acid or DNA can be utilized in an organic-metallic rectifying structure to detect radiation, especially alpha particles. This has become much more important in recent years due to crucial environmental detection needs in both peace and war. In this work, we fabricated an aluminum (Al)/DNA/Al structure and generated current–voltage characteristics upon exposure to alpha radiation. Two models were utilized to investigate these current profiles; the standard conventional thermionic emission model and Cheung and Cheung's method. Using these models, the barrier height, Richardson constant, ideality factor and series resistance of the metal-DNA-metal structure were analyzed in real time. The barrier height, Φ value calculated using the conventional method for non-radiated structure was 0.7149 eV, increasing to 0.7367 eV after 4 min of radiation. Barrier height values were observed to increase after 20, 30 and 40 min of radiation, except for 6, 8, and 10 min, which registered a decrease of about 0.67 eV. This was in comparison using Cheung and Cheung's method, which registered 0.6983 eV and 0.7528 eV for the non-radiated and 2 min of radiation, respectively. The barrier height values, meanwhile, were observed to decrease after 4 (0.61 eV) to 40 min (0.6945 eV). The study shows that conventional thermionic emission model could be practically utilized for estimating the diode parameters including the effect of series resistance. These changes in the electronic properties of the Al/DNA/Al junctions could therefore be utilized in the manufacture of sensitive alpha particle sensors.

  3. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))

    1993-08-01

    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  4. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  5. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  6. Phaeophytins from Thyrsacanthus ramosissimus Moric. with inhibitory activity on human DNA topoisomerase II-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Cabral, Analucia Guedes Silveira; Tenorio-Souza, Fabio Henrique; Moura, Marcelo Dantas; Mota, Sabrina Gondim Ribeiro; Silva Lins, Antonio Claudio da; Dias, Celidarque da Silva; Barbosa-Filho, Jose Maria [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Ciencias Frmaceuticas; Giulietti, Ana Maria [Universidade Estadual de Feira de Santana, Feira de Santana, BA (Brazil). Dept. de Ciencias Biologicas; Silva, Tania Maria Sarmento da [Universidade Federal Rural de Pernambuco, Recife, PE (Brazil). Dept. de Ciencias Moleculares; Santos, Creusioni Figueredo dos, E-mail: jbarbosa@ltf.ufpb.br [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Biologia Molecular

    2012-07-01

    Our study reports the extraction and isolation of a new phaeophytin derivative 15{sup 1}-hydroxy-(15{sup 1}-S)-porphyrinolactone, designated anamariaine (1) herein, isolated from the chloroform fraction of aerial parts of Thyrsacanthus ramosissimus Moric. along with the known 15{sup 1}-ethoxy-(15{sup 1}-S)-porphyrinolactone (2). These compounds were identified by usual spectroscopic methods. Both compounds were subjected to in vitro (inhibitory activity) tests by means of supercoiled DNA relaxation techniques and were shown to display inhibitory activity against human DNA topoisomerase II-{alpha} at 50 {mu}M. Interconversion of these two pigments under the mild conditions of the isolation techniques should be highly unlikely but cannot be entirely ruled out. (author)

  7. Synthesis of tritiated 1-alpha-methadol and 1-alpha-acetylmethadol

    Energy Technology Data Exchange (ETDEWEB)

    Thang, D.C.; Nam, N.H.; Pontikis, R. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital Fernand Widal, 75 - Paris (France)); Pichat, L. (CEA Centre d' Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Service des Molecules Marquees)

    1982-04-01

    dl-Methadone was resolved by crystallization of its ammonium d- ..cap alpha.. -bromocamphor-..pi..-sulfonate salt to give d-methadone. The latter in ethyl acetate solution was reduced with tritium gas to 1-..cap alpha..-methadol /sup 3/H in presence of Adams platinum oxide at normal temperature and pressure. Acetylation of 1-..cap alpha..-carbinol hydrochloride by means of acetyl chloride afforded 1-..cap alpha..-acetylmethadol /sup 3/H, specific activity: 20 Ci/mMole. The positions and extent of tritium labelling were determined by /sup 3/H NMR spectroscopy.

  8. Expression of active recombinant human alpha 1-antitrypsin in transgenic rabbits

    NARCIS (Netherlands)

    Massoud, M.; Bischoff, Rainer; Dalemans, W.; Pointu, H.; Attal, J.; Schultz, H.; Clesse, D.; Stinnakre, M.G.; Pavirani, A.; Houdebine, L.M.

    1991-01-01

    A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average

  9. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status

    DEFF Research Database (Denmark)

    Lyckesvärd, Madeleine Nordén; Delle, Ulla; Kahu, Helena

    2014-01-01

    Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same...... mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation...... and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity...

  10. Ap/sub 4/A interactions with a multiprotein form of DNA polymerase. cap alpha. - primase from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Baril, E F; Owen, M W; Vishwanatha, J K; Zamecnik, P C

    1984-06-01

    In previous studies, it was shown that Ap/sub 4/A can function as a primer for in vitro DNA synthesis by the multiprotein form of DNA polymerase ..cap alpha.. with single-stranded DNA and an octadecamer double-stranded DNA template. In these studies, the authors show that Ap/sub 4/A that is greater than 99% pure by high performance liquid chromatography also stimulates the incorporation of (..cap alpha../sup 32/P)ATP into the 10-15 oligoribonucleotide primer with poly(dT) template by the primase that is resolved from the polymerase ..cap alpha.. core enzyme. Other dinucleotides or dinucleotide polyphosphates (e.g. ApA, Ap/sub 2/A or Ap/sub 3/A) do not enhance the incorporation of (..cap alpha../sup 32/P)ATP in this reaction. The results from phosphate transfer experiments demonstrate a covalent linkage between (/sup 3/H)Ap/sub 4/A and the /sup 32/P-labeled oligoriboadenylate that is synthesized by the primase.

  11. Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus.

    OpenAIRE

    Kilbourne, E J; Galper, J B

    1994-01-01

    We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3' end. The 5' end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such...

  12. Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

    International Nuclear Information System (INIS)

    Licastro, F.; Sarafian, T.; Verity, A.M.; Walford, R.L.

    1985-01-01

    The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain

  13. Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability

    OpenAIRE

    Lambert, Muriel W

    2016-01-01

    Non-erythroid alpha spectrin (?IISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. ?IISp?s interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear ?IISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in ma...

  14. Electronic Properties of DNA-Based Schottky Barrier Diodes in Response to Alpha Particles.

    Science.gov (United States)

    Al-Ta'ii, Hassan Maktuff Jaber; Periasamy, Vengadesh; Amin, Yusoff Mohd

    2015-05-21

    Detection of nuclear radiation such as alpha particles has become an important field of research in recent history due to nuclear threats and accidents. In this context; deoxyribonucleic acid (DNA) acting as an organic semiconducting material could be utilized in a metal/semiconductor Schottky junction for detecting alpha particles. In this work we demonstrate for the first time the effect of alpha irradiation on an Al/DNA/p-Si/Al Schottky diode by investigating its current-voltage characteristics. The diodes were exposed for different periods (0-20 min) of irradiation. Various diode parameters such as ideality factor, barrier height, series resistance, Richardson constant and saturation current were then determined using conventional, Cheung and Cheung's and Norde methods. Generally, ideality factor or n values were observed to be greater than unity, which indicates the influence of some other current transport mechanism besides thermionic processes. Results indicated ideality factor variation between 9.97 and 9.57 for irradiation times between the ranges 0 to 20 min. Increase in the series resistance with increase in irradiation time was also observed when calculated using conventional and Cheung and Cheung's methods. These responses demonstrate that changes in the electrical characteristics of the metal-semiconductor-metal diode could be further utilized as sensing elements to detect alpha particles.

  15. Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

    Science.gov (United States)

    Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S

    2001-05-01

    The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

  16. Biological chemistry as a foundation of DNA genealogy: the emergence of "molecular history".

    Science.gov (United States)

    Klyosov, A A

    2011-05-01

    This paper presents the basis of DNA genealogy, a new field of science, which is currently emerging as an unusual blend of biochemistry, history, linguistics, and chemical kinetics. The methodology of the new approach is comprised of chemical (biological) kinetics applied to a pattern of mutations in non-recombinant fragments of DNA (Y chromosome and mtDNA, the latter not being considered in this overview). The goal of the analysis is to translate DNA mutation patterns into time spans to the most recent common ancestors of a given population or tribe and to the dating of ancient migration routes. To illustrate this approach, time spans to the common ancestors are calculated for ethnic Russians, that is Eastern Slavs (R1a1 tribe), Western Slavs (I1 and I2 tribes), and Northern (or Uralic) Slavs (N1c tribe), which were found to live around 4600 years before present (R1a1), 3650 ybp (I1), 3000 and 10,500 ybp (I2, two principal DNA lineages), and 3525 ybp (N1c) (confidence intervals are given in the main text). The data were compared with the respective dates for the nearest common ancestor of the R1a1 "Indo-European" population in India, who lived 4050 years before present, whose descendants represent the majority of the upper castes in India today (up to 72%). Furthermore, it was found that the haplotypes of ethnic Russians of the R1a1 haplogroup (up to 62% of the population in the Russian Federation) and those of the R1a1 Indians (more than 100 million today) are practically identical to each other, up to 67-marker haplotypes. This essentially solves a 200-year-old mystery of who were the Aryans who arrived in India around 3500 years before the present. Haplotypes and time spans to the ancient common ancestors were also compared for the ethnic Russians of haplogroups I1 and I2, on one hand, and the respective I1 and I2 populations in Eastern and Western Europe and Scandinavia, on the other. It is suggested that the approach described in this overview lays the

  17. Calculation of the Electronic Parameters of an Al/DNA/p-Si Schottky Barrier Diode Influenced by Alpha Radiation

    Directory of Open Access Journals (Sweden)

    Hassan Maktuff Jaber Al-Ta'ii

    2015-02-01

    Full Text Available Many types of materials such as inorganic semiconductors have been employed as detectors for nuclear radiation, the importance of which has increased significantly due to recent nuclear catastrophes. Despite the many advantages of this type of materials, the ability to measure direct cellular or biological responses to radiation might improve detector sensitivity. In this context, semiconducting organic materials such as deoxyribonucleic acid or DNA have been studied in recent years. This was established by studying the varying electronic properties of DNA-metal or semiconductor junctions when exposed to radiation. In this work, we investigated the electronics of aluminium (Al/DNA/silicon (Si rectifying junctions using their current-voltage (I-V characteristics when exposed to alpha radiation. Diode parameters such as ideality factor, barrier height and series resistance were determined for different irradiation times. The observed results show significant changes with exposure time or total dosage received. An increased deviation from ideal diode conditions (7.2 to 18.0 was observed when they were bombarded with alpha particles for up to 40 min. Using the conventional technique, barrier height values were observed to generally increase after 2, 6, 10, 20 and 30 min of radiation. The same trend was seen in the values of the series resistance (0.5889–1.423 Ω for 2–8 min. These changes in the electronic properties of the DNA/Si junctions could therefore be utilized in the construction of sensitive alpha particle detectors.

  18. Electronic Properties of DNA-Based Schottky Barrier Diodes in Response to Alpha Particles

    Directory of Open Access Journals (Sweden)

    Hassan Maktuff Jaber Al-Ta'ii

    2015-05-01

    Full Text Available Detection of nuclear radiation such as alpha particles has become an important field of research in recent history due to nuclear threats and accidents. In this context; deoxyribonucleic acid (DNA acting as an organic semiconducting material could be utilized in a metal/semiconductor Schottky junction for detecting alpha particles. In this work we demonstrate for the first time the effect of alpha irradiation on an Al/DNA/p-Si/Al Schottky diode by investigating its current-voltage characteristics. The diodes were exposed for different periods (0–20 min of irradiation. Various diode parameters such as ideality factor, barrier height, series resistance, Richardson constant and saturation current were then determined using conventional, Cheung and Cheung’s and Norde methods. Generally, ideality factor or n values were observed to be greater than unity, which indicates the influence of some other current transport mechanism besides thermionic processes. Results indicated ideality factor variation between 9.97 and 9.57 for irradiation times between the ranges 0 to 20 min. Increase in the series resistance with increase in irradiation time was also observed when calculated using conventional and Cheung and Cheung’s methods. These responses demonstrate that changes in the electrical characteristics of the metal-semiconductor-metal diode could be further utilized as sensing elements to detect alpha particles.

  19. Cytokine vaccination: neutralising IL-1alpha autoantibodies induced by immunisation with homologous IL-1alpha

    DEFF Research Database (Denmark)

    Svenson, M; Hansen, M B; Thomsen, Allan Randrup

    2000-01-01

    with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding...... in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients...

  20. DNA locus HLA-DQ alpha polymorphism in human population of the north-eastern Poland.

    Science.gov (United States)

    Pepiński, W; Skawrońska, M; Janica, J

    1996-01-01

    Investigations on DNA polymorphism locus HLA-DQ alpha were carried out on a sample of 117 adult unrelated inhabitants from the north-eastern Poland. The polymerase chain reaction and the reverse dot-blot hybridisation were employed to detect 6 different HLA-DQ alpha alleles. Population data on 20 different genotypes served as a basis for statistic evaluation. The results of genotype analysis were in Hardy-Weinberg equilibrium. Other population data were compared.

  1. Topoisomerase 3alpha and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frank Hartung

    2008-12-01

    Full Text Available Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3alpha (TOP3alpha functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3alpha in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3alpha-2, which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3alpha-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3alpha, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3alpha is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3alpha mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3alpha and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.

  2. Amplification of marine methanotrophic enrichment DNA with 16S rDNA PCR primers for type II alpha proteobacteria methanotrophs.

    Science.gov (United States)

    Rockne, Karl J; Strand, Stuart E

    2003-09-01

    Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.

  3. Classification of alpha 1-adrenoceptor subtypes

    NARCIS (Netherlands)

    Michel, M. C.; Kenny, B.; Schwinn, D. A.

    1995-01-01

    Two alpha 1-adrenoceptor subtypes (alpha 1A and alpha 1B) have been detected in various tissues by pharmacological techniques, and three distinct cDNAs encoding alpha 1-adrenoceptor subtypes have been cloned. The profile of an increasing number of subtype-selective compounds at cloned and endogenous

  4. Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

    Science.gov (United States)

    Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

    2004-06-01

    Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

  5. Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks

    International Nuclear Information System (INIS)

    Hofbauer, Daniela

    2010-01-01

    Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

  6. In-vitro DNA binding and cleavage studies with pBR322 of N,N-Bis(3{beta}-acetoxy-5{alpha}-cholest-6-yl-idene)hydrazine

    Energy Technology Data Exchange (ETDEWEB)

    Tabassum, Zishan [School of Industrial Technology, Universiti Sains Malaysia, 11800 USM, Penang (Malaysia); Muddassir, Mohd [Department of Chemistry, Aligarh Muslim University, Aligarh 202002, U.P. (India); Sulaiman, Othman [School of Industrial Technology, Universiti Sains Malaysia, 11800 USM, Penang (Malaysia); Arjmand, Farukh [Department of Chemistry, Aligarh Muslim University, Aligarh 202002, U.P. (India)

    2012-08-15

    The DNA binding studies of the triterpenoid derivative, cholesterol, N,N-Bis(3{beta}-acetoxy-5{alpha}-cholest-6-yl-idene)hydrazine (L) with CT DNA were carried out by employing different optical methods viz, UV-vis and fluorescence spectroscopy. The ligand binds to DNA through hydrophobic interaction with K{sub b} value found to be 4.7 Multiplication-Sign 10{sup 3} M{sup -1}. These observations have been validated also by fluorescence spectroscopy. (L) exhibits a remarkable DNA cleavage activity with pBR322 DNA in the presence of different activators and the DNA is probably cleaved by an other than oxidative mechanism, possibly by a discernable hydrolytic pathway. In the presence of major and minor groove binding agents, (L) prefers major groove binding of the DNA. - Highlights: Black-Right-Pointing-Pointer DNA binding studies of the triterpenoid derivative, cholesterol, N,N-Bis(3{beta}-acetoxy-5{alpha}-cholest-6-yl-idene)hydrazine. Black-Right-Pointing-Pointer The ligand binds to DNA through hydrophobic interaction with K{sub b} value found to be 4.7 Multiplication-Sign 10{sup 3} M{sup -1}. Black-Right-Pointing-Pointer DNA is probably cleaved by an other than oxidative mechanism, possibly by a discernable hydrolytic pathway. Black-Right-Pointing-Pointer In the presence of major and minor groove binding agents, the (L) prefers major groove binding of the DNA.

  7. Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

    DEFF Research Database (Denmark)

    Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

    2003-01-01

    decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

  8. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  9. Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells

    Directory of Open Access Journals (Sweden)

    Harvey Mario

    2010-01-01

    Full Text Available Abstract Background UDP-glucuronosyltransferase 1A1 (UGT1A1 is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4 is found close to a HNF1 response element (HRE, known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF binding site. Results Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1. Conclusions This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.

  10. Alpha 1A and alpha 1B-adrenoceptors enhance inositol phosphate generation in rat renal cortex

    NARCIS (Netherlands)

    Michel, M. C.; Büscher, R.; Philipp, T.; Brodde, O. E.

    1993-01-01

    We have studied the role of alpha 1A- and alpha 1B-adrenoceptors in noradrenaline- and methoxamine-stimulated inositol phosphate accumulation in rat renal cortical slices. [3H]Prazosin binding studies with and without inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment suggested

  11. Alpha 1 B- but not alpha 1 A-adrenoceptors mediate inositol phosphate generation

    NARCIS (Netherlands)

    Michel, M. C.; Hanft, G.; Gross, G.

    1990-01-01

    We used novel highly subtype-selective antagonists to study whether alpha 1A- and/or alpha 1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 microM) and prazosin (100 nM) completely abolished the stimulated inositol

  12. Assignment of casein kinase 2 alpha sequences to two different human chromosomes

    DEFF Research Database (Denmark)

    Boldyreff, B; Klett, C; Göttert, E

    1992-01-01

    Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter...

  13. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  14. Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

    Science.gov (United States)

    Buczek, Pawel; Horvath, Martin P

    2006-06-23

    The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

  15. Alpha1-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Stolk, Jan; Seersholm, Niels; Kalsheker, Noor

    2006-01-01

    The Alpha One International Registry (AIR), a multinational research program focused on alpha1-antitrypsin (AAT) deficiency, was formed in response to a World Health Organization recommendation. Each of the nearly 20 participating countries maintains a national registry of patients with AAT defic...

  16. A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

    Science.gov (United States)

    Steinberg, M H; Coleman, M B; Adams, J G; Hartmann, R C; Saba, H; Anagnou, N P

    1986-02-01

    A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

  17. Treatment of lung disease in alpha-1 antitrypsin deficiency: a systematic review

    Directory of Open Access Journals (Sweden)

    Edgar RG

    2017-05-01

    Full Text Available Ross G Edgar,1,2 Mitesh Patel,3 Susan Bayliss,4 Diana Crossley,2,5 Elizabeth Sapey,2,5 Alice M Turner4,6 1Therapy Services, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK; 2Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK; 3Division of Primary Care, University of Nottingham, Nottingham, UK; 4Institute of Applied Health Research, University of Birmingham, Birmingham, UK; 5Department of Respiratory Medicine, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK; 6Department of Respiratory Medicine, Heart of England NHS Foundation Trust, Birmingham, UK Background: Alpha-1 antitrypsin deficiency (AATD is a rare genetic condition predisposing individuals to chronic obstructive pulmonary disease (COPD. The treatment is generally extrapolated from COPD unrelated to AATD; however, most COPD trials exclude AATD patients; thus, this study sought to systematically review AATD-specific literature to assist evidence-based patient management.Methods: Standard review methodology was used with meta-analysis and narrative synthesis (PROSPERO-CRD42015019354. Eligible studies were those of any treatment used in severe AATD. Randomized controlled trials (RCTs were the primary focus; however, case series and uncontrolled studies were eligible. All studies had ≥10 participants receiving treatment or usual care, with baseline and follow-up data (>3 months. Risk of bias was assessed appropriately according to study methodology.Results: In all, 7,296 studies were retrieved from searches; 52 trials with 5,632 participants met the inclusion criteria, of which 26 studies involved alpha-1 antitrypsin augmentation and 17 concerned surgical treatments (largely transplantation. Studies were grouped into four management themes: COPD medical, COPD surgical, AATD specific, and other treatments. Computed tomography (CT density, forced expiratory volume in 1 s, diffusing capacity of the lungs for carbon monoxide

  18. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

    Science.gov (United States)

    Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

    2017-04-01

    Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor

  19. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from

  20. Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

    OpenAIRE

    Liebhaber, S A; Kan, Y W

    1981-01-01

    The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expressi...

  1. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  2. Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

    Science.gov (United States)

    Mahotka, C; Hansen-Hagge, T E; Bartram, C R

    1995-10-01

    Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

  3. Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

    Science.gov (United States)

    Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

    2003-08-01

    Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

  4. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

    Science.gov (United States)

    Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

    2004-10-01

    We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

  5. Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

    Science.gov (United States)

    Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

    1995-03-01

    A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

  6. Effects of alpha particles on zebrafish embryos

    International Nuclear Information System (INIS)

    Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

    2008-01-01

    Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

  7. Alpha-fetoprotein and Fanconi Anemia: Relevance to DNA Repair and Breast Cancer Susceptibility.

    Science.gov (United States)

    Lakhi, Nisha A; Mizejewski, Gerald J

    2017-02-01

    Elevations of serum alpha-fetoprotein (sAFP) have been reported in fetal and infant states of anemia. Fanconi anemia (FA) belongs to a family of genetic instability disorders which lack the capability to repair DNA breaks. The lesion occurs at a checkpoint regulatory step of the G2 to mitotic transition, allowing FA cells to override cell-cycle arrest. FA DNA repair pathways contain complementation groups known as FANC proteins. FANC proteins form multi-protein complexes with BRCA proteins and are involved in homologous DNA repair. An impaired cascade in these events imparts an increased breast cancer susceptibility to female FA patients. Elevations of sAFP have availed this fetal protein to serve as a biomarker for FA disease. However, the origin of the synthesis of sAFA has not been determined in FA patients. We hypothesize that hematopoietic multipotent progenitor stem cells in the bone marrow are the source of sAFP production in FA patients.

  8. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Hui-Hsuan [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chou, Fen-Pi [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Wang, Chau-Jong [Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Hsuan, Shu-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China); Wang, Cheng-Kun [E-Chyun Dermatology Clinic, No.70, Sec. 3, Jhonghua E. Rd., East District, Tainan, Taiwan (China); Chen, Jing-Hsien [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China)

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  9. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

    Science.gov (United States)

    Landvik, N E; Arlt, V M; Nagy, E; Solhaug, A; Tekpli, X; Schmeiser, H H; Refsnes, M; Phillips, D H; Lagadic-Gossmann, D; Holme, J A

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system. Copyright 2009 Elsevier B.V. All rights reserved.

  10. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Landvik, N.E. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Arlt, V.M.; Nagy, E. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Solhaug, A. [Section for Toxicology, Department of Feed and Food Safety, National Veterinary Institute Pb 750 Sentrum, N-0106 Oslo (Norway); Tekpli, X. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Schmeiser, H.H. [Research Group Genetic Alteration in Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Refsnes, M. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Phillips, D.H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Lagadic-Gossmann, D. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Holme, J.A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway)

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ({sup 32}P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of I{kappa}B-{alpha} (suggesting activation of NF-{kappa}B) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-{kappa}B play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

  11. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  12. Characterization of Makrofol ® DE 1-1 for alpha particle radiography

    Science.gov (United States)

    El Ghazaly, M.; Aydarous, Abdulkadir; Al-Thomali, Talal A.

    2017-09-01

    Makrofol ® DE 1-1 (bisphenol-A polycarbonate) was investigated for alpha particle radiography. The edge spread function (ESF) was measured by razor-blade's edge. Makrofol ® DE 1-1 detectors were irradiated with perpendicular incident alpha particles of energy 2.5, 4 and 5.4 MeV, thereafter they were etched in 75% 6N KOH+25% C2H5OH at a temperature of 50 °C for different durations. The etched Makrofol®DE 1-1 detectors were imaged with an optical microscope equipped with a CCD camera. The results revealed that the green channel of the original RGB image provides the highest contrast comparing with red and blue channel by a factor of 27.6% of the original RGB image. The image contrast of alpha particle-irradiated Makrofol®DE 1-1 detector was found to be inversely related to the etching time since the alpha particle tracks proceed from a conical phase to spherical phase. The spatial resolution of alpha particle-irradiated Makrofol®DE 1-1 detector, in terms of line spread function, was found to deteriorate as the etching time increases for all examined alpha particle energies. The results revealed the potential capability of Makrofol®DE 1-1 detector as an efficient detector for alpha particle radiography such as autoradiography.

  13. Alteration of alpha 1 Na+,K(+)-ATPase 86Rb+ influx by a single amino acid substitution

    International Nuclear Information System (INIS)

    Herrera, V.L.; Ruiz-Opazo, N.

    1990-01-01

    The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension

  14. Anti-IL-1alpha autoantibodies in early rheumatoid arthritis

    DEFF Research Database (Denmark)

    Forslind, K; Svensson, Birte; Svenson, M

    2001-01-01

    To investigate the potential predictive value of autoantibodies against IL1-alpha (anti-IL-1alpha) in patients with early rheumatoid arthritis (RA).......To investigate the potential predictive value of autoantibodies against IL1-alpha (anti-IL-1alpha) in patients with early rheumatoid arthritis (RA)....

  15. Alpha-1 Antitrypsin Deficiency (Inherited Emphysema)

    Science.gov (United States)

    ... antitrypsin inactivates elastase once it has finished its job. Without alpha 1 antitrypsin, elastase can destroy the air sacs of the lung. How is the diagnosis made? Because Alpha-1 related disease is COPD, the diagnosis is made by the same methods. Your doctor may have you do a number ...

  16. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

    Science.gov (United States)

    Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

    2004-01-16

    Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

  17. Evaluation on a radioimmunoassay of. alpha. /sub 1/ microglobulin (. alpha. /sub 1/-m) with simplified procedures

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Kazuyo; Moriuma, Hatsuko; Mishima, Chiho; Honda, Minoru; Tomonobu, Masahiro; Kanao, Keisuke; Fushimi, Hisako (Sumitomo Hospital, Osaka (Japan))

    1989-06-01

    A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25{mu}l, and thus 200 mg/l of {alpha}/sub 1/-m standard was prepared using 50 {mu}l of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intraassay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about {alpha}/sub 1/-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of {alpha}/sub 1/-m were less than 25 mg/l is serum and less than 10 mg/l in urine. The present results indicate that the determination of {alpha}/sub 1/-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function. (author).

  18. Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

    Science.gov (United States)

    Nagasawa, Hatsumi; Lin, Yu-Fen; Kato, Takamitsu A; Brogan, John R; Shih, Hung-Ying; Kurimasa, Akihiro; Bedford, Joel S; Chen, Benjamin P C; Little, John B

    2017-02-01

    The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single

  19. Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after {alpha}-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Han Wei [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Chen Shaopeng [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Wu Lijun [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)

    2010-02-03

    Low-dose {alpha}-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose {alpha}-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor {beta}1 (TGF-{beta}1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-{beta}1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

  20. Immunodetection of Thyroid Hormone Receptor (Alpha1/Alpha2) in the Rat Uterus and Oviduct

    International Nuclear Information System (INIS)

    Öner, Jale; Öner, Hakan

    2007-01-01

    The aim of this study was to investigate the immunolocalization and the existence of thyroid hormone receptors (THR) (alpha1/alpha2) in rat uterus and oviduct. For this purpose 6 female Wistar albino rats found in estrous period were used. Tissue samples fixed in 10% neutral formalin were examined immunohistochemically. Sections were incubated with primary mouse-monoclonal THR (alpha1/alpha2) antibody. In uterus, THR (alpha1/alpha2) immunoreacted strongly with uterine luminal epithelium, endometrial gland epithelium and endometrial stromal cells and, moderately with myometrial smooth muscle. In oviduct, they were observed moderately in the epithelium of the tube and the smooth muscle cells of the muscular layer. In conclusion, the presence of THR in uterus and oviduct suggests that these organs are an active site of thyroid hormones

  1. High frequency of HIF-1 alpha overexpression in BRCA1 related breast cancer

    NARCIS (Netherlands)

    van der Groep, Petra; Bouter, Alwin; Menko, Fred H.; van der Wall, Elsken; van Diest, Paul J.

    2008-01-01

    Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1 alpha (HIF-1 alpha) is the key regulator of the hypoxia response. HIF-1 alpha is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1 alpha in hereditary breast

  2. Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Morris, D R; Jorstad, C M; Seyfried, C E

    1977-09-01

    The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

  3. Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

    Science.gov (United States)

    Yamaguchi, Y; Crane, S; Zhou, L; Ochoa, S M; Falanga, V

    2000-12-01

    Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

  4. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  5. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  6. Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation of iron.

    Science.gov (United States)

    Park, Sung-Soo; Bae, Insoo; Lee, Yong J

    2008-04-15

    Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF-1alpha is continuously degraded by the ubiquitin-proteasome pathway through pVHL (von Hippel-Lindau tumor suppressor protein). Under hypoxic conditions, HIF-1alpha is stabilized and induces the transcription of HIF-1 target genes. Quercetin, a flavonoid with anti-oxidant, anti-inflammatory, and kinase modulating properties, has been found to induce HIF-1alpha accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin-mediated HIF-1alpha accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF-1alpha can be induced through the phosphatidylinositol 3-kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant-type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin-induced HIF-1alpha accumulation. And we observed that HIF-1alpha accumulated by quercetin is not ubiquitinated and the interaction of HIF-1alpha with pVHL is reduced, compared with HIF-1alpha accumulated by the proteasome inhibitor MG132. The use of quercetin's analogues showed that only quercetin and galangin induce HIF-1/2alpha accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF-1/2alpha proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF-1/2alpha in normoxia by hindering PHD through chelating iron ions.

  7. Hypothalamic PGC-1 alpha Protects Against High-Fat Diet Exposure by Regulating ER alpha

    NARCIS (Netherlands)

    Morselli, Eugenia; Fuente-Martin, Esther; Finan, Brian; Kim, Min; Frank, Aaron; Garcia-Caceres, Cristina; Navas, Carlos Rodriguez; Gordillo, Ruth; Neinast, Michael; Kalainayakan, Sarada P.; Li, Dan L.; Gao, Yuanqing; Yi, Chun-Xia; Hahner, Lisa; Palmer, Biff F.; Tschöp, Matthias H.; Clegg, Deborah J.

    2014-01-01

    High-fat diets (HFDs) lead to obesity and inflammation in the central nervous system (CNS). Estrogens and estrogen receptor alpha (ER alpha) protect premenopausal females from the metabolic complications of inflammation and obesity-related disease. Here, we demonstrate that hypothalamic PGC-1 alpha

  8. Construction and identification of eukaryotic expression vector of pcDNA3-UHRF1

    International Nuclear Information System (INIS)

    Li Xinli; Zhu Ran; Zhu Wei; Fan Saijun; Meng Qinghui

    2011-01-01

    Objective: To generate eukaryotic expression vector of pcDNA3-UHRF1(ubiquitin-like, containing PHD and RING finger domains 1, UHRF1) and testify its expression in breast cancer cells MDA-MB-231. Methods: A 2.3 kb cDNA fragment was amplified from the total RNA of the human breast cancer cells MCF-7 by the RT-PCR method and was cloned into the plasmid pcDNA3. The vector was identified by the double digestion with restriction enzymes Kpn I and Xho I and was sequenced. The cDNA of UHRF1 was transfected into human breast cancer cells MDA-MB-231 by Lipofactamin2000. The positive clones were selected by G418. The expression of the UHRF1 was detected by RT-PCR and Western blot analysis. Results: The recombinant eukaryotic expression vector pcDNA3-UHRF1 was digested with Kpn I and BamH I, and the electrophoresis of the digested products showed two fragments; 2.3kb fragment of UHRF1 and 5.4 kb fragment of pcDNA3, and the sequence inserted was identical to the published sequence. The MDA-MB-231 cells transfected with the pcDNA3-UHRF1 plasmid expressed a high level of the UHRF1 mRNA and protein. Conclusion: The recombinant eukaryotic cell expression vector of pcDNA3-UHRF1 is constructed successfully. The recombinant plasmid pcDNA3-UHRF1 can provide a very useful tool and lay an important foundation for the research on the function of UHRF1. (authors)

  9. Radiobiological Effects of Alpha-Particles from Astatine-211: From DNA Damage to Cell Death

    Energy Technology Data Exchange (ETDEWEB)

    Claesson, Kristina

    2011-05-15

    In recent years, the use of high linear energy transfer (LET) radiation for radiotherapeutic applications has gained increased interest. Astatine-211 (211At) is an alpha-particle emitting radionuclide, promising for targeted radioimmunotherapy of isolated tumor cells and microscopic clusters. To improve development of safe radiotherapy using 211At it is important to increase our knowledge of the radiobiological effects in cells. During radiotherapy, both tumors and adjacent normal tissue will be irradiated and therefore, it is of importance to understand differences in the radio response between proliferating and resting cells. The aim of this thesis was to investigate effects in fibroblasts with different proliferation status after irradiation with alpha-particles from 211At or X-rays, from inflicted DNA damage, to cellular responses and biological consequences. Throughout this work, irradiation was performed with alpha-particles from 211A or X-rays. The induction and repair of double-strand breaks (DSBs) in human normal fibroblasts were investigated using pulsed-field gel electrophoresis and fragment analysis. The relative biological effectiveness (RBE) of 211At for DSB induction varied between 1.4 and 3.1. A small increase of DSBs was observed in cycling cells compared to stationary cells. The repair kinetics was slower after 211At and more residual damage was found after 24 h. Comparison between cells with different proliferation status showed that the repair was inefficient in cycling cells with more residual damage, regardless of radiation quality. Activation of cell cycle arrests was investigated using immunofluorescent labeling of the checkpoint kinase Chk2 and by measuring cell cycle distributions with flow cytometry analysis. After alpha-particle irradiation, the average number of Chk2-foci was larger and the cells had a more affected cell cycle progression for several weeks compared with X-irradiated cells, indicating a more powerful arrest after 211At

  10. DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

    Science.gov (United States)

    Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

    1995-09-05

    Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

  11. The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1.

    Science.gov (United States)

    Chin, H; Krall, M; Kim, H L; Kozak, C A; Mock, B

    1992-12-01

    Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

  12. A discriminatory function for prediction of protein-DNA interactions based on alpha shape modeling.

    Science.gov (United States)

    Zhou, Weiqiang; Yan, Hong

    2010-10-15

    Protein-DNA interaction has significant importance in many biological processes. However, the underlying principle of the molecular recognition process is still largely unknown. As more high-resolution 3D structures of protein-DNA complex are becoming available, the surface characteristics of the complex become an important research topic. In our work, we apply an alpha shape model to represent the surface structure of the protein-DNA complex and developed an interface-atom curvature-dependent conditional probability discriminatory function for the prediction of protein-DNA interaction. The interface-atom curvature-dependent formalism captures atomic interaction details better than the atomic distance-based method. The proposed method provides good performance in discriminating the native structures from the docking decoy sets, and outperforms the distance-dependent formalism in terms of the z-score. Computer experiment results show that the curvature-dependent formalism with the optimal parameters can achieve a native z-score of -8.17 in discriminating the native structure from the highest surface-complementarity scored decoy set and a native z-score of -7.38 in discriminating the native structure from the lowest RMSD decoy set. The interface-atom curvature-dependent formalism can also be used to predict apo version of DNA-binding proteins. These results suggest that the interface-atom curvature-dependent formalism has a good prediction capability for protein-DNA interactions. The code and data sets are available for download on http://www.hy8.com/bioinformatics.htm kenandzhou@hotmail.com.

  13. Expansion of microsatellite in the thyroid hormone receptor-alpha1 gene linked to increased receptor expression and less aggressive thyroid cancer

    DEFF Research Database (Denmark)

    Onda, Masamitsu; Li, Daisy; Suzuki, Shinichi

    2002-01-01

    PURPOSE: The purpose of this study was to determine whether the length of the THRA1 microsatellite, which resides in a noncoding portion of the thyroid hormone receptor-alpha1 gene, affects receptor expression and is linked to clinicopathological parameters in thyroid cancer. EXPERIMENTAL DESIGN......: In 30 cases of surgically resected sporadic thyroid cancer, the length of the THRA1 microsatellite was determined by DNA sequence analysis, and expression of thyroid hormone receptor-alpha1 was assessed immunohistochemically in thin sections cut from tumor blocks. The length of THRA1 and expression...... of thyroid hormone receptor-alpha1 were also assessed in seven cancer cell lines. Regression analysis was used to gauge the correlation between the size of THRA1 and receptor expression. Multivariate analysis was used to test for links to the clinical parameters of gender, age, histology, stage, nodal...

  14. DNA polymerase. beta. reaction with ultraviolet-irradiated DNA incised by correndonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Nowak, R; Zarebska, Z [Instytut Onkologii, Warsaw (Poland); Zmudzka, B [Polska Akademia Nauk, Warsaw. Inst. Biochemii i Biofizyki

    1980-09-19

    Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J/m/sup 2/, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with Micrococcus luteus correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf thymus DNA polymerase ..cap alpha.. but was recognized as a template by DNA polymerase ..beta... The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules of dNMP) per one correndonuclease incision. The length of the DNA polymerase ..beta.. product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg/sup 2 +/ and four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate.

  15. Frequency of distribution of inflammatory cytokines IL-1, IL-6 and TNF-alpha gene polymorphism in patients with obstructive sleep apnea.

    Science.gov (United States)

    Popko, K; Gorska, E; Potapinska, O; Wasik, M; Stoklosa, A; Plywaczewski, R; Winiarska, M; Gorecka, D; Sliwinski, P; Popko, M; Szwed, T; Demkow, U

    2008-12-01

    Obesity is one of the most commonly identified factors for the obstructive sleep apnea syndrome (OSAS). Adipose tissue is the source of many cytokines, among them there are IL-6, IL-1, and TNF-alpha. The level of inflammatory cytokines increases in people with OSAS and obesity. The aim of this study was to evaluate the distribution of genotypes in inflammatory cytokine genes in people with obesity-related OSAS. The examined group consisted of 102 person with obesity related-OSAS and 77 normal weight person without OSAS. Genotyping of DNA sequence variation was carried out by restriction enzyme (IL-1: Taq I, IL-6: Lwe I, TNF-alpha: Nco I) analysis of PCR amplified DNA. The study revealed a significant correlation between polymorphism located in the promoter region of inflammatory cytokine genes and obesity-related OSAS.

  16. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.

    2005-01-01

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site...

  17. Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

    1994-09-01

    The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

  18. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

    Directory of Open Access Journals (Sweden)

    Ilker Kudret Sariyer

    Full Text Available PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV, which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs. We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF. SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen.Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins.Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to

  19. Serum concentrations of interleukin-1 alpha, interleukin-6 and tumor necrosis factor-alpha in neonatal sepsis and meningitis

    International Nuclear Information System (INIS)

    Fida, Nadia M.; Fadelallah, Mohamed F.; Al-Mughales, Jamil A.

    2006-01-01

    To investigate whether serum levels of interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor alpha (TNF-alpha), C-reactive protein (CRP) are useful in the diagnosis of neonatal sepsis and meningitis and differentiate them. Blood samples were collected from 35 full term neonates with suspected infection who admitted to the Neonatology Unit, Pediatric Department, King Abdul-Aziz University Hospital, Jeddah, Saudi Arabia during January 2002 - June 2003. On the basis of laboratory and bacteriological results, newborns were classified into: sepsis (n=28), meningitis (n=7), and healthy controls (n=16). Sepsis groups were further subdivided according to culture results into: group 1 = proven sepsis (n=6), group 2 = clinical sepsis (n=14), and group 3 = possible-infected (n=8). Serum levels of IL-1alpha, IL-6, TNF-alpha were measured using Enzyme-Linked Immunosorbent Assay while CRP by nephelometer: In sepsis and meningitis patients, serum levels of CRP (p<0.01, p<0.05,) and IL-1alpha (p<0.001, p<0.05) were elevated than controls. C-reactive protein levels elevated in proven sepsis (p<0.001) and IL-1alpha elevated in all subgroups of sepsis (groups 1, 2, 3) compared with (p<0.05, p<0.001, p<0.01) controls. Interleukin-6, TNF-alpha showed no significant differences between studied groups. In sepsis and meningitis, IL-1alpha had a highest sensitivity (89%, 86%), and negative predictive values (89% and 93%). Interleukin-1alpha and CRP increased in neonatal sepsis and meningitis, but cannot differentiate between them. Interleukin-1alpha had a highest sensitivity in prediction of neonatal infection and its assessment may improve accuracy of diagnosis. (author)

  20. BstXI RFLP in the human inter-alpha-trypsin inhibitor light chain gene

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T; Bourguignon, J; Sesbouee, R; Hanauer, A; Salier, J P; Diarra-Mehrpour, M; Martin, J P

    1988-03-25

    The 1.2 kb EcoRI/SmaI fragment of lambdaHuLITI2 was used as probe. lambdaHuLITI2 is a full length cDNA clone coding for human inter-alpha-trypsin inhibitor light chain isolated from immunochemical screening of a lambdagt11 library. Its sequence coding for HI-30 and alpha-1-microglobulin is in agreement. BstXI identifies five invariant bands at 5.0 kb, 2.3 kb, 1.5 kb, 1.1 kb, and 0.7 kb and a diallelic polymorphism with DNA fragments at 2.0 kb or 1.7 kb.

  1. In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

    Directory of Open Access Journals (Sweden)

    Ka L. Hong

    2015-01-01

    Full Text Available Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

  2. Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

    Science.gov (United States)

    Avni, A; Avital, S; Gromet-Elhanan, Z

    1991-04-25

    Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

  3. Alteration of alpha 1 Na+,K(+)-ATPase sup 86 Rb sup + influx by a single amino acid substitution

    Energy Technology Data Exchange (ETDEWEB)

    Herrera, V.L.; Ruiz-Opazo, N. (Boston Univ. School of Medicine, MA (USA))

    1990-08-31

    The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension.

  4. Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

    Science.gov (United States)

    Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

    2009-10-01

    1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

  5. Role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in acute lung injury in rats

    DEFF Research Database (Denmark)

    Shanley, T P; Schmal, H; Friedl, H P

    1995-01-01

    in bronchoalveolar lavage (BAL) fluids by Western blot analysis. Anti-MIP-1 alpha administered at commencement of IgG immune complex- or LPS-induced injury resulted in significant reductions in BAL neutrophils as well as in injury as measured by pulmonary vascular permeability. Under such conditions, in both models...... to production of TNF-alpha, which in turn up-regulates vascular adhesion molecules required for neutrophil influx....

  6. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

    Directory of Open Access Journals (Sweden)

    Freya M Mowat

    2010-06-01

    Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

  7. The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Y.; Vavougios, G.; Hinek, A. [Univ. of Toronto (Canada)] [and others

    1996-07-01

    Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.

  8. Complex rearrangements within the human J delta-C delta/J alpha-C alpha locus and aberrant recombination between J alpha segments

    NARCIS (Netherlands)

    Baer, R.; Boehm, T.; Yssel, H.; Spits, H.; Rabbitts, T. H.

    1988-01-01

    We have examined DNA rearrangements within a 120 kb cloned region of the human T cell receptor J delta-C delta/J alpha-C alpha locus. Three types of pattern emerge from an analysis of T cell lines and clones. Firstly, cells with two rearrangements within J delta-C delta; secondly, cells with one

  9. Intravenous alpha-1 antitrypsin augmentation therapy: systematic review

    DEFF Research Database (Denmark)

    Gøtzsche, Peter C; Johansen, Helle Krogh

    2010-01-01

    We reviewed the benefits and harms of augmentation therapy with alpha-1 antitrypsin in patients with alpha-1 antitrypsin deficiency and lung disease. We searched for randomised trials comparing augmentation therapy with placebo or no treatment in PubMed and ClinicalTrials (7 January 2010). Two...... (difference 1.14 g/l; 95% confidence interval 0.14 to 2.14; p = 0.03) over the total course of the trials. Augmentation therapy with alpha-1 antitrypsin cannot be recommended in view of the lack of evidence of clinical benefit and the cost of treatment....

  10. The prevalence of alpha-1 antitrypsin deficiency in Ireland.

    LENUS (Irish Health Repository)

    Carroll, Tomas P

    2011-07-13

    Abstract Background Alpha-1 antitrypsin deficiency (AATD) results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. Methods We present data from the first 3,000 individuals screened following ATS\\/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP). We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. Results The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. Conclusion Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.

  11. The prevalence of alpha-1 antitrypsin deficiency in Ireland.

    LENUS (Irish Health Repository)

    Carroll, Tomas P

    2012-02-01

    BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. METHODS: We present data from the first 3,000 individuals screened following ATS\\/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP). We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. RESULTS: The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. CONCLUSION: Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.

  12. PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

    DEFF Research Database (Denmark)

    Gillberg, Linn; Jacobsen, Stine; Rönn, Tina

    2014-01-01

    OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High...... and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA...... methylation was assessed with bisulfite sequencing and mRNA expression with quantitative real-time PCR. RESULTS: Following high-fat overfeeding, increased SAT PPARGC1A DNA methylation was observed in LBW subjects but not in NBW controls. Basal SAT PPARGC1A mRNA expression was unaffected by diet and similar...

  13. Correlation of repressed transcription of alpha-tocopherol transfer protein with serum alpha-tocopherol during hepatocarcinogenesis

    NARCIS (Netherlands)

    Wu, C. G.; Hoek, F. J.; Groenink, M.; Reitsma, P. H.; van Deventer, S. J.; Chamuleau, R. A.

    1997-01-01

    Using a subtraction-enhanced display technique, we identified a rodent alpha-tocopherol transfer protein (alpha-TTP) cDNA which exhibited markedly lower messenger RNA (mRNA) amounts in rat hepatocellular carcinoma (HCC) than in healthy controls. Several lines of evidence have substantiated that

  14. Reggeon field theory for alpha (0)>1

    CERN Document Server

    Amati, Daniele; Le Bellac, M; Marchesini, G

    1976-01-01

    The asymptotic behaviour of the scattering amplitude is obtained when the pomeron has intercept alpha (0) larger than one. The reggeon field theory is studied by introducing a lattice in impact parameter space. Use is made of a previous result showing that asymptotically the dynamics is controlled at each lattice site ( alpha '=0 case) by a two-level structure. This leads to a non-Hermitean Hamiltonian expressed in terms of spin operators in which the intersite interaction term is proportional to the pomeron slope alpha '. The spectrum of such a system shows a degenerate ground state for alpha (0)> alpha /sub c/>or approximately=1 and a continuum with vanishing excitation gap at alpha (0)= alpha /sub c/. The vacuum does not change structure at the critical value. The criticality is shown by an order parameter which is given by the matrix element of a field operator between the vacuum and its degenerate companion. The nature of this critical phenomenon is better understood by continuously transforming the Hami...

  15. Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA

    International Nuclear Information System (INIS)

    Setlow, B.; Hand, A.R.; Setlow, P.

    1991-01-01

    Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores

  16. Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion.

    OpenAIRE

    Liebhaber, S A; Cash, F E; Main, D M

    1985-01-01

    alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expressi...

  17. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

  18. Inhibition of HIF-2.alpha. heterodimerization with HIF1.beta. (ARNT)

    Science.gov (United States)

    Bruick, Richard K.; Caldwell, Charles G.; Frantz, Doug E.; Gardner, Kevin H.; MacMillan, John B.; Scheuermann, Thomas H.; Tambar, Uttam K.

    2017-09-12

    Provided is a method of inhibiting heterodimerization of HIF-2.alpha. to HIF1.beta. (ARNT) comprising binding certain small molecules to the HIF-2.alpha. PAS-B domain cavity but not to HIF1.alpha. and inhibiting HIF-2.alpha. heterodimerization to HIF1.beta. (ARNT) but not inhibiting HIF1.alpha. heterodimerization to HIF1.beta. (ARNT). Those certain small molecules are also referenced synonymously as HIF2-HDI and HIF2.alpha. heterodimerization inhibitors and also simply as certain small molecules.

  19. Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

    Science.gov (United States)

    Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

    2007-09-01

    Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

  20. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jianmin, E-mail: jmhuang@partners.org [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Levitsky, Lynne L. [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Rhoads, David B., E-mail: rhoads@helix.mgh.harvard.edu [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States)

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  1. Involvement of reversible binding to alpha 2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity.

    Science.gov (United States)

    Charbonneau, M; Strasser, J; Lock, E A; Turner, M J; Swenberg, J A

    1989-06-01

    Similarly to unleaded gasoline, 1,4-dichlorobenzene (1,4-DCB) administered for 2 years caused a dose-related increase in the incidence of renal tumors in male but not in female rats or in either sex of mice. Unleaded gasoline and 2,2,4-trimethylpentane (TMP), a component of unleaded gasoline, increased protein droplet formation and cell proliferation in male but not in female rat kidneys. These protein droplets contained, alpha 2u-globulin, a male rat-specific low-molecular-weight protein and 2,4,4-trimethyl-2-pentanol, a metabolite of TMP that was reversibly bound to this protein. Studies were undertaken to determine if 1,4-DCB produced similar effects; 1,2-DCB was used for comparison since it did not produce renal carcinogenesis in male rats. Gel filtration chromatography of a 116,000g supernatant prepared from kidneys of 1,4-[14C]DCB-treated rats showed that radiolabel coeluted with alpha 2u-globulin as one sharp peak as opposed to a multipeak pattern observed for 1,2-[14C]DCB; the maximal quantity of radiolabel for 1,4-DCB was twice that for 1,2-DCB. Equilibrium dialysis of kidney cytosol in the presence or absence of sodium dodecyl sulfate demonstrated that the radiolabel was reversibly bound to alpha 2u-globulin; the amount for 1,4-[14C]DCB-treated rats was almost twice as much as that for 1,2-[14C]DCB-treated rats. 1,2-DCB was also shown to be covalently bound to renal alpha 2u-globulin, and covalently bound to liver and plasma high-molecular-weight proteins. 1,4-DCB and, to a minor extent, 2,5-dichlorophenol, the major metabolite of 1,4-DCB, were reversibly bound to renal alpha 2u-globulin from 1,4-DCB-treated rats. 1,4-DCB increased protein droplet formation in male but not in female rat kidneys, whereas equimolar doses of 1,2-DCB showed no effect in either sex. Renal cell proliferation, measured by [3H]thymidine incorporation into renal DNA, was increased after 1,4-DCB but not after 1,2-DCB treatment. Nephrotoxicity and biochemical alterations induced by

  2. Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II

    DEFF Research Database (Denmark)

    Madsen, Jack Egelund; Petersen, Bent Larsen; Motawia, Mohammed Saddik

    2006-01-01

    in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2......Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-alpha-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative...

  3. A basic research of gadolinium hydrogen [alpha], [alpha]', [alpha]'', [alpha]'''-tetramethly- 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetate with high complex stability as a contrast agent for MRI

    Energy Technology Data Exchange (ETDEWEB)

    Seri, Shigemi; Hashiguchi, Yuji; Kubomura, Kan; Abe, Yukiko; Iguchi, Toshio; Iwai, Kumiko [Nihon Medi-Physics Co., Ltd., Sodegaura, Chiba (Japan); Watanabe, Tokuko

    1993-05-01

    Gadolinium hydrogen [alpha], [alpha]', [alpha]'', [alpha]'''-tetramethyl- 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetate (abbreviated Gd-DOTMA) was developed as a new contrast agent for magnetic resonance imaging. Our study focused on the evaluation of the pharmaceutical properties as in vivo agent. The new modified process by which Gd-DOTMA was synthesized resulted in high yields of this agent. A high stability constant of 10[sup 26] fro Gd-DOTMA was determined at physiological pH. It is more stable than Gd complex with tetraazacyclododecanetetraacetic acid (which is regarded as the most stable Gd complex). The strong T[sub 1] relaxivities of 4.0 and 3.7 (mM [center dot] s)[sup -1] at 0.5 tesla and 1.5 tesla were measured in the aqueous solution. The osmolarity of 0.5 M solution, dissolved with equal amounts of meglumine as a solubilizer is 1020 mOsmol/kg. This contrasting agent was studied in vivo by using rats as the experimental group. The agent showed strong enhancement of transplanted tumors within the rat population studied. This compound is rapidly excreted by the kidneys, and has a half-life of 26 min in blood. The median lethal dose (LD[sub 50] value) of the stable Gd-DOTMA has a favorable tolerance of over 12.3 mmol/kg. (author).

  4. Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

    Science.gov (United States)

    Qin, M; Oie, S

    1994-11-01

    The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

  5. Interaction of a gibberellin-induced factor with the upstream region of an alpha-amylase gene in rice aleurone tissue.

    OpenAIRE

    Ou-Lee, T M; Turgeon, R; Wu, R

    1988-01-01

    The interaction between the DNA sequences of an alpha-amylase (EC 3.2.1.1) gene and a tissue-specific factor induced in rice (Oryza sativa L.) aleurone tissue by gibberellin was studied. DNA mobility-shift during electrophoresis indicated that a 500-base-pair sequence (HS500) of a rice alpha-amylase genomic clone (OSamy-a) specifically interacted with a factor from gibberellin-induced rice aleurone tissue. The amount of complex formed between the HS500 DNA fragment and the gibberellin-induced...

  6. Immunoassay of serum alpha-1 antitrypsin level in uveitis.

    OpenAIRE

    Gupta, A K; Sarin, G S

    1984-01-01

    The serum alpha-1 antitrypsin level was measured in 60 patients with endogenous uveitis, 27 patients with phacoallergic endophthalmitis, 12 patients with phacolytic glaucoma, and 58 healthy subjects. Thirty-four patients with endogenous uveitis were also followed up for 6 months after treatment, and the serum alpha-1 antitrypsin level was measured again. There was a significant rise in the serum alpha-1 antitrypsin level in cases of endogenous uveitis and phacoallergic endophthalmitis but no ...

  7. Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta cell function

    DEFF Research Database (Denmark)

    Hansen, Sara K; Párrizas, Marcelina; Jensen, Maria L

    2002-01-01

    Mutations in the genes encoding hepatocyte nuclear factor 4alpha (HNF-4alpha) and HNF-1alpha impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4alpha is known to be an essential positive regulator of HNF-1alpha. More recent data demonstrates that HNF-4alpha...... in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G --> A mutation in a conserved nucleotide position of the HNF-1alpha binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1alpha...

  8. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S

    2001-01-01

    Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

  9. Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

    Science.gov (United States)

    Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

    2009-11-27

    Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

  10. DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

    International Nuclear Information System (INIS)

    Kosaka, T.; Kuwabara, M.; Koide, F.

    1992-01-01

    Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

  11. Therapeutics: Gene Therapy for Alpha-1 Antitrypsin Deficiency.

    Science.gov (United States)

    Gruntman, Alisha M; Flotte, Terence R

    2017-01-01

    This review seeks to give an overview of alpha-1 antitrypsin deficiency, including the different disease phenotypes that it encompasses. We then describe the different therapeutic endeavors that have been undertaken to address these different phenotypes. Lastly we discuss future potential therapeutics, such as genome editing, and how they may play a role in treating alpha-1 antitrypsin deficiency.

  12. Fibrinogen and alpha(1)-antitrypsin in COPD exacerbations

    DEFF Research Database (Denmark)

    Sylvan Ingebrigtsen, Truls; Marott, J. L.; Rode, L.

    2015-01-01

    Background We tested the hypotheses that fibrinogen and alpha(1)-antitrypsin are observationally and genetically associated with exacerbations in COPD. Methods We studied 13 591 individuals with COPD from the Copenhagen General Population Study (2003-2013), of whom 6857 were genotyped for FGB -455...... and exacerbations in instrumental variable analyses. Results Elevated fibrinogen and alpha(1)-antitrypsin levels were associated with increased risk of exacerbations in COPD, HR=1.14 (1.07 to 1.22, p...

  13. The experience with setting-up radioimmunoassay for alpha-1 fetoprotein

    International Nuclear Information System (INIS)

    Fingerova, H.

    1981-01-01

    The decisive factor in the preparation of radioimmunological alpha-1-fetoprotein determination, provided sufficient commercial or own antisera and standards are available for calibration, is the quality of the preparation for labelling. Alpha-1-fetoprotein was separated by affinity chromatography using Sepharose with alpha-1-fetoprotein-bound antibodies. The isolates thus obtained were labelled with 125 I using enzyme and chloramine T and Iodogen techniques. The labelled alpha-1-fetoprotein can be used for RIA. In view of reduced immunoreactivity of the preparation, however, the performance of the radioimmunological determination has so far not matched the quality of imported kits. The technique is currently being optimized. (author)

  14. Alpha-Lipoic acid counteracts the promoted oxidative DNA damage in the liver of septic rats

    International Nuclear Information System (INIS)

    Abd-Allah, Adel R.A.

    2006-01-01

    Viral, parasitic infections and chemical carcinogens are among the etiological factors of liver cancer. It seems important to study the initiating and promoting agents to evaluate the etiology and prevention of such life threatening disease. Intestine-derived bacteria product, lipopolysaccharide (LPS), is mainly detoxified by the liver. It has shown to induce a state of oxidative DNA damage is not fully investigated. Increased oxidative DNA damage and rate of cell proliferation may initiate or even promote cancer. In the present work, the capability of LPS to induce 8-hydroxydeoxyguanosine (8-HDG), a specific DNA adduct for oxidative DNA damage, in rat livers is tested. Furthermore, a possible protective effect of alpha lipoic acid (ALA) is also assessed. Investigated parameters are liver contents of glutathione (GSH), lipid peroxides (MDA), nitric oxide (NO) and 8-HDG in the liver-extracted DNA. Serum activities of ALT, AST and GGT as liver-function markers as well as IL2 are assessed. Moreover, liver histology is examined. LPS was given doses of 1, 3, 5, 7 and 9 mg/kg once i.p. while, the rat mortality was examined 24 hours later. ALA was given in doses of 50, 100 and 200 mg/kg once i.p. 3h before LPS is found to be 5mg/kg. LPS increased the level of 8-HDG, MDA and NO in the liver. It also induced acute liver necrosis and inflammatory cell infiltration as shown in liver-histopathology and in the significant increase in the activities of ALT, AST and GGT. LPS increased the serum level of IL2 as well. The dose 200mg/kg of ALA revealed a 100% protection against LPS-induced lethality. It also, prevented the LPS-induced increase in 8-HDG in liver extracted DNA, the liver contents of MDA and NO. ALA also rescued the LPS-induced GSH depletion. It corrected the liver function as shown by the prevention of increases in the activity of ALT, AST and GGT with a remarkable improvement in the liver histology. Moreover, it prevented the increase in serum level of IL2. These

  15. Downregulation of a tumor suppressor RECK by hypoxia through recruitment of HDAC1 and HIF-1alpha to reverse HRE site in the promoter.

    Science.gov (United States)

    Lee, Kyung Ju; Lee, Kwang Youl; Lee, You Mie

    2010-05-01

    Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor and the suppression of RECK is induced by Ras or Her-2/neu oncogenes. However, regulation of RECK under hypoxic microenvironment is largely unknown. Here, we identified that hypoxia significantly downregulates RECK mRNA and protein expression using semiquantitative RT-PCR, real-time RT-PCR and western blot analysis. This repression was reversed by the HDAC inhibitor, trichostatin A (TSA) and HIF-1 inhibitor, YC-1. Hypoxia-induced downregulation of RECK was abolished by knockdown of HDAC1 and HIF-1alpha with respective small interfering RNAs (siRNAs), whereas overexpression of HDAC1 and HIF-1alpha suppressed RECK expression similar to the level under hypoxic conditions. Transfection of a deletion mutant of the second reverse HRE (rHRE2, -2345 to -2333) site of RECK promoter completely removed RECK suppression under hypoxia, indicating that the rHRE2 site is responsible for the inhibition of RECK. Chromatin immunoprecipitation and DNA affinity precipitation assays demonstrated that HDAC1 and HIF-1alpha were recruited to the rHRE2 region of RECK promoter under hypoxic conditions, but the treatment of TSA or YC-1 inhibited their binding to the rHRE2 site. Moreover, TSA and YC-1 inhibited hypoxia-induced cancer cell migration, invasion and MMPs secretion. Taken together, we can conclude that hypoxia induces RECK downregulation through the recruitment of HDAC1 and HIF-1alpha to the rHRE2 site in the promoter and the inhibition of hypoxic RECK silencing would be a therapeutic and preventive target for early tumorigenesis. Copyright 2010 Elsevier B.V. All rights reserved.

  16. Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence.

    Science.gov (United States)

    Creekmore, Amy L; Ziegler, Yvonne S; Bonéy, Jamie L; Nardulli, Ann M

    2007-03-15

    We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

  17. Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene

    Science.gov (United States)

    Amy L. Ross-Davis; John W. Hanna; Mee-Sook Kim; Ned B. Klopfenstein

    2012-01-01

    The translation elongation factor-1 alpha gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The...

  18. Effect of alpha1-blockers on stentless ureteroscopic lithotripsy

    Directory of Open Access Journals (Sweden)

    Jianguo Zhu

    2016-02-01

    Full Text Available ABSTRACT Objective To evaluate the clinical efficiency of alpha1-adrenergic antagonists on stentless ureteroscopic lithotripsy treating uncomplicated lower ureteral stones. Materials and Methods From January 2007 to January 2013, 84 patients who have uncomplicated lower ureteral stones treated by ureteroscopic intracorporeal lithotripsy with the holmium laser were analyzed. The patients were divided into two groups, group A (44 patients received indwelled double-J stents and group B (40 patients were treated by alpha1-adrenergic antagonists without stents. All cases of group B were treated with alpha1 blocker for 1 week. Results The mean operative time of group A was significantly longer than group B. The incidences of hematuria, flank/abdominal pain, frequency/urgency after surgery were statistically different between both groups. The stone-free rate of each group was 100%. Conclusions The effect of alpha1-adrenergic antagonists is more significant than indwelling stent after ureteroscopic lithotripsy in treating uncomplicated lower ureteral stones.

  19. Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants

    NARCIS (Netherlands)

    Schulze, A.J.; Huber, R.; Degryse, E.; Speck, D.; Bischoff, Rainer

    1991-01-01

    Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg,

  20. Localization of pig Na[sup +], K[sup +]-ATPase [alpha] and [beta] subunit genes to chromosome 4 by radioactive in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Lahbib-Mansais, Y.; Yerle, M.; Dalens, M.; Chevalet, C.; Gellin, J. (Centre de Recherches de Toulouse (France))

    1993-01-01

    Two genes coding for Na[sup +],K[sup +] -ATPase [alpha] and [beta] subunits are localized on pig chromosome 4, to the q1.6[yields]q2.3 and 1.3[yields]q2.1 regions, respectively, by radioactive in situ hybridization. According to nucleotide and amino acid sequence comparisons with different human isoforms of Na[sup +] ,K[sup +]-ATPase, these pig [alpha] and [beta] ATPase genes show strong homologies with human [alpha]1 and [beta] subunit ATPase genes, respectively. These results are discussed with respect to comparative mapping data of conserved genes in mammalian species. We showed that the pig cDNA probes encoding ATPase [alpha] and, [beta] genes reveal DNA polymorphism in Meishan an Large White pigs. 35 refs., 4 figs., 2 tabs.

  1. A comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma

    Directory of Open Access Journals (Sweden)

    Mogilevski Grigori

    2004-09-01

    Full Text Available Abstract Background Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients. Methods Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6 or non-alpha-1 antitrypsin deficiency emphysema (n = 34 or wedge resection for hamartochondroma (n = 14 were examined by transmission electron microscopy and PCR. Results In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR. Conclusions These data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process

  2. Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

    Science.gov (United States)

    Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

    2017-08-17

    DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells

    Czech Academy of Sciences Publication Activity Database

    Sturzenegger, A.; Burdová, Kamila; Kanagaraj, R.; Levikova, M.; Pinto, C.; Cejka, P.; Janščák, Pavel

    2014-01-01

    Roč. 289, č. 39 (2014), s. 27314-27326 ISSN 0021-9258 R&D Projects: GA ČR GAP305/10/0281 Grant - others:Swiss National Science Foundation(CH) 31003A-129747; Swiss National Science Foundation(CH) 31003A_146206; Swiss National Science Foundation(CH) PP00P3 133636; University of Zurich(CH) FK-13-098 Institutional support: RVO:68378050 Keywords : DNA Damage * DNA Helicase * DNA Recombination * DNA Repair * Genomic Instability * RecQ Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.573, year: 2014

  4. Radial-velocity variations in Alpha Ori, Alpha Sco, and Alpha Her

    International Nuclear Information System (INIS)

    Smith, M.A.; Patten, B.M.; Goldberg, L.

    1989-01-01

    Radial-velocity observations of Alpha Ori, Alpha Sco A, and Alpha Her A are used to study radial-velocity periodicities in M supergiants. The data refer to several metallic lines in the H-alpha region and to H-alpha itself. It is shown that Alpha Ori and Alpha Sco A have cycle lengths of about 1 yr and semiamplitudes of 2 km/s. It is suggested that many semiregular red supergiant varibles such as Alpha Ori may be heading toward chaos. All three stars show short-term stochastic flucutations with an amplitude of 1-2 km/s. It is found that the long-term variability of H-alpha velocities may be a consequence of intermittent failed ejections. 58 refs

  5. Miscoding properties of 1,N{sup 6}-ethanoadenine, a DNA adduct derived from reaction with antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Bo; Guliaev, Anton B.; Chenna, Ahmed; Singer, B.

    2003-03-05

    1,N{sup 6}-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) {alpha}, {beta}, {eta} and {iota}. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using a primer extension assay, both pols {alpha} and {beta} were primarily blocked by EA or {var_epsilon}A with very minor extension. Pol {eta} a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol {eta} incorporated all four nucleotides opposite EA and {var_epsilon}A, but with differential preferences and mainly in an error-prone manner. Human pol {iota}, a paralog of human pol {eta}, was blocked by both adducts with a very small amount of synthesis past {var_epsilon}A. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. {var_epsilon}A, could affect the specificity of pol {iota} toward the template T immediately 3 feet to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or {var_epsilon}A showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to {var_epsilon}A, is a miscoding lesion.

  6. Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

    Science.gov (United States)

    Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

    2004-11-01

    GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

  7. Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

    Science.gov (United States)

    Hennemann, B; Tam, Y K; Tonn, T; Klingemann, H G

    1999-07-01

    Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

  8. A biallelic RFLP of the human. alpha. 2-C4 adrenergic receptor gene (ADRA2RL2) localized on the short arm of chromosome 4 and encoding the putative. alpha. 2B receptor is identified with Bsu 36 L using a 1. 5 kb probe (p ADRA2RL2)

    Energy Technology Data Exchange (ETDEWEB)

    Hoeche, M.R.; Berrettini, W.H. (Clinical Neurogenetics Branch, Bethesda, MD (USA)); Regan, J.W. (Duke Univ. Medical Center, Durham, NC (USA))

    1989-12-11

    A 1.5 kb Eco RI cDNA fragment representing the human alpha2-C4 adrenergic receptor (AR) gene encoding the putative alpha2B-AR, containing approximately 1270 bp of the coding and 240 bp of the 3{prime}flanking region, inserted into pSP65, was used as a probe (p ADRA2RL2). This clone was obtained by screening a human kidney lambda GT10 cDNA library with the 0.95 kb Pst I restriction fragment derived from the coding block of the gene for the human platelet alpha2-AR. Hybridization of human genomic DNA digested with Bsu 36 I identifies a two allele polymorphism with bands at 12 kb and 5.8 kb. 20 unrelated North American caucasian subjects were evaluated with frequencies of: A allele, 0.45; B allele, 0.55, heterozygosity (obs), 0.5. This alpha2-AR gene has been mapped in a separation effort in 59 CEPH reference pedigrees to the tip of the short arm of chromosome 4 just proximal to GB (4p 16.3) reported to be linked to the Huntingston's disease gene. Codominant inheritance was observed in seven families with two and three generations, respectively. The number of meioses scored was 95.

  9. A randomized clinical trial of alpha(1)-antitrypsin augmentation therapy.

    Science.gov (United States)

    Dirksen, A; Dijkman, J H; Madsen, F; Stoel, B; Hutchison, D C; Ulrik, C S; Skovgaard, L T; Kok-Jensen, A; Rudolphus, A; Seersholm, N; Vrooman, H A; Reiber, J H; Hansen, N C; Heckscher, T; Viskum, K; Stolk, J

    1999-11-01

    We have investigated whether restoration of the balance between neutrophil elastase and its inhibitor, alpha(1)-antitrypsin, can prevent the progression of pulmonary emphysema in patients with alpha(1)-antitrypsin deficiency. Twenty-six Danish and 30 Dutch ex-smokers with alpha(1)-antitrypsin deficiency of PI*ZZ phenotype and moderate emphysema (FEV(1) between 30% and 80% of predicted) participated in a double-blind trial of alpha(1)-antitrypsin augmentation therapy. The patients were randomized to either alpha(1)-antitrypsin (250 mg/kg) or albumin (625 mg/kg) infusions at 4-wk intervals for at least 3 yr. Self-administered spirometry performed every morning and evening at home showed no significant difference in decline of FEV(1) between treatment and placebo. Each year, the degree of emphysema was quantified by the 15th percentile point of the lung density histogram derived from computed tomography (CT). The loss of lung tissue measured by CT (mean +/- SEM) was 2.6 +/- 0.41 g/L/yr for placebo as compared with 1.5 +/- 0.41 g/L/yr for alpha(1)-antitrypsin infusion (p = 0.07). Power analysis showed that this protective effect would be significant in a similar trial with 130 patients. This is in contrast to calculations based on annual decline of FEV(1) showing that 550 patients would be needed to show a 50% reduction of annual decline. We conclude that lung density measurements by CT may facilitate future randomized clinical trials of investigational drugs for a disease in which little progress in therapy has been made in the past 30 yr.

  10. Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity.

    Science.gov (United States)

    Parasuram, Ramya; Coulther, Timothy A; Hollander, Judith M; Keston-Smith, Elise; Ondrechen, Mary Jo; Beuning, Penny J

    2018-02-20

    The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 10 5 nucleotide incorporations. The goal of this research is to determine the residues that contribute to the activity of the polymerase subunit. Homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity.

  11. Alpha-particle radiobiological experiments using thin CR-39 detectors

    International Nuclear Information System (INIS)

    Chan, K. F.; Siu, S. Y. M.; McClella, K. E.; Tse, A. K. W.; Lau, B. M. F.; Nikezic, D.; Richardson, B. J.; Lam, P. K. S.; Fong, W. F.; Yu, K. N.

    2006-01-01

    The present paper studied the feasibility of applying comet assay to evaluate the DNA damage in individual HeLa cervix cancer cells after alpha-particle irradiation. We prepared thin CR-39 detectors (<20 μm) as cell-culture substrates, with UV irradiation to shorten the track formation time. After irradiation of the HeLa cells by alpha particles, the tracks on the underside of the CR-39 detector were developed by chemical etching in (while floating on) a 14 N KOH solution at 37 deg. C. Comet assay was then applied. Diffusion of DNA out of the cells could be generally observed from the images of stained DNA. The alpha-particle tracks corresponding to the comets developed on the underside of the CR-39 detectors could also be observed by just changing the focal plane of the confocal microscope. (authors)

  12. alpha,beta-unsaturated 2-acyl imidazoles as a practical class of dienophiles for the DNA-Based catalytic asymmetric diels-alder reaction in water

    NARCIS (Netherlands)

    Boersma, A.J.; Feringa, B.L.; Roelfes, G.

    2007-01-01

    alpha,beta-Unsaturated 2-acyl imidazoles are a novel and practical class of dienophiles for the DNA-based catalytic asymmetric Diels-Alder reaction in water. The Diels-Alder products are obtained with very high diastereoselectivities and enantioselectivities in the range of 83-98%. The catalytic

  13. Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins.

    Science.gov (United States)

    Duque, Hernando; LaRocco, Michael; Golde, William T; Baxt, Barry

    2004-09-01

    At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus

  14. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...... subunit and 71% identity to the alpha' subunit of human casein kinase 2....

  15. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522 (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  16. Alpha radiation detection using silicon memory chips - preliminary studies

    International Nuclear Information System (INIS)

    Pace, R.; Paix, D.; Haskard, M.

    1993-01-01

    Alpha radiation dosage is an important occupational health factor in the mining of uranium and mineral sands. Alpha radiation induced errors in the data of silicon based memory chips provide the foundation for a new type of sensor, with the potential for affordable and prompt measurement of personal alpha doses. With particular reference to Dynamic Random Access Memories (DRAM) this paper introduces the operating principle of a memory based radiation sensor, which is the error mechanism in silicon integrated circuits. 14 refs., 3 figs

  17. Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

    Science.gov (United States)

    Rudd, M Katharine; Mays, Robert W; Schwartz, Stuart; Willard, Huntington F

    2003-11-01

    Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

  18. Nucleotide sequence of a cDNA coding for the amino-terminal region of human prepro. alpha. 1(III) collagen

    Energy Technology Data Exchange (ETDEWEB)

    Toman, P D; Ricca, G A [Rorer Biotechnology, Inc., Springfield, VA (USA); de Crombrugghe, B [National Institutes of Health, Bethesda, MD (USA)

    1988-07-25

    Type III Collagen is synthesized in a variety of tissues as a precursor macromolecule containing a leader sequence, a N-propeptide, a N-telopeptide, the triple helical region, a C-telopeptide, and C-propeptide. To further characterize the human type III collagen precursor, a human placental cDNA library was constructed in gt11 using an oligonucleotide derived from a partial cDNA sequence corresponding to the carboxy-terminal part of the 1(III) collagen. A cDNA was identified which contains the leader sequence, the N-propeptide and N-telopeptide regions. The DNA sequence of these regions are presented here. The triple helical, C-telopeptide and C-propeptide amino acid sequence for human type III collagen has been determined previously. A comparison of the human amino acid sequence with mouse, chicken, and calf sequence shows 81%, 81%, and 92% similarity, respectively. At the DNA level, the sequence similarity between human and mouse or chicken type III collagen sequences in this area is 82% and 77%, respectively.

  19. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  20. alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

    Directory of Open Access Journals (Sweden)

    M.R.S.C. Wenning

    2000-09-01

    Full Text Available Seven unrelated patients with hemoglobin (Hb H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aaT]. Among the 27 patients with structural alterations, 15 (of Italian descent had Hb Hasharon (alpha47Asp->His associated with the -alpha3.7 deletion, 4 (of Italian descent were heterozygous for Hb J-Rovigo (alpha53Ala->Asp, 4 (3 Blacks and 1 Caucasian were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys associated with the alpha+-thalassemia, 1 (Black was heterozygous for Hb G-Pest (alpha74Asp->Asn, 1 (Caucasian was heterozygous for Hb Kurosaki (alpha7Lys->Glu, 1 (Caucasian was heterozygous for Hb Westmead (alpha122His->Gln, and 1 (Caucasian was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val. Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

  1. Stromal cell-derived factor-1 alpha (SDF-1 alpha) improves neural recovery after spinal cord contusion in rats

    NARCIS (Netherlands)

    Zendedel, A.; Nobakht, M.; Bakhtiyari, M.; Beyer, C.; Kipp, M.; Baazm, M.; Joghataie, M.T.

    2012-01-01

    Stromal cell-derived factor-1 alpha (SDF-1α) is an important cytokine, implicated in the control of stem cell trafficking and bone marrow-derived stem cell mobilization. Generally, SDF-1α regulates multiple physiological processes such as embryonic development and organ homeostasis. There is also

  2. Enthalpies of combustion and formation of {alpha}-D-glucoheptono-1,4-lactone and {alpha},{beta}-glucooctanoic-1,4-lactone

    Energy Technology Data Exchange (ETDEWEB)

    Amador, Patricia [Facultad de Ciencias Qui' micas, Benemerita Universidad Autonoma de Puebla, 14 Sur y Av. San Claudio, Col. Manuel, C.P. 72570 Puebla Pue (Mexico)], E-mail: cs000721@siu.buap.mx; Mata, Marian Y.; Flores, Henoc [Facultad de Ciencias Qui' micas, Benemerita Universidad Autonoma de Puebla, 14 Sur y Av. San Claudio, Col. Manuel, C.P. 72570 Puebla Pue (Mexico)

    2008-05-15

    The standard molar energies of combustion, {delta}{sub c}U{sub m}{sup 0}(cr,298.15K), of {alpha}-D-glucoheptono-1,4-lactone (GH) and {alpha},{beta}-glucooctanoic-1,4-lactone (GO) were obtained by micro-combustion calorimetry. The obtained values are -(2924.6 {+-} 2.3) kJ . mol{sup -1} and -(3459.5 {+-} 2.5) kJ . mol{sup -1}, respectively. From combustion energies, the standard molar enthalpies of formation in crystalline phase, {delta}{sub f}H{sub m}{sup 0}(cr,298.15K), for GH and GO were determined as -(1546.2 {+-} 2.5) kJ . mol{sup -1} and -(1690.6 {+-} 2.7) kJ . mol{sup -1}, respectively. Also it was found that when the hydroxyl group number increases in the aldonolactones their standard molar enthalpies of formation increase too.

  3. Differential vascular alpha1-adrenoceptor antagonism by tamsulosin and terazosin

    NARCIS (Netherlands)

    Schäfers, R. F.; Fokuhl, B.; Wasmuth, A.; Schumacher, H.; Taguchi, K.; de Mey, C.; Philipp, T.; Michel, M. C.

    1999-01-01

    AIMS: In patients with lower urinary tract symptoms suggestive of benign prostatic obstruction the alpha1-adrenoceptor antagonist terazosin lowers blood pressure whereas only very small if any alterations were reported with the alpha1-adrenoceptor antagonist tamsulosin. Therefore, we have compared

  4. MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

    Science.gov (United States)

    Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

    2017-07-01

    Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Mechanical design of DNA nanostructures

    Science.gov (United States)

    Castro, Carlos E.; Su, Hai-Jun; Marras, Alexander E.; Zhou, Lifeng; Johnson, Joshua

    2015-03-01

    Structural DNA nanotechnology is a rapidly emerging field that has demonstrated great potential for applications such as single molecule sensing, drug delivery, and templating molecular components. As the applications of DNA nanotechnology expand, a consideration of their mechanical behavior is becoming essential to understand how these structures will respond to physical interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mechanical properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mechanical stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mechanically active nanomachines that can generate, transmit, and respond to physical cues in molecular systems.Structural DNA nanotechnology is a rapidly emerging field that has demonstrated great potential for applications such as single molecule sensing, drug delivery, and templating molecular components. As the applications of DNA nanotechnology expand, a consideration of their mechanical behavior is becoming essential to understand how these structures will respond to physical interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mechanical properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mechanical stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mechanically active nanomachines that can generate, transmit, and respond to physical cues in molecular systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07153k

  6. Alpha1-antitrypsin deficiency: a clinical-genetic overview

    Directory of Open Access Journals (Sweden)

    Abboud RT

    2011-03-01

    Full Text Available Raja T Abboud1, Tanya N Nelson2, Benjamin Jung2, Andre Mattman31Department of Medicine, Respiratory Division, University of British Columbia, Vancouver, BC, Canada; 2Department of Pathology and Laboratory Medicine, Children's and Women's Health Centre of British Columbia, University of British Columbia, Vancouver, BC, Canada; 3Department of Pathology and Laboratory Medicine, St. Paul's Hospital, University of British Columbia, Vancouver, BC, CanadaAbstract: Severe α1-antitrypsin deficiency (AATD is an inherited disorder, leading to development of emphysema in smokers at a relatively young age with disability in their forties or fifties. The emphysema results from excessive elastin degradation by neutrophil elastase as a result of the severe deficiency of its major inhibitor α1-antitrypsin (AAT. The AAT expression is determined by the SERPINA1 gene which expresses codominant alleles. The three most common alleles are the normal M, the S with plasma levels of 60% of normal, and the severely deficient Z with levels of about 15% of normal. Homozygosity for the Z mutant allele is associated with retention of abnormal AAT in the liver, which may lead to neonatal hepatitis, liver disease in children, and liver disease in adults. Regular intravenous infusions of purified human AAT (AAT augmentation therapy have been used to partially correct the biochemical defect and protect the lung against further injury. Two randomized controlled trials showed a trend of slower progression of emphysema by chest computerized tomography. Integrated analysis of these two studies indicated significantly slower progression of emphysema. AAT is quantified by immunologic measurement of AAT in serum, the phenotype characterized by isoelectric focusing, the common genotypes by targeted DNA analysis, and by sequencing the coding region of the gene when the AAT abnormality remains undefined. AATD is often unrecognized, and diagnosis delayed. Testing for AATD is recommended

  7. Evaluation of pGL1-TNF-alpha therapy in combination with radiation

    Science.gov (United States)

    Li, J.; Andres, M. L.; Fodor, I.; Nelson, G. A.; Gridley, D. S.

    1998-01-01

    Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. In this study a new plasmid-based human tumor necrosis factor-alpha (TNF-alpha) expression vector was synthesized (pGL1-TNF-alpha) and evaluated together with radiation in the aggressive, rapidly growing C6 rat glioma model. pGL1-TNF-alpha was successfully transfected into C6 cells in vitro using a cationic polyamine method. Expression was detected up to 7 days and averaged 0.4 ng of TNF-alpha in the culture medium from 1x10(5) cells. The expressed protein was biologically functional, as evidenced by growth inhibition of L929, a TNF-alpha-susceptible cell line. Using fluorescence-labeled monoclonal antibodies and laser scanning cytometry, we confirmed that both the P55 and P75 receptors for TNF-alpha were present on the C6 cell membrane. However, the receptors were present at low density and P55 was expressed more than the P75 receptor. These findings were in contrast to results obtained with TNF-alpha-susceptible L929 cells. Tests in athymic mice showed that pGL1-TNF-alpha administered intratumorally 16-18 h before radiation (each modality given three times) significantly inhibited C6 tumor progression (Palpha alone did not slow tumor growth and radiation alone had little effect on tumor growth. These results indicate that pGL1-TNF-alpha has potential to augment the antitumor effects of radiation against a tumor type that is virtually incurable.

  8. Collagen Type I alpha1 (COL1A1 Gene Polymorphism and Bone Mineral Density in Postmenopausal Kazakh Women

    Directory of Open Access Journals (Sweden)

    Akbota Aitkulova

    2014-12-01

    Full Text Available Introduction: Single nucleotide polymorphism (SNP at the collagen type I alpha 1 gene (COL1A1 rs1800012 has been widely studied and has shown an association with bone mineral density (BMD and fractures. A minor allele TT of this SNP was found to be greatly overrepresented in individuals with fractures compared to controls, thus becoming a good predictor of  increased fracture risk. The aim of this investigation was to evaluate potential association between COL1A1 gene polymorphism and osteoporosis in Kazakh postmenopausal women.Methods: The study population included 103 postmenopausal women recruited from Pavlodar and Almaty clinics. BMD was measured using DEXA. Genomic DNA was extracted from peripheral venous blood of study participants with Wizard® Genomic DNA Purification Kit (Promega, USA. Detection of COL1A1 +1245G/T (Sp1 polymorphism was done by the TaqMan® SNP Genotyping Assay of real-time PCR.Results: Densitometry results revealed 36 osteoporotic, 42 osteopenic, and 25 normal postmenopausal women. Data analysis of 1245G>T polymorphism in COL1A1 gene in the group of women with osteopenia and osteoporosis revealed deviation from Hardy-Weinberg equilibrium. The mutant TT genotype was prevalent compared to the heterozygous genotype GT in both groups. Distributions were 83% GG, 3% GT, and 14% TT in the group with osteopenia and 80% GG, 6% GT, and 14% TT in the group with osteoporosis. The distribution of genotypes frequency in the group of normal postmenopausal women was 76% GG, 16% GT, and 8% TT.Conclusion: These results suggest that TT genotype of COL1A1 +1245G/T (Sp1 polymorphism is associated with risk of postmenopausal osteoporosis in Kazakh women. Further studies involving a larger number of women are needed to clarify the relationship of this polymorphism with risk of osteoporosis. 

  9. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  10. Inhibition of HIF-1{alpha} activity by BP-1 ameliorates adjuvant induced arthritis in rats

    Energy Technology Data Exchange (ETDEWEB)

    Shankar, J. [Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago (United States); Thippegowda, P.B., E-mail: btprabha@uic.edu [Department of Pharmacology, (M/C 868), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612 (United States); Kanum, S.A. [Department of Chemistry, Yuvaraj' s College, University of Mysore, Mysore (India)

    2009-09-18

    Rheumatoid arthritis (RA) is a chronic inflammatory, angiogenic disease. Inflamed synovitis is a hallmark of RA which is hypoxic in nature. Vascular endothelial growth factor (VEGF), one of the key regulators of angiogenesis, is overexpressed in the pathogenesis of RA. VEGF expression is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a master regulator of homeostasis which plays a pivotal role in hypoxia-induced angiogenesis. In this study we show that synthetic benzophenone analogue, 2-benzoyl-phenoxy acetamide (BP-1) can act as a novel anti-arthritic agent in an experimental adjuvant induced arthritis (AIA) rat model by targeting VEGF and HIF-1{alpha}. BP-1 administered hypoxic endothelial cells and arthritic animals clearly showed down regulation of VEGF expression. Further, BP-1 inhibits nuclear translocation of HIF-1{alpha}, which in turn suppresses transcription of the VEGF gene. These results suggest a further possible clinical application of the BP-1 derivative as an anti-arthritic agent in association with conventional chemotherapeutic agents.

  11. Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets.

    Directory of Open Access Journals (Sweden)

    Reini F Luco

    2008-05-01

    Full Text Available DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3. We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

  12. Study on cellular genotoxicities induced by alpha particles irradiation in combination with NNK treatment

    International Nuclear Information System (INIS)

    Li Ping; Yang Zhihua; Pan Xiujie; Cao Zhenshan; Mi Na; Chen Zhongmin; Liu Gang; Wei Han; Li Huiying; Zhu Maoxiang

    2006-01-01

    Objective: To investigate cellular genotoxicities of aplha particles irradiation in combination with NNK treatment. Methods: Exponentially growing immortalized human bronchial epithelial cells were divided into the normal control group (NC), alpha particles irradiation (α), NNK administration group (NNK), NNK administration (100 μg/ml) followed by alpha particles irradiation group (NNK + α), and alpha particles irradiation followed by NNK administration (100 μg/ml) group (μ + NNK). DNA damage were detected by single cell gel electrophoresis (SCGE); multinuclear cell assay was used to detect the frequency of the HPRT gene mutation; cell micronucleus frequency were detected by cytogenetic methods. Results: In the group exposed to both alpha particles irradiation and NNK, DNA damage, HPRT gene mutation frequency, and cell micronucleus frequency were significantly higher than those in the same dose groups irradiated with alpha particles or NNK administration alone. Subtracted the NNK effect, DNA damage, HPRT gene mutation frequency and cell micronucleus frequency in the group irradiated by alpha particles in combination with NNK administration were significantly higher than those of alpha particles irradiation alone. Conclusion: The genotoxicity of alpha particles irradiation in combination with NNK administration had synergistic effect. (authors)

  13. Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

    Science.gov (United States)

    Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

    1998-08-01

    This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT

  14. Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

    Science.gov (United States)

    Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

    1999-02-15

    We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

  15. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  16. Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

    Science.gov (United States)

    Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

    2008-10-01

    Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

  17. Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

    Science.gov (United States)

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2016-08-22

    Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Nanodosimetry and nanodosimetric-based models of radiation action for radon alpha particles

    International Nuclear Information System (INIS)

    1992-01-01

    The objective of our research work is to provide -- with the aid of biophysical models of radiation action -- information on human risks following exposure to radon alpha particles. The approach proposed consists of (1) developing appropriate models (parametric and non-parametric) for alpha radiation induction of relevant end points (survival, cellular transformation), (2) providing an accurate physical characterization of the particle tracks in terms of nanodosimetric distributions, (3) supporting the models by detailed, molecular studies of the direct and indirect effects of alpha particles on DNA. Activities in the second year of this project are described

  19. Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX

    Science.gov (United States)

    Dong, Lili; Tan, Qiwen; Ye, Wei; Liu, Dongli; Chen, Haifeng; Hu, Hongwei; Wen, Duo; Liu, Yang; Cao, Ya; Kang, Jingwu; Fan, Jia; Guo, Wei; Wu, Weizhong

    2015-01-01

    Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed. PMID:26497223

  20. Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization.

    Science.gov (United States)

    Haq, Imran; Irving, James A; Saleh, Aarash D; Dron, Louis; Regan-Mochrie, Gemma L; Motamedi-Shad, Neda; Hurst, John R; Gooptu, Bibek; Lomas, David A

    2016-01-01

    Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

  1. Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

    Science.gov (United States)

    Barbosa, Aulus E A D; Albuquerque, Erika V S; Silva, Maria C M; Souza, Djair S L; Oliveira-Neto, Osmundo B; Valencia, Arnubio; Rocha, Thales L; Grossi-de-Sa, Maria F

    2010-06-17

    Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

  2. PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

    DEFF Research Database (Denmark)

    Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

    2009-01-01

    The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO......) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1alpha KO mice, VEGF protein...... expression was approximately 60-80% lower and the capillary-to-fiber ratio approximately 20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1alpha KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased...

  3. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. Crown Copyright © 2012. Published by Elsevier India Pvt Ltd. All rights reserved.

  4. Effect of N-Acetyl-L-Cysteine and alpha-Tocopherol Administration on Endogenous Antioxidant Protection of Liver DNA and RNA and plasma Lipid Profile in gamma-Irradiated Rats

    International Nuclear Information System (INIS)

    Abou-Safi, H.M.; Ashry, O.M.; Kafafy, Y.A.

    2005-01-01

    The present study wasundertaken to evaluate the combined antioxidative capacity of N-acetyl-L-cysteine (NAC, 120 mg/100g b. wt) and alpha tocopherol (10mg/100g b. wt.) injected intra peritoneally one h before irradiation of male rats. Whole body gamma irradiation (2Gy) induced significant elevation in liver DNA and significant drop in liver protein content, while liver RNA showed no significant changes. Triglycerides and LDL-cholesterol elevated significantly after irradiation, whereas no significant changes were observed in total cholesterol, while HDL-cholesterol significantly decreased. Blood and liver glutathione were significantly decreased, whereas plasma MDA was significantly increased. NAC and alpha-tocopherol injection elevated RNA and blood glutathione levels compared to control and depressed total cholesterol and LDL-cholesterol levels, as well as MDA in the liver. The combined treatment prior to irradiation decreased DNA, elevated RNA and normalized liver protein content. Triglycerides were decreased after 1 and 3 days and total cholesterol dropped significantly on the 1 st and 7 th days. LDL was ameliorated while HDL was significantly declined then elevated after 7 days. Blood glutathione was normalized while liver glutathione was significantly elevated and MDA was reduced both in liver and plasma. This combined treatment has proven to be recommended to enhance the natural defenses against deleterious effects of oxidative stress

  5. 5 alpha-reductase inhibitors and prostatic disease.

    Science.gov (United States)

    Schröder, F H

    1994-08-01

    5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. A G {r_arrow} A transition at position IVS-11 +1 of the HEX A {alpha}-chain gene in a non-Ashkenazic Mexican Tay-Sachs infant

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, S.R.P.; Gwon, S.; DeGasperi, R. [New York Univ. Medical Center, NY (United States)] [and others

    1994-09-01

    Tay-Sachs disease (TSD) is an autosomal recessive storage disorder caused by a deficiency of the lysosomal enzyme, {beta}-N-acetylhexosaminidase A (Hex A), a heteropolymer composed of two polypeptides, {alpha} and {beta}. Mutations in the {alpha}-chain gene render the enzyme defective, resulting in the accumulation of g{sub m2} ganglioside in the nervous system. Deficiency of Hex A was detected in a non-Ashkenazic girl of Mexican origin indicating infantile onset of TSD. Molecular investigation of the {alpha}-chain gene excluded the typical Ashkenazic 4 bp insertion in the exon 11 and the intron 12 splice-junction mutations by Hae III and Dde I restriction analysis, respectively. Single strand conformation polymorphism (SSCP) analysis showed a different pattern in the sample where exon 11 and flanking regions were amplified in the patient DNA as compared to the migration of control DNA. Sequencing of PCR amplified genomic DNA containing exon 11 and flanking intronic regions showed a single base substitution (G {r_arrow} A) at position IVS-11 +1. This mutation creates a recognition site for the restriction enzyme Mbo II. Digestion of exon 11 and flanking regions with Mbo II demonstrated homozygosity of the patient for this mutation and heterozygosity in the mother. mRNA from cultured fibroblasts obtained from a normal control and from the propositus was reverse transcribed. The cDNAs coding for Hex A {alpha}-chain were amplified in four overlapping fragments. In the patient sample it was not possible to amplify the fragment containing the exon 11/intron 11 junction, indicating that this mutation alters normal RNA processing of the Hex A pre-mRNA resulting in the deficiency of Hex A activity.

  7. Association of alpha-1 antitrypsin level and lung function in patients with chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Serapinas Danielius

    2017-01-01

    Full Text Available Introduction/Objective. Alpha-1 antitrypsin deficiency is a well established inherited risk factor for chronic obstructive pulmonary disease (COPD; however, alpha-1 antitrypsin level may result in different lung function reduction. The aim of our study was to evaluate possible associations of alpha-1 antitrypsin level and lung function in COPD patients with different alpha-1 antitrypsin phenotypes. Methods. Serum alpha-1 antitrypsin concentration from patients (n = 1,167 with COPD, defined according to the GOLD criteria, were analyzed by nephelometry, and alpha-1 antitrypsin phenotype was determined by means of isoelectric-focusing. Results. In COPD patients without alpha-1 antitrypsin deficiency (MM, a significant negative association of lung function (FEV1 with serum alpha-1 antitrypsin (r = -0.511; p < 0.05 and C-reactive protein (CRP concentrations (r = -0.583; p < 0.05 was detected; moreover, the level of alpha-1 antitrypsin positively correlated with CRP concentration (r = 0.667; p < 0.05. Conclusions. In patients without alpha-1 antitrypsin deficiency, detected negative association of alpha-1 antitrypsin level with FEV1 and positive association with the CRP level defined the importance of alpha-1 antitrypsin for lung function in COPD patients.

  8. Rational design, synthesis, biologic evaluation, and structure-activity relationship studies of novel 1-indanone alpha(1)-adrenoceptor antagonists.

    Science.gov (United States)

    Li, Minyong; Xia, Lin

    2007-11-01

    In the present report, a novel series of 1-indanone alpha(1)-adrenoceptor antagonists were designed and synthesized based on 3D-pharmacophore model. Their in vitro alpha(1)-adrenoceptor antagonistic assay showed that three compounds (2a, 2m, and 2o) had similar or improved alpha(1)-adrenoceptor antagonistic activities relative to the positive control prazosin. Based on these results, a three-dimensional quantitative structure-activity relationship study was performed using a Self-Organizing Molecular Field Analysis method to provide insight for the future development of alpha(1)-adrenoceptor antagonists.

  9. Hypoxia-inducible factor-1 alpha has a key role in hypoxic preconditioning.

    Science.gov (United States)

    Taie, Satoshi; Ono, Junichiro; Iwanaga, Yasuyuki; Tomita, Shuhei; Asaga, Takehiko; Chujo, Kosuke; Ueki, Masaaki

    2009-08-01

    Sublethal hypoxia induces tolerance to subsequent hypoxic insults in a process known as hypoxic preconditioning (HP). Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key transcription protein involved in the mechanism of HP. In this study, we investigated the effects of HP on tissue oxygenation and expression of HIF-1 alpha gene targets in the brain using neural cell-specific HIF-1 alpha-deficient mice. The animals were exposed to 8% oxygen for 3 hours. Twenty-four hours later, the oxygen partial pressure (pO(2)) of brain tissue and gene expression were measured during hypoxia. HP improved the pO(2) of brain tissue during subsequent hypoxia with upregulated inducible nitric oxide synthase in wild-type mice, whereas HP had no detectable effect in the mutant mice. Our results indicate that the protective effects of HP may be partially mediated by improving tissue oxygenation via HIF-1 alpha and inducible nitric oxide synthase.

  10. Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen

    Directory of Open Access Journals (Sweden)

    Sperling Rhoda

    2011-08-01

    Full Text Available Abstract Background During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response. Methods We compare culture-generated monocyte derived DCs (MDDCs with directly isolated myeloid dendritic cells (mDCs and plasmacytoid dendritic cells (pDCs and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy. Results We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients. Conclusions Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells.

  11. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

    DEFF Research Database (Denmark)

    Berquist, Brian R; Singh, Dharmendra Kumar; Fan, Jinshui

    2010-01-01

    XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R...... mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S...

  12. Transcriptional switch from albumin to alpha-fetoprotein and changes in transcription of other genes during carbon tetrachloride induced liver regeneration

    International Nuclear Information System (INIS)

    Panduro, A.; Shalaby, F.; Weiner, F.R.; Biempica, L.; Zern, M.A.; Shafritz, D.A.

    1986-01-01

    During liver regeneration induced by CCl 4 administration to rats, changes in the relative transcription rates of albumin and alpha-fetoprotein genes have been measured in conjunction with other liver-specific and general cellular function genes. Within 24 h following CCl 4 administration, albumin gene transcription decreases by 85%, whereas alpha-fetoprotein transcription increases from undetectable levels to 50% of that observed for albumin. These changes precede maximal [ 3 H]thymidine incorporation into DNA which peaks at 48 h. Other genes related to liver-specific functions, such as ligandin, alpha 1-antitrypsin, and cytochrome P-450's, as well as general cellular genes pro alpha 1- and pro alpha 2-collagen, beta-actin, and alpha-tubulin, respond in kinetic patterns often distinct from each other and from albumin and alpha-fetoprotein. Changes in the steady-state levels of albumin and alpha-fetoprotein mRNA correlate with changes in transcription, but there is a lag in alpha-fetoprotein mRNA accumulation, which peaks at 72 h following CCl 4 administration. These studies indicate that reciprocal changes in albumin and alpha-fetoprotein gene transcription occur during CCl 4 -induced liver regeneration, leading to changes in the level of these specific mRNAs. These changes precede DNA synthesis and would appear to represent an alteration in differentiated function of hepatocytes in conjunction with the liver regenerative process

  13. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

    Directory of Open Access Journals (Sweden)

    Alessandra eMaresca

    2015-03-01

    Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

  14. The role of DNA repair in herpesvirus pathogenesis.

    Science.gov (United States)

    Brown, Jay C

    2014-10-01

    In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

  15. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Toyo; Inagaki, Kenjiro [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji [Genomic Science Laboratories, DainipponSumitomo Pharma Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-8555 (Japan); Kasuga, Masato [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655 (Japan)

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  16. Calcineurin signaling and PGC-1alpha expression are suppressed during muscle atrophy due to diabetes.

    Science.gov (United States)

    Roberts-Wilson, Tiffany K; Reddy, Ramesh N; Bailey, James L; Zheng, Bin; Ordas, Ronald; Gooch, Jennifer L; Price, S Russ

    2010-08-01

    PGC-1alpha is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1alpha expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1alpha participates in the regulation of muscle mass. PGC-1alpha gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1alpha in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1alpha expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21days, the levels of PGC-1alpha protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1alpha transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1alpha regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1alpha expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1alpha were also decreased in muscles of CnAalpha-/- and CnAbeta-/- mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1alpha expression. These findings demonstrate that Cn activity is a major determinant of PGC-1alpha expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

  17. Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection

    DEFF Research Database (Denmark)

    Madsen, Andreas N; Nansen, Anneline; Christensen, Jan P

    2003-01-01

    The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1alpha (MIP-1alpha) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1alpha-deficient mice. Furthermore, MIP-1alpha is not required for T-cell-mediated virus...... control or virus-induced T-cell-dependent inflammation. Thus, MIP-1alpha is not mandatory for T-cell-mediated antiviral immunity....

  18. The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity.

    Science.gov (United States)

    Kroczynska, Barbara; Evangelista, Christina M; Samant, Shalaka S; Elguindi, Ebrahim C; Blond, Sylvie Y

    2004-03-19

    The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

  19. Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.

    Science.gov (United States)

    Yamamoto, E; Zeng, L; Baird, W V

    1998-02-01

    Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass.

  20. Alpha-1-antitrypsin studies: canine serum and canine surfactant protein

    International Nuclear Information System (INIS)

    Tuttle, W.C.; Slauson, D.O.; Dahlstrom, M.; Gorman, C.

    1974-01-01

    Canine serum alpha-1-antitrypsin was isolated by gel filtration and affinity chromatography and characterized by polyacrylamide gel electrophoresis and immunoelectrophoresis. Measurement of the trypsin inhibitory capacity of the separated protein indicated a ninefold concentration of functional trypsin inhibitor during the isolation procedure. Electrophoresis demonstrated the presence of a single protein with alpha-globulin mobility and a molecular weight near that of human alpha-1-antitrypsin. The trypsin inhibitory capacity of pulmonary surfactant protein from five Beagle dogs was measured, related to total surfactant protein concentration, and compared with similar measurements on whole serum from the same animals. Results indicated a variable concentration of trypsin inhibitor in the canine pulmonary surfactant protein. However, the concentration in the surfactant protein was always significantly higher than that in the corresponding serum sample. Preliminary experiments designed to separate the trypsin inhibitory fraction(s) from the other surfactant proteins by gel filtration chromatography indicated that the trypsin inhibitor was probably a single protein with a molecular weight near that of alpha-1-antitrypsin. (U.S.)

  1. SDF-1 alpha expression during wound healing in the aged is HIF dependent.

    Science.gov (United States)

    Loh, Shang A; Chang, Edward I; Galvez, Michael G; Thangarajah, Hariharan; El-ftesi, Samyra; Vial, Ivan N; Lin, Darius A; Gurtner, Geoffrey C

    2009-02-01

    Age-related impairments in wound healing are associated with decreased neovascularization, a process that is regulated by hypoxia-responsive cytokines, including stromal cell-derived factor (SDF)-1 alpha. Interleukin-1 beta is an important inflammatory cytokine involved in wound healing and is believed to regulate SDF-1 alpha expression independent of hypoxia signaling. Thus, the authors examined the relative importance of interleukin (IL)-1 beta and hypoxia-inducible factor (HIF)-1 alpha on SDF-1 alpha expression in aged wound healing. Young and aged mice (n = 4 per group) were examined for wound healing using a murine excisional wound model. Wounds were harvested at days 0, 1, 3, 5, and 7 for histologic analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot. An engineered wild-type and mutated SDF luciferase reporter construct were used to determine HIF transactivation. Aged mice demonstrated significantly impaired wound healing, reduced granulation tissue, and increased epithelial gap compared with young controls. Real-time polymerase chain reaction demonstrated reduced SDF-1 alpha levels in aged wounds that correlated with reduced CD31+ neovessels. Western blots revealed decreased HIF-1 alpha protein in aged wounds. However, both IL-1 beta and macrophage infiltrate were unchanged between young and aged animals. Using the wild-type and mutated SDF luciferase reporter construct in which the hypoxia response element was deleted, only young fibroblasts were able to respond to IL-1 beta stimulation, and this response was abrogated by mutating the HIF-binding sites. This suggests that HIF binding is essential for SDF-1 transactivation in response to both inflammatory and hypoxic stimuli. SDF-1 alpha deficiency observed during aged wound healing is attributable predominantly to decreased HIF-1 alpha levels rather than impaired IL-1 beta expression.

  2. Quantitation of alpha 1-adrenergic receptors in porcine uterine and mesenteric arteries

    International Nuclear Information System (INIS)

    Farley, D.B.; Ford, S.P.; Reynolds, L.P.; Bhatnagar, R.K.; Van Orden, D.E.

    1984-01-01

    The activation of vascular alpha-adrenergic receptors may be involved in the control of uterine blood flow. A radioligand binding assay with the use of the alpha 1-adrenergic antagonist 3 H-WB-4101 was established to characterize the alpha-adrenergic receptors in uterine and mesenteric arterial membranes obtained from nonpregnant pigs. Specific binding of 3 H-WB-4101 was rapid, saturable, and exhibited the alpha-adrenergic agonist potency order of (-)-epinephrine inhibition constant [Ki] . 0.6 mumol/L greater than (-)-norepinephrine (Ki . 1.5 mumol/L) much greater than (-)-isoproterenol (Ki . 120 mumol/L). The alpha-adrenergic antagonist phentolamine (Ki . 6.0 nmol/L) was 200 times more potent than the beta-adrenergic antagonist (+/-)-propranolol (Ki . 1,200 nmol/L); the alpha 1-selective antagonist prazosin (Ki . 1.2 nmol/L) was 130 times more potent than the alpha 2-selective antagonist yohimbine (Ki . 160 nmol/L). Scatchard analysis, as well as iterative curve-fitting analysis, demonstrated that 3 H-WB-4101 binding by arterial membranes was to a single class of binding sites. Uterine arteries exhibited greater maximal binding capacity (BMax) than that of mesenteric arteries (47.5 +/- 3.2 versus 30.9 +/- 3.6 fmol per milligram of protein, p less than 0.01), but the uterine artery dissociation constant (Kd) was higher, thus indicating a lower affinity, when compared with mesenteric artery (0.43 +/- 0.04 versus 0.33 +/- 0.04 nmol/L, p less than 0.05)

  3. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    International Nuclear Information System (INIS)

    Tao Weitao; Budd, Martin; Campbell, Judith L.

    2003-01-01

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Δ double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Δ alone. However, surprisingly, the dna2-2 sgs1Δ lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Δ lethality is only partially suppressed by deletion of rad51Δ. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

  4. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    Energy Technology Data Exchange (ETDEWEB)

    Tao Weitao; Budd, Martin; Campbell, Judith L

    2003-11-27

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1{delta} double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1{delta} alone. However, surprisingly, the dna2-2 sgs1{delta} lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1{delta} lethality is only partially suppressed by deletion of rad51{delta}. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

  5. Do saw palmetto extracts block human alpha1-adrenoceptor subtypes in vivo?

    Science.gov (United States)

    Goepel, M; Dinh, L; Mitchell, A; Schäfers, R F; Rübben, H; Michel, M C

    2001-02-15

    To test whether saw palmetto extracts, which act as alpha1-adrenoceptor antagonists in vitro, also do so in vivo in man. In a placebo-controlled, double-blind, four-way cross-over study 12 healthy young men were treated with three different saw palmetto extract preparations (320 mg o.d.) for 8 days each. On the last day, before and 2, 4 and 6 hr after drug intake blood pressure and heart rate were determined and blood samples obtained, which were used in an ex vivo radioreceptor assay with cloned human alpha1-adrenoceptor subtypes. Saw palmetto extract treatment did not result in alpha1-adrenoceptor subtype occupancy in the radioreceptor assay. Although the saw palmetto extracts caused minor reductions of supine blood pressure, they did not affect blood pressure during orthostatic stress testing and did not alter heart rate under either condition. Moreover, plasma catecholamines remained largely unaltered. Despite their alpha1-adrenoceptor antagonist effects in vitro, therapeutically used doses of saw palmetto extracts do not cause alpha1-adrenoceptor antagonism in man in vivo. Copyright 2001 Wiley-Liss, Inc.

  6. Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF-kappaB1 DNA binding domain.

    Science.gov (United States)

    Lambert, Cherie; Li, Jimei; Jonscher, Karen; Yang, Teng-Chieh; Reigan, Philip; Quintana, Megan; Harvey, Jean; Freed, Brian M

    2007-07-06

    Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The alpha,beta-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-kappaB DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-kappaB1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-kappaB consensus sequence. Matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.

  7. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

    Science.gov (United States)

    Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

    2016-01-01

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

  8. Studies of the Gly482Ser polymorphism of the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) gene in Danish subjects with the metabolic syndrome

    DEFF Research Database (Denmark)

    Ambye, L; Rasmussen, S; Fenger, Mogens

    2005-01-01

    The peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) is a novel transcriptional co-activator that holds an important role in lipid and glucose metabolism. PGC-1alpha is a candidate gene for the metabolic syndrome (MS) as well as type 2 diabetes. Recent studies...... related to this syndrome. The variant was examined, using PCR-RFLP, in the DanMONICA cohort comprising a population-based sample of 2349 subjects. MS was defined using the National Cholesterol Education Program -- Adult Treatment Panel III (NCEP-ATPIII) criteria. The allelic frequency of the Ser482 allele...... and insulin secretion, 24-ambulatory blood pressure or left ventricular mass index. In conclusion, the Gly482Ser polymorphism of the PGC-1alpha gene is not associated with the metabolic syndrome, related quantitative traits or cardiac hypertrophy among Danish Caucasian subjects...

  9. [125Iodo]BE 2254, a new radioligand for alpha 1-adrenoceptors

    International Nuclear Information System (INIS)

    Engel, G.; Hoyer, D.

    1982-01-01

    [ 125 cIodo]2-[beta-(14-hydroxyphenyl)-ethyl-aminomethyl]-tetralone([ 125 Iodo]BE 2254 or IBE 2254), a new iodinated radioligand of high specific radioactivity (2175 Ci/mmol), was synthesized and used to characterize alpha 1-adrenoceptors in rat lung and cerebral cortex membranes. The binding constants of IBE 2254, using rat lung and cortex membranes, were Kd . 53 +/- 10 pM, Bmax . 53 +/- 8 fmol/mg; and Kd . 78 +/- 14 pM, Bmas . 210 +/- 26 fmol/mg, respectively (Kd . dissociation constant of IBE 2254 determined in saturation experiments). In equilibrium binding experiments with IBE 2254, at concentrations of the free ligand up to 1.2 nM, only one class of binding sites could be detected. In kinetic experiments, the association and dissociation rate constants were 2.3 X 10(9) M-1 min-1 and 0.10 min-1, respectively. In rat cerebral cortex membranes, alpha-adrenoceptor antagonists competed for IBE 2254 binding in the following order: prazosin greater than BE 2254 greater than WB 4101 greater than phentolamine greater than corynanthine greater than yohimbine greater than rauwolscine, indicating strongly that IBE 2254 binds to alpha 1-adrenoceptors. The calculated affinities of different alpha-adrenoceptor blocking agents from inhibition of IBE 2254 binding were nearly identical in rat lung and cerebral cortex. The low dissociation constant of the ligand together with its high specific radioactivity allows binding studies to be carried out with tissue samples where only small densities of alpha 1-adrenoceptors are present

  10. Ubiquitous hazardous metal lead induces TNF-{alpha} in human phagocytic THP-1 cells: Primary role of ERK 1/2

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Mohd Imran [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Islam, Najmul [Department of Biochemistry, J.N Medical College, Aligarh Muslim University, Aligarh (India); Sahasrabuddhe, Amogh A. [Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow (India); Mahdi, Abbas Ali [Department of Biochemistry, C.S.M. Medical University, Lucknow (India); Siddiqui, Huma; Ashquin, Mohd [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Ahmad, Iqbal, E-mail: ahmadi@sify.com [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India)

    2011-05-15

    Induction of tumor necrosis factor-{alpha} (TNF-{alpha}) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-{alpha} is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-{alpha} induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-{alpha} m-RNA and protein secretion. Moreover, the stability of TNF-{alpha} m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-{alpha} m-RNA. However, SB 203580 partially inhibited production and stability of TNF-{alpha} m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-{alpha} m-RNA. Data tends to suggest that expression and stability of TNF-{alpha} induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

  11. Theoretical foundation and experimental proof of the accumulating transfer of tritium from water into DNA and other biomolecules in vitro and in vivo

    International Nuclear Information System (INIS)

    Baumgartner, F.

    2000-01-01

    Theoretical foundation and experimental proof of the accumulating transfer of tritium from water into DNA and other biomolecules were presented using the data of experiments on fish sperm DNA and piglet's liver (in vitro tests) and maize and barley (in vivo tests). Heisenberg's uncertainty principle, which describes a fundamental property of matter, predicts the light hydrogen isotope to prefer strong hydrogen bridge positions (with large local uncertainty). In contrast, the heavy hydrogen isotope tritium originally localized in water, should finally be found in the exchangeable hydrogen bridge positions of proteins, carbohydrates and nucleotides which are definitely less strong than those of water. Fractionation factor of tritium and protonium measured between water and DNA as well as the mixture of biomolecules of liver confirm this conclusion and show tritium accumulation biomolecules. Furthermore, the elevated rate of natural increase in tritium concentration in tissue solids of growing maize and barley confirm the accumulating tritium transfer from water to biomolecules taking place in vivo [ru

  12. Accurate measurement of gene copy number for human alpha-defensin DEFA1A3.

    Science.gov (United States)

    Khan, Fayeza F; Carpenter, Danielle; Mitchell, Laura; Mansouri, Omniah; Black, Holly A; Tyson, Jess; Armour, John A L

    2013-10-20

    Multi-allelic copy number variants include examples of extensive variation between individuals in the copy number of important genes, most notably genes involved in immune function. The definition of this variation, and analysis of its impact on function, has been hampered by the technical difficulty of large-scale but accurate typing of genomic copy number. The copy-variable alpha-defensin locus DEFA1A3 on human chromosome 8 commonly varies between 4 and 10 copies per diploid genome, and presents considerable challenges for accurate high-throughput typing. In this study, we developed two paralogue ratio tests and three allelic ratio measurements that, in combination, provide an accurate and scalable method for measurement of DEFA1A3 gene number. We combined information from different measurements in a maximum-likelihood framework which suggests that most samples can be assigned to an integer copy number with high confidence, and applied it to typing 589 unrelated European DNA samples. Typing the members of three-generation pedigrees provided further reassurance that correct integer copy numbers had been assigned. Our results have allowed us to discover that the SNP rs4300027 is strongly associated with DEFA1A3 gene copy number in European samples. We have developed an accurate and robust method for measurement of DEFA1A3 copy number. Interrogation of rs4300027 and associated SNPs in Genome-Wide Association Study SNP data provides no evidence that alpha-defensin copy number is a strong risk factor for phenotypes such as Crohn's disease, type I diabetes, HIV progression and multiple sclerosis.

  13. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

    Science.gov (United States)

    Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

    2016-03-04

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

    Directory of Open Access Journals (Sweden)

    French Frank S

    2004-08-01

    Full Text Available Abstract Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed

  15. Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver

    International Nuclear Information System (INIS)

    Lutz, W.K.; Buesser, M.T.; Sagelsdorff, P.

    1984-01-01

    In order to investigate the role of the stimulation of cell division for the initiation (and possibly promotion) of liver tumors by chemical carcinogens, the incorporation of radiolabelled thymidine into liver DNA was determined in male rats. Single doses of various levels of aflatoxin B1, benzidine and carbon tetrachloride (all known to be genotoxic via DNA binding) did not affect cell division, whereas several hepatocarcinogens known not to bind to DNA (alpha-HCH, clofibrate, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) gave rise to a dose-dependent stimulation of liver DNA synthesis within 24 h. An equation combining the influences of mitotic stimulation, expressed as dose required to double the control level of DNA synthesis, and DNA binding potency, expressed as the Covalent Binding Index, correlated well with the carcinogenic potency for both classes of hepatocarcinogens

  16. Macrophage inflammatory protein-1alpha: a link between innate immunity and familial Mediterranean fever?

    Science.gov (United States)

    Dizdar, Omer; Kalyoncu, Umut; Karadag, Omer; Akdogan, Ali; Kiraz, Sedat; Ertenli, Ihsan; Barista, Ibrahim; Calguneri, Meral

    2007-01-01

    The aim of this study is to investigate the relationship between chemokines and the inflammation in Familial Mediterranean Fever (FMF). Forty-nine patients with FMF (41 in remission and 8 in acute attack period) and 20 healthy controls were included in the study. Serum levels of macrophage inflammatory protein-1alpha (MIP-1alpha) were assessed in the patients and the controls, along with other parameters of disease activity, i.e., fibrinogen, C-reactive protein and erythrocyte sedimentation rate. Serum MIP-1alpha levels of the patients with FMF in acute attack period were significantly higher than the patients in remission and healthy controls (p=0.02 and p=0.038, respectively). MIP-1alpha levels were weakly correlated with CRP (r=0.32, p=0.032) levels. MIP-1alpha may have a role in the pathogenesis of FMF attacks. MIP-1alpha and other chemokines may constitute a link between the innate immune system and FMF.

  17. DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

    2009-01-01

    Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures......Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...

  18. DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

    OpenAIRE

    Couldrey, Christine; Wells, David N.

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT bla...

  19. Whole-body irradiation transiently diminishes the adrenocorticotropin response to recombinant human interleukin-1{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Perlstein, R.S.; Mehta, N.R.; Neta, R.; Whitnall, M.H. [Armed Forces Radiobiology Research Institute, Bethesda, MD (United States); Mougey, E.H. [Walter Reed Army Institute of Research, Washington, DC (United States)

    1995-03-01

    Recombinant human interleukin-1{alpha} (rhIL-1{alpha}) has significant potential as a radioprotector and/or treatment for radiation-induced hematopoietic injury. Both IL-1 and whole-body ionizing irradiation acutely stimulate the hypothalamic-pituitary-adrenal axis. We therefore assessed the interaction of whole-body irradiation and rhIL-1{alpha} in altering the functioning of the axis in mice. Specifically, we determined the adrenocorticotropin (ACTH) and corticosterone responses to rhIL-1{alpha} administered just before and hours to days after whole-body or sham irradiation. Our results indicate that whole-body irradiation does not potentiate the rhIL-1{alpha}-induced increase in ACTH levels at the doses used. In fact, the rhIL-1{alpha}-induced increase in plasma ACTH is transiently impaired when the cytokine is administered 5 h after, but not 1 h before, exposure to whole-body irradiation. The ACTH response may be inhibited by elevated corticosterone levels after whole-body irradiation, or by other radiation-induced effects on the pituitary gland and hypothalamus. 36 refs., 3 figs.

  20. Genetics Home Reference: alpha-1 antitrypsin deficiency

    Science.gov (United States)

    ... rapid heartbeat upon standing. Affected individuals often develop emphysema, which is a lung disease caused by damage to the small air ... exposure to tobacco smoke accelerates the appearance of emphysema symptoms and damage to the lungs. About 10 percent of infants with alpha-1 ...

  1. Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

    Science.gov (United States)

    Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

    1996-01-19

    Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

  2. Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Xia, E-mail: zhongxia1977@126.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Li, Xiaonan; Liu, Fuli; Tan, Hui [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Shang, Deya, E-mail: wenhuashenghuo1@163.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.

  3. ADD1/SREBP1c activates the PGC1-alpha promoter in brown adipocytes

    DEFF Research Database (Denmark)

    Hao, Qin; Hansen, Jacob B; Petersen, Rasmus K

    2010-01-01

    Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol...

  4. Binding of alpha2ML1 to the low density lipoprotein receptor-related protein 1 (LRP1 reveals a new role for LRP1 in the human epidermis.

    Directory of Open Access Journals (Sweden)

    Marie-Florence Galliano

    Full Text Available BACKGROUND: The multifunctional receptor LRP1 has been shown to bind and internalize a large number of protein ligands with biological importance such as the pan-protease inhibitor alpha2-macroglobulin (alpha2M. We recently identified Alpha2ML1, a new member of the alpha2M gene family, expressed in epidermis. alpha2ML1 might contribute to the regulation of desquamation through its inhibitory activity towards proteases of the chymotrypsin family, notably KLK7. The expression of LRP1 in epidermis as well as its ability to internalize alpha2ML1 was investigated. METHODS AND PRINCIPAL FINDINGS: In human epidermis, LRP1 is mainly expressed within the granular layer of the epidermis, which gathers the most differentiated keratinocytes, as shown by immunohistochemistry and immunofluorescence using two different antibodies. By using various experimental approaches, we show that the receptor binding domain of alpha2ML1 (RBDl is specifically internalized into the macrophage-like cell line RAW and colocalizes with LRP1 upon internalization. Coimmunoprecipitation assays demonstrate that RBDl binds LRP1 at the cell surface. Addition of RAP, a universal inhibitor of ligand binding to LRP1, prevents RBDl binding at the cell surface as well as internalization into RAW cells. Silencing Lrp1 expression with specific siRNA strongly reduces RBDl internalization. CONCLUSIONS AND SIGNIFICANCE: Keratinocytes of the upper differentiated layers of epidermis express LRP1 as well as alpha2ML1. Our study also reveals that alpha2ML1 is a new ligand for LRP1. Our findings are consistent with endocytosis by LRP1 of complexes formed between alpha2ML1 and proteases. LRP1 may thus control desquamation by regulating the biodisponibility of extracellular proteases.

  5. Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

    Energy Technology Data Exchange (ETDEWEB)

    Sansing, Hope A. [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States); Sarkeshik, Ali; Yates, John R. [Department of Chemical Physiology, Scripps Research Institute, La Jolla, CA (United States); Patel, Vyomesh; Gutkind, J. Silvio [Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Yamada, Kenneth M. [Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Berrier, Allison L., E-mail: allison.berrier@gmail.com [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States)

    2011-03-11

    Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.

  6. The Structure of Neurexin 1[alpha] Reveals Features Promoting a Role as Synaptic Organizer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fang; Venugopal, Vandavasi; Murray, Beverly; Rudenko, Gabby (Michigan)

    2014-10-02

    {alpha}-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The {alpha}-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how {alpha}-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1{alpha} extracellular domain (n1{alpha}) to 2.65 {angstrom}. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 {angstrom} structure of n1{alpha} carrying splice insert SS3 in LNS4 reveals that SS3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables {alpha}-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.

  7. Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

    Science.gov (United States)

    O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

    2014-01-01

    Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

  8. Characterization of class II alpha genes and DLA-D region allelic associations in the dog.

    Science.gov (United States)

    Sarmiento, U M; Storb, R F

    1988-10-01

    Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

  9. DNA polymerases in the rat pituitary gland. Effect of oestrogens and sulpiride.

    Science.gov (United States)

    Jahn, G A; Kalbermann, L E; Machiavelli, G; Szijan, I; Burdman, J A

    1980-06-01

    Changes in the activity of DNA polymerase and [3H]thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats. The activities of DNA polymerases alpha and beta, extracted in Tris--HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively alpha, while in the phosphate soluble fraction it is a mixture of alpha and beta. The administration of oestrogens to male rats increases [3H]thymidine incorporation and enhances the activity of DNA polymerases in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further increment of [3H]thymidine incorporation and of DNA polymerase activity in the Tris-soluble fraction. In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction. Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase alpha. This activity changes in parallel with [3H]thymidine incorporation into DNA which is an indication of cell proliferation in the gland. This is discussed with respect to a negative feedback mechanism between intracellular prolactin concentration and DNA synthesis in the APG.

  10. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr; Sirma-Ekmekci, Sema, E-mail: semasirma@gmail.com; Yildirim, Ozlem, E-mail: ozlm-yildirim@hotmail.com; Erginel-Unaltuna, Nihan, E-mail: nihanerginel@yahoo.com

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  11. Prognostic impact of HIF-1{alpha} expression in patients with definitive radiotherapy for cervical cancer

    Energy Technology Data Exchange (ETDEWEB)

    Dellas, K.; Bache, M.; Kappler, M.; Haensgen, G. [Halle-Wittenberg Univ., Halle (Germany). Dept. of Radiotherapy; Pigorsch, S.U. [Technische Univ. Muenchen (Germany). Dept. of Radiotherapy and Radiation Oncology; Taubert, H.; Holzhausen, H.J. [Halle-Wittenberg Univ., Halle (Germany). Inst. of Pathology; Holzapfel, D.; Zorn, E. [Halle-Wittenberg Univ., Halle (Germany). Dept. of Radiotherapy; Halle-Wittenberg Univ., Halle (Germany). Inst. of Pathology

    2008-03-15

    Purpose: To investigate the relationship between the hypoxia-inducible factor-(HIF-)1{alpha} expression in tumor tissue, tumor oxygenation and hemoglobin levels in patients with advanced cervical cancers prior to radiotherapy and the effect on clinical outcome. Patients and Methods: The investigation included 44 patients who underwent definitive radiotherapy for advanced cervical cancers between May 1995 and March 1999. Tumor biopsies were taken prior to treatment, and HIF-1{alpha} expression was determined by immunohistochemistry. In the same tumor area, tumor tissue oxygenation (pO{sub 2}) was measured using the Eppendorf device. Results: The 5-year cancer-specific survival of all patients was 60%. Twelve of 44 tumor specimens were HIF-1{alpha}-negative with a significantly better 5-year survival (92 {+-} 8%) versus 32 patients who were HIF-1{alpha}-positive (45 {+-} 10%; p < 0.02). There was no correlation between HIF-1{alpha} expression and tumor oxygenation (p = 0.57 both for pO{sub 2} median and hypoxic fraction < 5 mmHg vs. HIF-1{alpha} expression). However, patients with hemoglobin levels < 11 g/dl showed elevated HIF-1{alpha} expression compared to patients with hemoglobin levels > 12.5 g/dl (p = 0.04). Furthermore, HIF-1{alpha} correlated with vascular endothelial growth factor expression (p = 0.002). In a multivariate Cox regression model, HIF-1{alpha} expression (relative risk [RR] = 7.5; p = 0.05) revealed an increased risk of tumor-related death. Conclusion: The study indicates, that endogenous tumor markers such as HIF-1{alpha} may serve as prognostic markers of clinical outcome concerning cervical cancer after primary radiotherapy. (orig.)

  12. The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

    Science.gov (United States)

    Bolduc, Gilles R; Madoff, Lawrence C

    2007-12-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

  13. Age-dependent changes in expression of alpha1-adrenergic receptors in rat myocardium

    International Nuclear Information System (INIS)

    Schaffer, W.; Williams, R.S.

    1986-01-01

    The expression of alpha 1 -adrenergic receptors within ventricular myocardium of rats ranging in age from 21 days of fetal life to 24 months after birth was measured from [ 125 I] 2-(β hydroxy phenyl) ethylaminomethyl tetralone binding isotherms. No difference was observed in binding affinity between any of the age groups studied. The number of alpha 1 -adrenergic receptors was found to be 60-120% higher in membranes from fetal or immature rats up to 25 days of age when compared with adult animals. The increased expression of alpha 1 -adrenergic receptors in the developing heart relative to that observed in adult heart is consistent with the hypothesis that alpha 1 -adrenergic receptor stimulation may modulate protein synthesis and growth in mammalian myocardium

  14. Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Weiping, E-mail: weiping.qin@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Pan, Jiangping; Wu, Yong [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Bauman, William A. [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Department of Rehabilitation Medicine, Mount Sinai School of Medicine, NY (United States); Cardozo, Christopher, E-mail: Chris.Cardozo@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Department of Rehabilitation Medicine, Mount Sinai School of Medicine, NY (United States)

    2010-12-17

    Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis, REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius

  15. ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

    DEFF Research Database (Denmark)

    Lafuste, Peggy; Sonnet, Corinne; Chazaud, Bénédicte

    2005-01-01

    of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion...

  16. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  17. A double mutation in exon 6 of the [beta]-hexosaminidase [alpha] subunit in a patient with the B1 variant of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Ainsworth, P.J. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada)); Coulter-Mackie, M.B. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada) Children' s Psychiatric Research Institute, London, Ontario (Canada))

    1992-10-01

    The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the [alpha] subunit of [beta]-hexosaminidase A without altering its ability to associate with the [beta] subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the [alpha] subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G[sub 574][yields]C transversion causing a val[sub 192][yields]leu change and a G[sub 598][yields] A transition resulting in a val[sub 200][yields]met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G[sub 574][yields]C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5. 31 refs., 2 figs., 2 tabs.

  18. Alpha-1 antitrypsin Pi*SZ genotype: estimated prevalence and number of SZ subjects worldwide

    Directory of Open Access Journals (Sweden)

    Blanco I

    2017-06-01

    Full Text Available Ignacio Blanco,1 Patricia Bueno,2 Isidro Diego,3 Sergio Pérez-Holanda,4 Beatriz Lara,5 Francisco Casas-Maldonado,6 Cristina Esquinas,7 Marc Miravitlles7,8 1Alpha1-Antitrypsin Deficiency Spanish Registry (REDAAT, Lung Foundation Breathe, Spanish Society of Pneumology (SEPAR, Barcelona, Spain; 2Internal Medicine Department, County Hospital of Jarrio, Principality of Asturias, Spain; 3Materials and Energy Department, School of Mining Engineering, Oviedo University, Principality of Asturias, Spain; 4Surgical Department, University Central Hospital of Asturias, Oviedo, Spain; 5Respiratory Medicine Department, Coventry and Warwickshire University Hospital, Coventry, UK; 6Pneumology Department, University Hospital San Cecilio, Granada, Spain; 7Pneumology Department, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 8CIBER de Enfermedades Respiratorias (CIBERES, Barcelona, Spain Abstract: The alpha-1 antitrypsin (AAT haplotype Pi*S, when inherited along with the Pi*Z haplotype to form a Pi*SZ genotype, can be associated with pulmonary emphysema in regular smokers, and less frequently with liver disease, panniculitis, and systemic vasculitis in a small percentage of people, but this connection is less well established. Since the detection of cases can allow the application of preventive measures in patients and relatives with this congenital disorder, the objective of this study was to update the prevalence of the SZ genotype to achieve accurate estimates of the number of Pi*SZ subjects worldwide, based on studies performed according to the following criteria: 1 samples representative of the general population, 2 AAT phenotyping characterized by adequate methods, and 3 selection of studies with reliable results assessed with a coefficient of variation calculated from the sample size and 95% confidence intervals. Studies fulfilling these criteria were used to develop tables and maps with an inverse distance-weighted (IDW interpolation method, to

  19. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  20. Analysis of the local kinetics and localization of interleukin-1 alpha, tumour necrosis factor-alpha and transforming growth factor-beta, during the course of experimental pulmonary tuberculosis.

    Science.gov (United States)

    Hernandez-Pando, R; Orozco, H; Arriaga, K; Sampieri, A; Larriva-Sahd, J; Madrid-Marina, V

    1997-01-01

    A mouse model of pulmonary tuberculosis induced by the intratracheal instillation of live and virulent mycobacteria strain H37-Rv was used to examine the relationship of the histopathological findings with the local kinetics production and cellular distribution of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta). The histopathological and immunological studies showed two phases of the disease: acute or early and chronic or advanced. The acute phase was characterized by inflammatory infiltrate in the alveolar-capillary interstitium, blood vessels and bronchial wall with formation of granulomas. During this acute phase, which lasted from 1 to 28 days, high percentages of TNF-alpha and IL-1 alpha immunostained activated macrophages were observed principally in the interstium-intralveolar inflammatory infiltrate and in granulomas. Electron microscopy studies of these cells, showed extensive rough endoplasmic reticulum, numerous lysosomes and occasional mycobacteria. Double labelling with colloid gold showed that TNF-alpha and IL-1 alpha were present in the same cells, but were confined to separate vacuoles near the Golgi area, and mixed in larger vacuoles near to cell membrane. The concentration of TNF-alpha and IL-1 alpha as well as their respective mRNAs were elevated in the early phase, particularly at day 3 when the bacillary count decreased. A second peak was seen at days 14 and 21-28 when granulomas appeared and evolved to full maturation. In contrast, TGF-beta production and numbers of immunoreactive cells were low in comparison with the advanced phase of the disease. The chronic phase was characterized by histopathological changes indicative of more severity (i.e. pneumonia, focal necrosis and extensive interstitial fibrosis) with a decrease in the TNF-alpha and IL-1 alpha production that coincided with the highest level of TGF-beta. The bacillary counts were highest as the macrophages

  1. Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel.

    Science.gov (United States)

    Sherry, A M; Stroffekova, K; Knapp, L M; Kupert, E Y; Cuppoletti, J; Malinowska, D H

    1997-08-01

    A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

  2. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    International Nuclear Information System (INIS)

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B.

    1989-01-01

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue

  3. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

    DEFF Research Database (Denmark)

    Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

    2005-01-01

    and 2 (HIFs) are clearly related heterodimeric transcription factors that consist of an oxygen-depended alpha-subunit and a constitutive beta-subunit. With hypoxic exposure, HIF-1alpha and HIF-2alpha protein are stabilized. Upon heterodimerization, HIFs induce the transcription of a variety of genes......Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... including erythropoietin (EPO), transferrin and its receptor, as well as vascular endothelial growth factor (VEGF) and its receptor. Considering that several of these genes are also induced with exercise, we tested the hypothesis that the mRNA level of HIF-1alpha and HIF-2alpha subunits increases...

  4. Effect of hypoxia-inducible factor 1-alpha (HIF-1α) on proliferation ...

    African Journals Online (AJOL)

    Jane

    2011-07-25

    Jul 25, 2011 ... Full Length Research Paper. Effect of hypoxia-inducible factor 1-alpha ... 1Department of Neurosurgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025,. China. 2Department of Neurosurgery, Chaoyang Hospital, Huainan, Anhui, China. 3Department of Neurosurgery ...

  5. Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pan

    Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

  6. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

    Science.gov (United States)

    Cline, J; Braman, J C; Hogrefe, H H

    1996-09-15

    The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

  7. ''Spare'' alpha 1-adrenergic receptors and the potency of agonists in rat vas deferens

    International Nuclear Information System (INIS)

    Minneman, K.P.; Abel, P.W.

    1984-01-01

    The existence of ''spare'' alpha 1-adrenergic receptors in rat vas deferens was examined directly using radioligand binding assays and contractility measurements. Alpha 1-adrenergic receptors in homogenates of rat vas deferens were labeled with [ 125 I]BE 2254 ( 125 IBE). Norepinephrine and other full alpha 1-adrenergic receptor agonists were much less potent in inhibiting 125 IBE binding than in contracting the vas deferens in vitro. Treatment with 300 nM phenoxybenzamine for 10 min to irreversibly inactivate alpha 1-adrenergic receptors caused a large decrease in the potency of full agonists in causing contraction of this tissue and a 23-48% decrease in the maximal contraction observed. Using those data, equilibrium constants for activation (Kact values) of the receptors by agonists were calculated. These Kact values agreed well with the equilibrium binding constants (KD values) determined from displacement of 125 IBE binding. The reduction in alpha 1-adrenergic receptor density following phenoxybenzamine treatment was determined by Scatchard analysis of specific 125 IBE binding sites and compared with the expected reduction (q values) calculated from the agonist dose-response curves before and after phenoxybenzamine treatment. This suggests that phenoxybenzamine functionally inactivates alpha 1-adrenergic receptors at or near the receptor binding site. These experiments suggest that the potencies of agonists in activating alpha 1-adrenergic receptors in rat vas deferens agree well with their potencies in binding to the receptors. The greater potency of agonists in causing contraction may be due to spare receptors in this tissue. The data also demonstrate that phenoxybenzamine irreversibly inactivates alpha 1-adrenergic receptors in rat vas deferens, but that the decrease in receptor density is much smaller than that predicted from receptor theory

  8. IgG autoantibodies against interleukin 1 alpha in sera of normal individuals

    DEFF Research Database (Denmark)

    Svenson, M; Poulsen, L K; Fomsgaard, A

    1989-01-01

    A pool of human sera from healthy blood donors was found to interfere competitively with the binding of 125I-labelled human recombinant interleukin 1 alpha (rIL-1 alpha) to the murine T-cell line EL4. The interference was reversible at the cellular level, and direct binding of the ligand to serum...

  9. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Science.gov (United States)

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  10. Efficacy of lamivudine and thymosin alpha-1 combination therapy in treatment of HBeAg-positive chronic hepatitis B: a meta-analysis

    Directory of Open Access Journals (Sweden)

    QI Youtao

    2014-07-01

    Full Text Available ObjectiveTo evaluate the efficacy of lamivudine and thymosin alpha-1 combination therapy in the treatment of HBeAg-positive chronic hepatitis B (CHB by meta-analysis. MethodsRandomized controlled trials (RCTs of lamivudine and thymosin alpha-1 combination therapy in treatment of HBeAg-positive CHB (follow-up for at least 24 weeks, from January 1998 to date, were identified by searching Cochrane Library, PubMed, EMBASE, EBSCO, CNKI, Wanfang Data, and CQVIP. Lamivudine monotherapy RCTs were searched for in the same way as control tests. Efficacy was measured by odds ratio. Meta-analysis was carried out with RevMan 5.2 software. ResultsNine RCTs involving 600 patients were included, with 320 cases in the combination therapy group and 280 in the control group. At the end of follow-up, the combination therapy group had significantly higher serum ALT recovery rate, HBV-DNA negative conversion rate, HBeAg negative conversion rate, and HBeAg seroconversion rate than the control group (P<0.01 for all, with pooled odds ratios (95% confidence intervals of 4.84 (3.28, 7.16, 2.09 (1.45, 3.01, 5.32 (3.35, 8.46, and 6.22 (3.78, 10.25, respectively. ConclusionLamivudine and thymosin alpha-1 combination therapy is more likely to achieve sustained response rate than lamivudine monotherapy for HBeAg-positive CHB. More RCTs of high quality and large scale are required to verify this conclusion.

  11. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States); Rozengurt, Enrique, E-mail: erozengurt@mednet.ucla.edu [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Metformin inhibits cancer cell growth but the mechanism(s) are not understood. Black-Right-Pointing-Pointer We show that the potency of metformin is sharply dependent on glucose in the medium. Black-Right-Pointing-Pointer AMPK activation was enhanced in cancer cells incubated in physiological glucose. Black-Right-Pointing-Pointer Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. Black-Right-Pointing-Pointer Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser{sup 79} and Raptor at Ser{sup 792}, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the {alpha}{sub 1} and {alpha}{sub 2} catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  12. Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

    Science.gov (United States)

    Waye, J S; Willard, H F

    1986-09-01

    The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

  13. PGC-1alpha Deficiency Causes Multi-System Energy Metabolic Derangements: Muscle Dysfunction, Abnormal Weight Control and Hepatic Steatosis

    Directory of Open Access Journals (Sweden)

    Leone Teresa C

    2005-01-01

    Full Text Available The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha was targeted in mice. PGC-1alpha null (PGC-1alpha-/- mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1alpha-/- mice. With age, the PGC-1alpha-/- mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1alpha-/- mice, leading to reduced muscle performance and exercise capacity. PGC-1alpha-/- mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1alpha-/- mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1alpha-/- mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1alpha-/- mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1alpha-/- mice. These results demonstrate that PGC-1alpha is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.

  14. PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis.

    Directory of Open Access Journals (Sweden)

    Teresa C Leone

    2005-04-01

    Full Text Available The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha was targeted in mice. PGC-1alpha null (PGC-1alpha(-/- mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1alpha(-/- mice. With age, the PGC-1alpha(-/- mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1alpha(-/- mice, leading to reduced muscle performance and exercise capacity. PGC-1alpha(-/- mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1alpha(-/- mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1alpha(-/- mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1alpha(-/- mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1alpha(-/- mice. These results demonstrate that PGC-1alpha is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.

  15. HIF-1{alpha} is necessary to support gluconeogenesis during liver regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Tajima, Toshihide [Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Goda, Nobuhito, E-mail: goda@waseda.jp [Department of Life Science and Medical Bio-Science, School of Advanced Science and Engineering, Waseda University, TWIns 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Fujiki, Natsuko; Hishiki, Takako; Nishiyama, Yasumasa [Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Senoo-Matsuda, Nanami [Department of Life Science and Medical Bio-Science, School of Advanced Science and Engineering, Waseda University, TWIns 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Shimazu, Motohide [Department of Surgery, Tokyo Medical University Hachioji Medical Center, 1163 Tatemachi, Hachioji, Tokyo 193-0998 (Japan); Soga, Tomoyoshi [The Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata 997-0052 (Japan); Yoshimura, Yasunori [Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Johnson, Randall S. [Molecular Biology Section, Division of Biology, University of California, San Diego, La Jolla, CA 92093 (United States); Suematsu, Makoto [Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2009-10-02

    Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1{alpha} (HIF-1{alpha}) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1{alpha} in the regulation of gluconeogenesis under liver regeneration.

  16. Next generation DNA led technologies

    CERN Document Server

    Jyothsna, G; Kashyap, Amita

    2016-01-01

    This brief highlights advances in DNA technologies and their wider applications. DNA is the source of life and has been studied since a generation, but very little is known as yet. Several sophisticated technologies of the current era have laid their foundations on the principle of DNA based mechanisms. DNA based technologies are bringing a new revolution of Advanced Science and Technology. Forensic Investigation, Medical Diagnosis, Paternity Disputes, Individual Identity, Health insurance, Motor Insurance have incorporated the DNA testing and profiling technologies for settling the issues.

  17. Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

    Science.gov (United States)

    Thiersch, Markus; Lange, Christina; Joly, Sandrine; Heynen, Severin; Le, Yun Zheng; Samardzija, Marijana; Grimm, Christian

    2009-06-01

    Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

  18. p53 binding protein 1 foci as a biomarker of DNA double strand breaks induced by ionizing radiation

    International Nuclear Information System (INIS)

    Ng, C.K.M.; Wong, M.Y.P.; Lam, R.K.K.; Ho, J.P.Y.; Chiu, S.K.; Yu, K.N.

    2011-01-01

    Foci of p53 binding protein 1 (53 BP1) have been used as a biomarker of DNA double-strand breaks (DSBs) in cells induced by ionizing radiations. 53 BP1 was shown to relocalize into foci shortly after irradiation, with the number of foci closely paralleling the number of DNA DSBs. However, consensus on criteria in terms of the numbers of 53 BP1 foci to define cells damaged by direct irradiation or by bystander signals has not been reached, which is partly due to the presence of 53 BP1 also in normal cells. The objective of the present work was to study the changes in the distribution of cells with different numbers of 53 BP1 foci in a cell population after low-dose ionizing irradiation (<0.1 Gy) provided by alpha particles, with a view to propose feasible criteria for defining cells damaged by direct irradiation or by bystander signals. It was proposed that the change in the percentage of cells with 1-3 foci should be used for such purposes. The underlying reasons were discussed.

  19. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNFalpha, anti-IL-1alpha/beta and IL-1Ra.

    NARCIS (Netherlands)

    Joosten, L.A.B.; Helsen, M.M.A.; Loo, F.A.J. van de; Berg, W.B. van den

    2008-01-01

    OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated

  20. Radiation and biophysical studies on cells and viruses. Progress report, April 1, 1976--June 30, 1977. [Gamma radiation, alpha particles

    Energy Technology Data Exchange (ETDEWEB)

    Cole, A.

    1977-01-01

    Progress is reported on the following research projects: genetic structure of DNA, chromosomes, and nucleoproteins; particle beam studies of radiosensitive sites; division delay in CHO cells induced by partly penetrating alpha particles; location of cellular sites for mutation induction; sites for radioinduced cell transformation using partly penetrating particle beams; gamma-ray and particle irradiation of nucleoproteins and other model systems; quantitation of surface antigens on normal and neoplastic cells by x-ray fluorescence; hyperthermic effects on cell survival and DNA repair mechanisms; and studies on radioinduced cell transformation. (HLW)

  1. Two food-borne heterocyclic amines: Metabolism and DNA adduct formation of amino-alpha-carbolines

    DEFF Research Database (Denmark)

    Frederiksen, Hanne

    2005-01-01

    or proteins of animal or vegetable origin, furthermore they are found in many cooked foods, such as fish, meat, and chicken. The specific mutagenicity of the amino-a-carbolines are lower in the Ames Salmonella assay than other heterocyclic amines, but in rodent studies the carcinogenicity of the aminoa, alpha......The amino-alpha-carbolines 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and 2-amino-3-methyl-9H-pyrido-[2,3-b]indole (MeA alpha C) are two mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. Amino-alpha-carbolines can be formed in model systems by pyrolyzing tryptophan...

  2. Cell motility in chronic lymphocytic leukemia: defective Rap1 and alphaLbeta2 activation by chemokine.

    Science.gov (United States)

    Till, Kathleen J; Harris, Robert J; Linford, Andrea; Spiller, David G; Zuzel, Mirko; Cawley, John C

    2008-10-15

    Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.

  3. Estrogen alters the diurnal rhythm of alpha 1-adrenergic receptor densities in selected brain regions

    International Nuclear Information System (INIS)

    Weiland, N.G.; Wise, P.M.

    1987-01-01

    Norepinephrine regulates the proestrous and estradiol-induced LH surge by binding to alpha 1-adrenergic receptors. The density of alpha 1-receptors may be regulated by estradiol, photoperiod, and noradrenergic neuronal activity. We wished to determine whether alpha 1-receptors exhibit a diurnal rhythm in ovariectomized and/or estradiol-treated female rats, whether estradiol regulates alpha 1-receptors in those areas of brain involved with LH secretion and/or sexual behavior, and whether the concentrations of alpha-receptors vary inversely relative to previously reported norepinephrine turnover patterns. Young female rats, maintained on a 14:10 light-dark cycle were ovariectomized. One week later, half of them were outfitted sc with Silastic capsules containing estradiol. Groups of animals were decapitated 2 days later at 0300, 1000, 1300, 1500, 1800, and 2300 h. Brains were removed, frozen, and sectioned at 20 micron. Sections were incubated with [ 3 H]prazosin in Tris-HCl buffer, washed, dried, and exposed to LKB Ultrofilm. The densities of alpha 1-receptors were quantitated using a computerized image analysis system. In ovariectomized rats, the density of alpha 1-receptors exhibited a diurnal rhythm in the suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), and pineal gland. In SCN and MPN, receptor concentrations were lowest during the middle of the day and rose to peak levels at 1800 h. In the pineal gland, the density of alpha 1-receptors was lowest at middark phase, rose to peak levels before lights on, and remained elevated during the day. Estradiol suppressed the density of alpha 1 binding sites in the SCN, MPN, median eminence, ventromedial nucleus, and the pineal gland but had no effect on the lateral septum. Estrogen treatment altered the rhythm of receptor densities in MPN, median eminence, and the pineal gland

  4. Estrogen alters the diurnal rhythm of alpha 1-adrenergic receptor densities in selected brain regions

    Energy Technology Data Exchange (ETDEWEB)

    Weiland, N.G.; Wise, P.M.

    1987-11-01

    Norepinephrine regulates the proestrous and estradiol-induced LH surge by binding to alpha 1-adrenergic receptors. The density of alpha 1-receptors may be regulated by estradiol, photoperiod, and noradrenergic neuronal activity. We wished to determine whether alpha 1-receptors exhibit a diurnal rhythm in ovariectomized and/or estradiol-treated female rats, whether estradiol regulates alpha 1-receptors in those areas of brain involved with LH secretion and/or sexual behavior, and whether the concentrations of alpha-receptors vary inversely relative to previously reported norepinephrine turnover patterns. Young female rats, maintained on a 14:10 light-dark cycle were ovariectomized. One week later, half of them were outfitted sc with Silastic capsules containing estradiol. Groups of animals were decapitated 2 days later at 0300, 1000, 1300, 1500, 1800, and 2300 h. Brains were removed, frozen, and sectioned at 20 micron. Sections were incubated with (/sup 3/H)prazosin in Tris-HCl buffer, washed, dried, and exposed to LKB Ultrofilm. The densities of alpha 1-receptors were quantitated using a computerized image analysis system. In ovariectomized rats, the density of alpha 1-receptors exhibited a diurnal rhythm in the suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), and pineal gland. In SCN and MPN, receptor concentrations were lowest during the middle of the day and rose to peak levels at 1800 h. In the pineal gland, the density of alpha 1-receptors was lowest at middark phase, rose to peak levels before lights on, and remained elevated during the day. Estradiol suppressed the density of alpha 1 binding sites in the SCN, MPN, median eminence, ventromedial nucleus, and the pineal gland but had no effect on the lateral septum. Estrogen treatment altered the rhythm of receptor densities in MPN, median eminence, and the pineal gland.

  5. Coordinated balancing of muscle oxidative metabolism through PGC-1{alpha} increases metabolic flexibility and preserves insulin sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Summermatter, Serge [Biozentrum, Division of Pharmacology/Neurobiology, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel (Switzerland); Troxler, Heinz [Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University Children' s Hospital, University of Zurich, Steinwiesstrasse 75, CH-8032 Zurich (Switzerland); Santos, Gesa [Biozentrum, Division of Pharmacology/Neurobiology, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel (Switzerland); Handschin, Christoph, E-mail: christoph.handschin@unibas.ch [Biozentrum, Division of Pharmacology/Neurobiology, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel (Switzerland)

    2011-04-29

    Highlights: {yields} PGC-1{alpha} enhances muscle oxidative capacity. {yields} PGC-1{alpha} promotes concomitantly positive and negative regulators of lipid oxidation. {yields} Regulator abundance enhances metabolic flexibility and balances oxidative metabolism. {yields} Balanced oxidation prevents detrimental acylcarnitine and ROS generation. {yields} Absence of detrimental metabolites preserves insulin sensitivity -- Abstract: The peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) enhances oxidative metabolism in skeletal muscle. Excessive lipid oxidation and electron transport chain activity can, however, lead to the accumulation of harmful metabolites and impair glucose homeostasis. Here, we investigated the effect of over-expression of PGC-1{alpha} on metabolic control and generation of insulin desensitizing agents in extensor digitorum longus (EDL), a muscle that exhibits low levels of PGC-1{alpha} in the untrained state and minimally relies on oxidative metabolism. We demonstrate that PGC-1{alpha} induces a strictly balanced substrate oxidation in EDL by concomitantly promoting the transcription of activators and inhibitors of lipid oxidation. Moreover, we show that PGC-1{alpha} enhances the potential to uncouple oxidative phosphorylation. Thereby, PGC-1{alpha} boosts elevated, yet tightly regulated oxidative metabolism devoid of side products that are detrimental for glucose homeostasis. Accordingly, PI3K activity, an early phase marker for insulin resistance, is preserved in EDL muscle. Our findings suggest that PGC-1{alpha} coordinately coactivates the simultaneous transcription of gene clusters implicated in the positive and negative regulation of oxidative metabolism and thereby increases metabolic flexibility. Thus, in mice fed a normal chow diet, over-expression of PGC-1{alpha} does not alter insulin sensitivity and the metabolic adaptations elicited by PGC-1{alpha} mimic the beneficial effects of endurance training

  6. Topical N-acetylcysteine reduces interleukin-1-alpha in tear fluid after laser subepithelial keratectomy.

    Science.gov (United States)

    Urgancioglu, Berrak; Bilgihan, Kamil; Engin, Doruk; Cirak, Meltem Yalinay; Hondur, Ahmet; Hasanreisoglu, Berati

    2009-01-01

    To evaluate the effect of topical N-acetylcysteine (NAC) on interleukin 1-alpha (IL-1alpha) levels in tear fluid after myopic laser subepithelial keratectomy (LASEK) and its possible role in modulating corneal wound healing. Twenty-six eyes of 13 patients who underwent myopic LASEK were divided into 2 groups. Group 1 (n=10 eyes) was used as a control group. All patients received topical lomefloxacin and dexamethasone postoperatively. Additionally, patients in Group 2 received topical NAC for 1 month postoperatively. Tear fluid samples were collected with microcapillary tubes preoperatively, on the first and on the fifth postoperative day, and the release of IL-1alpha in tear fluid was calculated. Haze grading and confocal microscopic examination were performed at 1 and 3 months postoperatively. The mean IL-1-alpha release values were 0.285-/+0.159 pg/min in Group 1 and 0.235-/+0.142 pg/min in Group 2 preoperatively. In Group 1, the values were 0.243-/+0.155 pg/min on day 1 and 0.164-/+0.125 pg/min on day 5. In Group 2, the mean IL-1alpha release values were 0.220-/+0.200 pg/min on day 1 and 0.080-/+0.079 pg/min on day 5. The difference between the groups was significant only for day 5 (p0.05). NAC seems to have an additive effect to steroids in suppressing IL-1alpha levels in tear fluid and may be clinically advantageous in modulating corneal wound healing during the early postoperative period after LASEK.

  7. Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

    Directory of Open Access Journals (Sweden)

    Bin Shan

    Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

  8. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    Science.gov (United States)

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  9. TopBP1-mediated DNA processing during mitosis.

    Science.gov (United States)

    Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

    2016-01-01

    Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

  10. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  11. Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

    Science.gov (United States)

    Pace-Asciak, C R; Domazet, Z; Carrara, M

    1977-05-25

    Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.

  12. NMDA receptor dependent PGC-1alpha up-regulation protects the cortical neuron against oxygen-glucose deprivation/reperfusion injury.

    Science.gov (United States)

    Luo, Yun; Zhu, Wenjing; Jia, Jia; Zhang, Chenyu; Xu, Yun

    2009-09-01

    The peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1alpha) is a nuclear transcriptional coactivator that is widely expressed in the brain areas. Over-expression of PGC-1alpha can protect neuronal cells from oxidant-induced injury. The purpose of the current study is to investigate the role of PGC-1alpha in the oxygen (anoxia) deprivation (OGD) neurons. The PGC-1alpha mRNA and protein level between control and OGD neurons were examined by real-time PCR and Western blot. More PGC-1alpha expression was found in the OGD neurons compared with the normal group. Over-expression of PGC-1alpha suppressed cell apoptosis while inhibition of the PGC-1alpha expression induced cell apoptosis in OGD neurons. Furthermore, increase of PGC-1alpha resulted in activation of N-methyl-D-aspartate (NMDA) receptor, p38, and ERK mitogen-activated protein kinase (MAPK) pathway. The blocking of the NMDA receptor by its antagonists MK-801 reduced PGC-1alpha mRNA expression in OGD neurons, while NMDA itself can directly induce the expression of PGC-1alpha in neuronal cells. At the same time, PD98059 (ERK MAPK inhibitor) and SB203580 (P38 MAPK inhibitor) also prevented the up-regulation of PGC-1alpha in OGD neurons and MK801 can inhibit the expression of P38 and ERK MAPK. These data suggested that the expression of PGC-1alpha was up-regulated in OGD mice cortical neurons, which protected the neurons against OGD injury. Moreover, this effect was correlated to the NMDA receptor and the ERK and P38 MAPK pathway. The protective effect of PGC-1alpha on OGD cortical neurons may be useful for stroke therapy.

  13. Upregulation of alpha cell glucagon-like peptide 1 (GLP-1) in Psammomys obesus--an adaptive response to hyperglycaemia?

    DEFF Research Database (Denmark)

    Hansen, A M K; Bödvarsdottir, T B; Nordestgaard, D N E

    2011-01-01

    of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth. Methods Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore...... from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge...

  14. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair

    Science.gov (United States)

    Chapman, J. Ross; Sossick, Alex J.; Boulton, Simon J.; Jackson, Stephen P.

    2012-01-01

    Summary Following irradiation, numerous DNA-damage-responsive proteins rapidly redistribute into microscopically visible subnuclear aggregates, termed ionising-radiation-induced foci (IRIF). How the enrichment of proteins on damaged chromatin actually relates to DNA repair remains unclear. Here, we use super-resolution microscopy to examine the spatial distribution of BRCA1 and 53BP1 proteins within single IRIF at subdiffraction-limit resolution, yielding an unprecedented increase in detail that was not previously apparent by conventional microscopy. Consistent with a role for 53BP1 in promoting DNA double-strand break repair by non-homologous end joining, 53BP1 enrichment in IRIF is most prominent in the G0/G1 cell cycle phases, where it is enriched in dense globular structures. By contrast, as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, leading to an overall reduction of 53BP1 occupancy at DNA damage sites. Our data suggest that the BRCA1-associated IRIF core corresponds to chromatin regions associated with repair by homologous recombination, and the enrichment of BRCA1 in IRIF represents a temporal switch in the DNA repair program. We propose that BRCA1 antagonises 53BP1-dependent DNA repair in S phase by inhibiting its interaction with chromatin proximal to damage sites. Furthermore, the genomic instability exhibited by BRCA1-deficient cells might result from a failure to efficiently exclude 53BP1 from such regions during S phase. PMID:22553214

  15. Development of a defined-sequence DNA system for use in DNA misrepair studies

    International Nuclear Information System (INIS)

    Sutton, S.; Tobias, C.A.

    1984-01-01

    The authors have developed a system that allows them to study cellular DNA repair processes at the molecular level. In particular, the authors are using this system to examine the consequences of a misrepair of radiation-induced DNA damage, as a function of dose. The cells being used are specially engineered haploid yeast cells. Maintained in the cells, at one copy per cell, is a cen plasmid, a plasmid that behaves like a functional chromosome. This plasmid carries a small defined sequence of DNA from the E. coli lac z gene. It is this lac z region (called the alpha region) that serves as the target for radiation damage. Two copies of the complimentary portion of the lac z gene are integrated into the yeast genome. Irradiated cells are screened for possible mutation in the alpha region by testing the cells' ability to hydrolyze xgal, a lactose substrate. The DNA of interest is then extracted from the cells, sequenced, and the sequence is compared to that of the control. Unlike the usual defined-sequence DNA systems, theirs is an in vivo system. A disadvantage is the relatively high background mutation rate. Results achieved with this system, as well as future applications, are discussed

  16. Human pro. cap alpha. 1(III) collagen: cDNA sequence for the 3' end

    Energy Technology Data Exchange (ETDEWEB)

    Mankoo, B S; Dalgleish, R

    1988-03-25

    The authors have previously isolated two overlapping cDNA clones, pIII-21 and pIII-33, which encode the C-terminal end of human type III procollagen. They now present the sequence of 2520 bases encoded in these cDNAs which overlaps other previously published sequences for the same gene. The sequence presented differs from previously published sequences at five positions.

  17. DNA Repair, Redox Regulation and Modulation of Estrogen Receptor Alpha Mediated Transcription

    Science.gov (United States)

    Curtis-Ducey, Carol Dianne

    2009-01-01

    Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…

  18. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Koji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Munetsuna, Eiji [Department of Biochemistry, Fujita Health University School of Medicine, Toyoake (Japan); Yamada, Hiroya, E-mail: hyamada@fujita-hu.ac.jp [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan); Ando, Yoshitaka [Department of Joint Research Laboratory of Clinical Medicine, Fujita Health University Hospital, Toyoake (Japan); Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Suzuki, Koji [Department of Public Health, Fujita Health University School of Health Sciences, Toyoake (Japan); Teradaira, Ryoji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Hashimoto, Shuji [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan)

    2015-12-04

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  19. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    International Nuclear Information System (INIS)

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki; Suzuki, Koji; Teradaira, Ryoji; Hashimoto, Shuji

    2015-01-01

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  20. Reoxygenation of human coronary smooth muscle cells suppresses HIF-1{alpha} gene expression and augments radiation-induced growth delay and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Grumann, T.; Arab, A.; Bode, C.; Hehrlein, C. [Dept. of Cardiology, Univ. Clinic of Freiburg (Germany); Guttenberger, R. [Dept. of Radiotherapy, Univ. Clinic of Freiburg (Germany)

    2006-01-01

    Background and Purpose: Catheter-based coronary brachytherapy with {beta}- and {gamma}-radiation is an evidence-based method to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) and stent implantation, but the outcome may be PTCA are hypoxic. A lack of oxygen decreases the effect of low LET (linear energy transfer) irradiation. The authors assumed that reoxygenation of hypoxic human coronary smooth muscle cells (HCSMCs) improves the results of coronary brachytherapy. The expression of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) gene, and the rates of growth and apoptosis of hypoxic and reoxygenated HCSMCs after {gamma}-iradiation were therefore analyzed. Material and Methods: An in vitro model of megacolonies of HCSMCs was developed. After exposure to chronic hypoxia the HCSMCs were irradiated with graded doses of 2, 4, 8, and 16 Gy using a {sup 60}Co source either under hypoxia (pO{sub 2}<3 mmHg) or after reoxygenation (pO{sub 2}{approx}150 mmHg). RT-PCR (reverse transcription-polymerase chain reaction) analysis was used to quantify HIF-1{alpha} gene expression and the growth of HCSMC megacolonies was measured serially. The oxygen enhancement ratio (OER) was calculate from the specific growth delay. Apoptosis of HCSMCs was quantified by counting cells with specific DNA strand breaks using the TUNEL assy. Results: HIF-1{alpha} gene expression was markedly suppressed in reoxygenated cells versus hypoxic cells 30 min after {gamma}-irradiation at all radiation doses (158{+-}46% vs. 1,675{+-}1,211%; p<0.01). Apoptosis was markedly increased in reoxygenated HCSMCs. The OER was 1.8(95% CI[confidence interval]1.3-2.4). Therefore, reoxygenated HCSMCs require 44% less radiation dose to achieve the equivalent biological radiation effect compared to hypoxic HCSMCs. Conclusion: Reoxygenation of coronary smooth muscle cells should be considered an option to increase efficacy of coronary brachytherapy. This could be used to reduce radiation dose

  1. The role of PGC-1alpha on mitochondrial function and apoptotic susceptibility in muscle

    DEFF Research Database (Denmark)

    Adhihetty, Peter J; Uguccioni, Giulia; Leick, Lotte

    2009-01-01

    Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome......-c oxidase activity, an indicator of mitochondrial content, in muscle and other tissues (brain, liver, and pancreas). Lack of PGC-1alpha reduced mitochondrial content in all muscles (17-44%; P liver, and pancreas. However, the tissue expression of proteins involved...

  2. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale s....... As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures....

  3. Effects of tamsulosin metabolites at alpha-1 adrenoceptor subtypes

    NARCIS (Netherlands)

    Taguchi, K.; Saitoh, M.; Sato, S.; Asano, M.; Michel, M. C.

    1997-01-01

    We have investigated the affinity and selectivity of tamsulosin and its metabolites, M1, M2, M3, M4 and AM1, at the tissue and the cloned alpha-1 adrenoceptor subtypes in the radioligand binding and the functional studies. In the radioligand binding studies, the compounds competed for [3H]prazosin

  4. Exploring the role of CT densitometry: a randomised study of augmentation therapy in alpha1-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Dirksen, A; Piitulainen, E; Parr, D G

    2009-01-01

    for the assessment of the therapeutic effect of augmentation therapy in subjects with alpha(1)-antitrypsin (alpha(1)-AT) deficiency. In total, 77 subjects (protease inhibitor type Z) were randomised to weekly infusions of 60 mg x kg(-1) human alpha(1)-AT (Prolastin) or placebo for 2-2.5 yrs. The primary end...... was unaltered by treatment, but a reduction in exacerbation severity was observed. In patients with alpha(1)-AT deficiency, CT is a more sensitive outcome measure of emphysema-modifying therapy than physiology and health status, and demonstrates a trend of treatment benefit from alpha(1)-AT augmentation....

  5. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

    Science.gov (United States)

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-09-06

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

  6. Phase 1 study of pandemic H1 DNA vaccine in healthy adults.

    Directory of Open Access Journals (Sweden)

    Michelle C Crank

    Full Text Available A novel, swine-origin influenza A (H1N1 virus was detected worldwide in April 2009, and the World Health Organization (WHO declared a global pandemic that June. DNA vaccine priming improves responses to inactivated influenza vaccines. We describe the rapid production and clinical evaluation of a DNA vaccine encoding the hemagglutinin protein of the 2009 pandemic A/California/04/2009(H1N1 influenza virus, accomplished nearly two months faster than production of A/California/07/2009(H1N1 licensed monovalent inactivated vaccine (MIV.20 subjects received three H1 DNA vaccinations (4 mg intramuscularly with Biojector at 4-week intervals. Eighteen subjects received an optional boost when the licensed H1N1 MIV became available. The interval between the third H1 DNA injection and MIV boost was 3-17 weeks. Vaccine safety was assessed by clinical observation, laboratory parameters, and 7-day solicited reactogenicity. Antibody responses were assessed by ELISA, HAI and neutralization assays, and T cell responses by ELISpot and flow cytometry.Vaccinations were safe and well-tolerated. As evaluated by HAI, 6/20 developed positive responses at 4 weeks after third DNA injection and 13/18 at 4 weeks after MIV boost. Similar results were detected in neutralization assays. T cell responses were detected after DNA and MIV. The antibody responses were significantly amplified by the MIV boost, however, the boost did not increased T cell responses induced by DNA vaccine.H1 DNA vaccine was produced quickly, was well-tolerated, and had modest immunogenicity as a single agent. Other HA DNA prime-MIV boost regimens utilizing one DNA prime vaccination and longer boost intervals have shown significant immunogenicity. Rapid and large-scale production of HA DNA vaccines has the potential to contribute to an efficient response against future influenza pandemics.Clinicaltrials.gov NCT00973895.

  7. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

    Science.gov (United States)

    Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M

    2002-08-15

    The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

  8. The determination of $\\alpha_s$ by the ALPHA collaboration

    CERN Document Server

    Bruno, Mattia

    2016-01-01

    We review the ALPHA collaboration strategy for obtaining the QCD coupling at high scale. In the three-flavor effective theory it avoids the use of perturbation theory at $\\alpha > 0.2$ and at the same time has the physical scales small compared to the cutoff $1/a$ in all stages of the computation. The result $\\Lambda_\\overline{MS}^{(3)}=332(14)$~MeV is translated to $\\alpha_\\overline{MS}(m_Z)=0.1179(10)(2)$ by use of (high order) perturbative relations between the effective theory couplings at the charm and beauty quark "thresholds". The error of this perturbative step is discussed and estimated as $0.0002$.

  9. Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Desai, Amar; Qing, Yulan; Gerson, Stanton L

    2014-02-01

    Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

  10. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M

    2010-05-01

    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  11. DNA-based identification and phylogeny of North American Armillaria species

    Science.gov (United States)

    Amy L. Ross-Davis; John W. Hanna; Ned B. Klopfenstein

    2011-01-01

    Because Armillaria species display different ecological behaviors across diverse forest ecosystems, it is critical to identify Armillaria species accurately for any assessment of forest health. To further develop DNA-based identification methods, partial sequences of the translation elongation factor-1 alpha (EF-1α) gene were used to examine the phylogenetic...

  12. Actin isoform and alpha 1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.

    Science.gov (United States)

    Lundberg, M S; Sadhu, D N; Grumman, V E; Chilian, W M; Ramos, K S

    1995-09-01

    The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.

  13. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

    Science.gov (United States)

    Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

    2015-10-01

    Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

  14. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Shamaileh, Hadi A.; Edwards, Stacey L.

    2012-01-01

    Background: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen (R), an inhibitor of vascular endothelial growth factor (VEGF......) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly...... in one-step, technique is required for developing aptamers in limited time period. Principal Findings: Herein, we present a simple one-step selection of DNA aptamers against alpha-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed...

  15. Characterization of pancreatic lesions from MT-tgf alpha, Ela-myc and MT-tgf alpha/Ela-myc single and double transgenic mice.

    Science.gov (United States)

    Liao, Dezhong Joshua; Wang, Yong; Wu, Jiusheng; Adsay, Nazmi Volkan; Grignon, David; Khanani, Fayyaz; Sarkar, Fazlul H

    2006-07-05

    In order to identify good animal models for investigating therapeutic and preventive strategies for pancreatic cancer, we analyzed pancreatic lesions from several transgenic models and made a series of novel findings. Female MT-tgf alpha mice of the MT100 line developed pancreatic proliferation, acinar-ductal metaplasia, multilocular cystic neoplasms, ductal adenocarcinomas and prominent fibrosis, while the lesions in males were less severe. MT-tgf alpha-ES transgenic lines of both sexes developed slowly progressing lesions that were similar to what was seen in MT100 males. In both MT100 and MT-tgf alpha-ES lines, TGF alpha transgene was expressed mainly in proliferating ductal cells. Ela-myc transgenic mice with a mixed C57BL/6, SJL and FVB genetic background developed pancreatic tumors at 2-7 months of age, and half of the tumors were ductal adenocarcinomas, similar to what was reported originally by Sandgren et al 1. However, in 20% of the mice, the tumors metastasized to the liver. MT100/Ela-myc and MT-tgf alpha-ES/Ela-myc double transgenic mice developed not only acinar carcinomas and mixed carcinomas as previously reported but also various ductal-originated lesions, including multilocular cystic neoplasms and ductal adenocarcinomas. The double transgenic tumors were more malignant and metastasized to the liver at a higher frequency (33%) compared with the Ela-myc tumors. Sequencing of the coding region of p16ink4, k-ras and Rb cDNA in small numbers of pancreatic tumors did not identify mutations. The short latency for tumor development, the variety of tumor morphology and the liver metastases seen in Ela-myc and MT-tgf alpha/Ela-myc mice make these animals good models for investigating new therapeutic and preventive strategies for pancreatic cancer.

  16. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of); Baek, Won-Ki, E-mail: wonki@dsmc.or.kr [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of)

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  17. Neutron transition multipole moment for /sup 88/Sr(. cap alpha. ,. cap alpha. ')/sup 88/Sr (2/sup +/, 1. 84 MeV)

    Energy Technology Data Exchange (ETDEWEB)

    Datta, S.K.; Ray, S.; Majumdar, H.; Ghosh, S.K.; Samanta, C.; Dasgupta, P.; Chintalapudi, S.N.; Banerjee, S.R.

    1989-04-01

    The neutron transition multipole moment, M/sub n/, for (0/sup +/..-->..2/sup +/, 1.84 MeV) transition is inferred by measuring the (..cap alpha..,..cap alpha..') angular distribution at E/sub ..cap alpha../ = 50 MeV and comparing it with a microscopic distorted-wave Born approximation calculation. Proton transition densities are taken from electron scattering data. M/sub n//M/sub p/ is found to be substantially less than N/Z in agreement with the (p,p') result.

  18. Evaluation of primary DNA damage, cytogenetic biomarkers and genetic polymorphisms for CYP1A1 and GSTM1 in road tunnel construction workers.

    Science.gov (United States)

    Villarini, M; Moretti, M; Fatigoni, C; Agea, E; Dominici, L; Mattioli, A; Volpi, R; Pasquini, R

    2008-01-01

    In tunnel construction workers, occupational exposure to dust (alpha-quartz and other particles from blasting), gases (nitrogen dioxide, NO(2)), diesel exhausts, and oil mist has been associated with lung function decline, induction of inflammatory reactions in the lungs with release of mediators that may influence blood coagulation, and increased risk of chronic obstructive pulmonary disease. The present molecular epidemiology study was designed to evaluate whether occupational exposure to indoor pollutants during road tunnel construction might result in genotoxic effects. A study group of 39 underground workers and a reference group of 34 unexposed subjects were examined. Primary and oxidative DNA damage, sister-chromatid exchanges (SCE), and micronuclei (MN) were measured in peripheral blood cells. The possible influences of polymorphisms in gene encoding for CYP1A1 and GSTM1 xenobiotic-metabolizing enzymes were also investigated. Exposure assessment was performed with detailed interviews and questionnaires. There were no significant differences in the level of primary and oxidative DNA damage and frequency of SCE between the tunnel workers and controls, whereas the frequency of MN showed a significant increase in exposed subjects compared to controls. No effects of CYP1A1 or GSTM1 variants were observed for the analyzed biomarkers. Since MN in peripheral blood lymphocytes are recognized as a predictive biomarker of cancer risk within a population of healthy subjects, the genotoxic risk of occupational exposure to various indoor environmental pollutants during road tunnel construction cannot be excluded by this biomonitoring study.

  19. Mice with deleted multimerin 1 and alpha-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1.

    Science.gov (United States)

    Reheman, Adili; Tasneem, Subia; Ni, Heyu; Hayward, Catherine P M

    2010-05-01

    Multimerin 1 is a stored platelet and endothelial cell adhesive protein that shows significant conservation. In vitro, multimerin 1 supports platelet adhesion and it also binds to collagen and enhances von Willebrand factor-dependent platelet adhesion to collagen. As selective, multimerin 1 deficient mice have not been generated, we investigated multimerin 1 effects on platelet adhesion using a subpopulation of C57BL/6J mice with tandem deletion of the genes for multimerin 1 and alpha-synuclein, a protein that inhibits alpha-granule release in vitro. We postulated that multimerin 1/alpha-synuclein deficient mice might show impaired platelet adhesive function from multimerin 1 deficiency and increased alpha-granule release from alpha-synuclein deficiency. Platelet function was assessed by intravital microscopy, after ferric chloride injury, using untreated and human multimerin 1-transfused multimerin 1/alpha-synuclein deficient mice, and by in vitro assays of adhesion, aggregation and thrombin-induced P-selectin release. Multimerin 1/alpha-synuclein deficient mice showed impaired platelet adhesion and their defective thrombus formation at sites of vessel injury improved with multimerin 1 transfusion. Although multimerin 1/alpha-synuclein deficient platelets showed increased P-selectin release at low thrombin concentrations, they also showed impaired adhesion to collagen, and attenuated aggregation with thrombin, that improved with added multimerin 1. Our data suggest that multimerin 1 supports platelet adhesive functions and thrombus formation, which will be important to verify by generating and testing selective multimerin 1 deficient mice. Copyright (c) 2010. Published by Elsevier Ltd.

  20. DNA methylation of loci within ABCG1 and PHOSPHO1 in blood DNA is associated with future type 2 diabetes risk

    DEFF Research Database (Denmark)

    Dayeh, Tasnim; Tuomi, Tiinamaija; Almgren, Peter

    2016-01-01

    Identification of subjects with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention of the disease. Consequently, it is essential to search for new biomarkers that can improve the prediction of T2D. The aim of this study was to examine whether 5 DNA methylation loci...... muscle from diabetic vs. non-diabetic subjects. DNA methylation at the ABCG1 locus cg06500161 in blood DNA was associated with an increased risk for future T2D (OR = 1.09, 95% CI = 1.02-1.16, P-value = 0.007, Q-value = 0.018), while DNA methylation at the PHOSPHO1 locus cg02650017 in blood DNA...... was associated with a decreased risk for future T2D (OR = 0.85, 95% CI = 0.75-0.95, P-value = 0.006, Q-value = 0.018) after adjustment for age, gender, fasting glucose, and family relation. Furthermore, the level of DNA methylation at the ABCG1 locus cg06500161 in blood DNA correlated positively with BMI, HbA1c...

  1. The effect of dietary protein restriction on the secretory dynamics of 1 alpha-hydroxycorticosterone and urea in the dogfish, Scyliorhinus canicula: a possible role for 1 alpha-hydroxycorticosterone in sodium retention.

    Science.gov (United States)

    Armour, K J; O'Toole, L B; Hazon, N

    1993-08-01

    The putative osmoregulatory role of the unique elasmobranch corticosteroid, 1 alpha-hydroxycorticosterone (1 alpha-OH-B), was investigated using dietary protein restriction as a means of limiting urea biosynthetic ability. Groups of dogfish (Scyliorhinus canicula) were adapted to either a high or a low protein diet (HPD and LPD respectively) and the secretory dynamics of urea and 1 alpha-OH-B were determined following acclimation to normal (100%), 130% and 50% sea water. In normal sea water, LPD fish showed significantly decreased blood production of urea compared with fish fed a HPD (P strategy adopted by these animals was the retention of high plasma concentrations of Na+ and Cl-, which increased plasma osmolality and tended to decrease osmotic water loss. Concomitant with the increased ion concentrations, plasma 1 alpha-OH-B concentration was also greatly elevated in LPD fish indicating that the steroid may be acting to minimize Na+ (and Cl-) excretion at osmoregulatory sites such as the rectal gland, kidney and gills. This and a previous study have also demonstrated that 1 alpha-OH-B concentration is elevated in 50% sea water. Decreases in plasma Na+ concentration are tolerated down to 75% sea water, whereafter Na+ is preferentially retained and further decreases in osmolality are achieved by reductions in plasma urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Purification and properties of a thermostable pullulanase from Clostridium thermosulfurgenes EM1 which hydrolyses both. alpha. -1,6 and. alpha. -1,4-glycosidic linkages

    Energy Technology Data Exchange (ETDEWEB)

    Spreinat, A [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the {alpha}-1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an {alpha}-1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K{sub m} values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60deg-65deg C and a pH optimum at 5.5-6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. (orig.).

  3. Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

    Science.gov (United States)

    Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

    2017-04-01

    To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

  4. Performance of Alpha Fetoprotein in Combination with Alpha-1-acid Glycoprotein for Diagnosis of Hepatocellular Carcinoma Among Liver Cirrhosis Patients

    Directory of Open Access Journals (Sweden)

    Rino A Gani

    2016-05-01

    Full Text Available Aim: to evaluate the use of alpha-1-acid glycoprotein (AAG for diagnosing hepatocellular carcinoma (HCC, and to combine with alpha fetoprotein (AFP as part of routine examination in liver cirrhosis patients. Methods: this is a diagnostic study using cross-sectional design. A hundred and six patients were included in this study. Baseline data such as age, gender, AFP, AAG, peripheral blood count, AST and ALT were consecutively collected from liver cirrhosis patients with or without HCC. Serum AAG were measured quantitatively using immunoturboditimetric assay and AFP with enzyme immune assay (EIA. Statistical analysis were done using SPSS 13.0. Data comparisons between group were done using Mann-Whitney test. Diagnostic performance for each marker alone was compared to the surrogate use of both markers (combined parallel approach in HCC cases. Results: receiver operating characteristic (ROC analysis showed that area under the curve for AFP AAG combination was 88.1% and higher than AFP only (86.2% or AAG only (76.5% with sensitivity of 83%, 73% and 44%, respectively, at specificity of >80%. Conclusion: our study showed that combination of AFP and AAG is superior than either marker alone in diagnosing HCC in liver cirrhosis patients. Combination of AFP and AAG may be used to prompt early diagnosis screening of HCC. Key words: alpha fetoprotein, alpha-1-acid glycoprotein, biomarker, liver cancer

  5. Genomic organization of the rat alpha 2u-globulin gene cluster.

    Science.gov (United States)

    McFadyen, D A; Addison, W; Locke, J

    1999-05-01

    The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.

  6. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States); Rebecchi, Mario, E-mail: Mario.rebecchi@SBUmed.org [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States)

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  7. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    2009-06-01

    Full Text Available Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  8. PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Min, E-mail: chenminyx@gmail.com [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Yunnan Centers for Diseases Prevention and Control, Kunming 650022 (China); Wang, Yanru [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Qu, Aijuan [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2010-06-11

    Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+} transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.

  9. Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Warburton, P.E.; Gosden, J.; Lawson, D. [Western General Hospital, Edinburgh (United Kingdom)] [and others

    1996-04-15

    Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.

  10. PGC-1alpha and PGC-1beta have both similar and distinct effects on myofiber switching toward an oxidative phenotype

    DEFF Research Database (Denmark)

    Mortensen, Ole Hartvig; Frandsen, Lis; Schjerling, Peter

    2006-01-01

    Peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta (PGC-1alpha and PGC-1beta) were overexpressed by adenovirus-mediated gene transfer in cultures of primary rat skeletal muscle cells derived from neonatal myoblasts. Effects on muscle fiber type transition and metabolism...... an increase in the mRNA expression of TRB3, a negative regulator of insulin signaling. These results show that both PGC-1alpha and PGC-1beta are involved in the regulation of skeletal muscle fiber transition and metabolism and that they have both overlapping and differing effects....... were studied from days 5 to 22 of culture. PGC-1alpha and PGC-1beta overexpression caused a three- to fourfold increase in mRNA level, a doubling of enzymatic activity of citrate synthase, a slight increase in short-chain acyl-CoA dehydrogenase mRNA, a doubling of the mRNA level, and a 30-50% increase...

  11. 6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

    Science.gov (United States)

    Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

    2007-05-31

    Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

  12. Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

    Energy Technology Data Exchange (ETDEWEB)

    Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.; Shyamala, G.; Moses, Harold L.; Barcellos-Hoff, Mary Helen

    2005-03-03

    Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha} co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.

  13. Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Nielsen, Finn Cilius; Vinther, Lena

    2007-01-01

    interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer...... that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity....

  14. TOGAF version 9 foundation study guide preparation for the TOGAF 9 Part 1 examination

    CERN Document Server

    Harrison, Rachel

    2009-01-01

    This document is a Study Guide for TOGAF™ 9 Foundation.It gives an overview of every learning objective for the TOGAF 9 Foundation Syllabus and in-depth coverage on preparing and taking the TOGAF 9 Part 1 Examination. It is specifically designed to help individuals prepare for certification.

  15. Coenzyme O*U1*UO, Alpha-Tocopherol and Free Cholesterol in HDL and LDL Fractions

    DEFF Research Database (Denmark)

    Johansen, Kurt; Theorell, Henning; Karlsson, Jan

    1991-01-01

    Farmakologi, Alpha-tocopherol, Coenzyme Q*U1*U0, free cholesterol, LDL, Antioxidants, Lipoproteins, HDL......Farmakologi, Alpha-tocopherol, Coenzyme Q*U1*U0, free cholesterol, LDL, Antioxidants, Lipoproteins, HDL...

  16. Blood pressure, risk of ischemic cerebrovascular and ischemic heart disease, and longevity in alpha(1)-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Dahl, Morten; Tybjaerg-Hansen, Anne; Sillesen, Henrik

    2003-01-01

    Because elastase in alpha(1)-antitrypsin deficiency may attack elastin in the arterial wall, we tested whether alpha(1)-antitrypsin deficiency is associated with reduced blood pressure, risk of ischemic cerebrovascular (ICVD) and ischemic heart disease (IHD), and longevity.......Because elastase in alpha(1)-antitrypsin deficiency may attack elastin in the arterial wall, we tested whether alpha(1)-antitrypsin deficiency is associated with reduced blood pressure, risk of ischemic cerebrovascular (ICVD) and ischemic heart disease (IHD), and longevity....

  17. Mineralization of alpha-1-antitrypsin inclusion bodies in Mmalton alpha-1-antitrypsin deficiency.

    Science.gov (United States)

    Callea, Francesco; Giovannoni, Isabella; Francalanci, Paola; Boldrini, Renata; Faa, Gavino; Medicina, Daniela; Nobili, Valerio; Desmet, Valeer J; Ishak, Kamal; Seyama, Kuniaki; Bellacchio, Emanuele

    2018-05-16

    Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype. This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations. Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins. Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the

  18. Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies

    NARCIS (Netherlands)

    Yang, M.; Reese, J.; Cotecchia, S.; Michel, M. C.

    1998-01-01

    Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with

  19. Change of expression of renal alpha1-adrenergic receptor and angiotensin II receptor subtypes with aging in rats.

    Science.gov (United States)

    Li, Yan-Fang; Cao, Xiao-Jing; Bai, Xue-Yuan; Lin, Shu-Peng; Shi, Shu-Tian

    2010-04-01

    It has been considered that the functional decline of renal vasoconstriction during senescence is associated with an alteration in renal alpha1-adrenergic receptor (alpha1-AR) expression. While alterations in renal angiotensin II receptor (ATR) expression was considered to have an effect on renal structure and function, until now little information has been available concerning alpha1-AR and ATR expression variations over the entire aging continuum. The present study was undertaken to examine the expression levels of alpha1-AR and ATR subtypes in renal tissue during the spectrum running from young adulthood, to middle age, to the presenium, and to the senium. Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot were used to quantify the messenger RNA (mRNA) and protein levels of alpha1-AR and ATR subtypes in renal tissue in 3-month-old (young adult), 12-month-old (middle age), 18-month-old (presenium) and 24-month-old (senium) Wistar rats. alpha1A-AR expression decreased gradually with aging: it was decreased during middle age, the presenium and the senium, compared, respectively, with young adult values (page and in the senium with respect to the presenium. alpha1B-AR and alpha1D-AR expression were unmodified during senescence. AT1R expression was unaffected by aging during young adulthood and middle age, but exhibited a remarkable downregulation in the presenium and senium periods (prenal alpha1-AR and ATR subtypes during aging. alpha1A-AR expression downregulation may account for the reduced reactivity of renal alpha1-AR to vasoconstrictors and to renal function decline in the senium. Both the downregulation of AT1R and the upregulation of AT2R may be influential in maintaining normal physiological renal function during aging.

  20. Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

    Science.gov (United States)

    Feng, Allen L; Xiang, Yun-Yan; Gui, Le; Kaltsidis, Gesthika; Feng, Qingping; Lu, Wei-Yang

    2017-06-01

    This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca 2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

  1. Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

    Science.gov (United States)

    Saher, G; Hildt, E

    1999-09-24

    Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

  2. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  3. Alpha and beta-adrenoceptors in hypertension. I. Cardiac and renal alpha 1-, beta 1-, and beta 2-adrenoceptors in rat models of acquired hypertension

    NARCIS (Netherlands)

    Michel, M. C.; Kanczik, R.; Khamssi, M.; Knorr, A.; Siegl, H.; Beckeringh, J. J.; Brodde, O. E.

    1989-01-01

    To determine whether adrenoceptor changes in genetic hypertension occur primary or secondary to blood pressure elevation, we measured cardiac and renal alpha 1- (by [125I]Be 2254 binding) and beta 1- and beta 2-adrenoceptors (by (-)-[125I]iodocyanopindolol binding) densities in various rat models of

  4. Experimental study on dynamic behavior of large scale foundation, 1

    International Nuclear Information System (INIS)

    Hanada, Kazufumi; Sawada, Yoshihiro; Esashi, Yasuyuki; Ueshima, Teruyuki; Nakamura, Hideharu

    1983-01-01

    The large-sized, high performance vibrating table in the Nuclear Power Engineering Test Center is installed on a large-scale concrete foundation of length 90.9 m, width 44.8 m and maximum thickness 21 m, weighing 150,000 tons. Through the experimental study on the behavior of the foundation, which is set on gravel ground, useful information should be obtained on the siting of a nuclear power plant on the Quaternary stratum ground. The objective of research is to grasp the vibration characteristics of the foundation during the vibration of the table to evaluate the interaction between the foundation and the ground, and to evaluate an analytical method for numerically simulating the vibration behavior. In the present study, the vibration behavior of the foundation was clarified by measurement, and in order to predict the vibration behavior, the semi-infinite theory of elasticity was applied. The accuracy of this analytical method was demonstrated by comparison with the measured results. (Mori, K.)

  5. Crystalline anhydrous {alpha},{alpha}-trehalose (polymorph {beta}) and crystalline dihydrate {alpha},{alpha}-trehalose: A calorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, Susana S. [Centro de Quimica Estrutural, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail: susanapinto@ist.utl.pt; Diogo, Herminio P. [Centro de Quimica Estrutural, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail: hdiogo@ist.utl.pt; Moura-Ramos, Joaquim J. [Centro de Quimica-Fisica Molecular, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail: mouraramos@ist.utl.pt

    2006-09-15

    The mean values of the standard massic energy of combustion of crystalline anhydrous {alpha},{alpha}-trehalose (C{sub 12}H{sub 22}O{sub 11}, polymorph {beta}) and crystalline dihydrate {alpha},{alpha}-trehalose (C{sub 12}H{sub 26}O{sub 13}) measured by static-bomb combustion calorimetry in oxygen, at the temperature T=298.15K, are {delta}{sub c}u{sup o}=-(16434.05+/-4.50)J.g{sup -1} and {delta}{sub c}u{sup o}=-(14816.05+/-3.52)J.g{sup -1}, respectively. The standard (p{sup o}=0.1MPa) molar enthalpy of formation of these compounds were derived from the corresponding standard molar enthalpies of combustion, respectively, {delta}{sub f}H{sub m}{sup o} (C{sub 12}H{sub 22}O{sub 11},cr)=-(2240.9+/-3.9)kJ.mol{sup -1}, and {delta}{sub f}H{sub m}{sup o} (C{sub 12}H{sub 26}O{sub 13},cr)=-(2832.6+/-3.6)kJ.mol{sup -1}. The values of the standard enthalpies of formation obtained in this work, together with data on enthalpies of solution at infinite dilution ({delta}{sub sol}H{sup {approx}}) for crystalline dihydrate and amorphous anhydrous trehalose, allow a better insight on the thermodynamic description of the trehalose system which can provide, together with the future research on the subject, a contribution for understanding the metabolism in several organisms, as well as the phase transition between the different polymorphs.

  6. Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

    Science.gov (United States)

    Schausi, Diane; Tiffoche, Christophe; Thieulant, Marie-Lise

    2003-07-01

    We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

  7. Photoaffinity cross-linking of a radioiodinated probe, 125I-A55453, into alpha 1-adrenergic receptors

    International Nuclear Information System (INIS)

    Dickinson, K.E.; Leeb-Lundberg, L.M.; Heald, S.L.; Wikberg, J.E.; DeBernardis, J.F.; Caron, M.G.; Lefkowitz, R.J.

    1984-01-01

    We have synthesized and characterized a high-affinity alpha 1-adrenergic receptor probe, 4-amino-6,7-dimethoxy-2[4'- [5''(3'''- 125 I-iodo-4'''-aminophenyl)pentanoyl]-1'-piperazinyl] quinazoline ( 125 I-A55453). This ligand binds reversibly to rat hepatic plasma membranes with high affinity (KD . 77 +/- 6 pM), and it labels the same number of specific prazosin-competable sites as the alpha 1-adrenergic receptor-selective radioligand [ 125 I] iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone. Specific binding is stereoselective and competed for by alpha-adrenergic agents with an alpha 1-adrenergic receptor specificity. 125 I-A55453 can be covalently photoincorporated into peptides of rat hepatic and splenic membranes using the bifunctional photoactive cross-linker, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. Following photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled hepatic membranes reveals a major specifically labeled peptide of Mr . 82,000 (+/- 1,000) with minor peptides at Mr . 50,000 (+/- 500), and 40,000 (+/- 300). Covalent incorporation of 125 I-A55453 into the Mr . 82,000 peptide is inhibited by adrenergic drugs with an alpha 1-adrenergic receptor specificity. Labeled splenic membranes demonstrate a broad band of photoincorporated radioactivity centered at Mr . 82,000, and covalent incorporation into this peptide is also attenuated with an alpha 1-adrenergic receptor specificity. This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues

  8. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

    Science.gov (United States)

    Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

    2016-06-01

    Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

  9. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth facto...

  10. SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Waaqo Daddacha

    2017-08-01

    Full Text Available DNA double-strand break (DSB repair by homologous recombination (HR is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.

  11. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    DEFF Research Database (Denmark)

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork

    2008-01-01

    challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...... for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol....

  12. Exonuclease 1 and its versatile roles in DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

    2016-01-01

    Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...... maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions...

  13. Capturing Snapshots of APE1 Processing DNA Damage

    Science.gov (United States)

    Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

    2015-01-01

    DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

  14. Serum concentrations of interleukin (IL-)1alpha, 1beta, 6 and tumor necrosis factor (TNF-) alpha in patients with thyroid eye disease (TED).

    Science.gov (United States)

    Laban-Guceva, Nevenka; Bogoev, Milko; Antova, Magdalena

    2007-01-01

    Serum proinflamatory cytokines were found to be altered in Graves disease (GD) and in TED. Serum values of IL1alpha, IL-1beta, IL-6, TNF-alpha were assessed in 22 patients with TED before and after treatment (aged 46.82 +/- 12.47, M:F=16:6). Free thyroxin was high, TSH low, thyroid ultrasound showed diffuse thyroid enlargement, treatment with antithyroid drugs propylthyouracil (PTU) or methymasol (MMI) resulted in clinical and hormonal remission. Several months after the initiation of the signs of hyperthyroidism, a progression in the ophthalmopathy was observed (Hertel up to 25 mm: normal 15-17 mm) while patients were clinically and hormonally euthyroid. Blood was collected in euthyroid state (with TED signs present, before corticosteroid therapy (CS) treatment) and after 3 months of treatment (patients without TED and without TED treatment). CS resulted in response of 8/22 patients. Ophthalmic irradiation (01) given with CS therapy, resulted in a response in twelve patients (12/12). Lack of response to CS treatment, with rapid increase in proptosis, and loss of visual acuity prompted ophthalmic decompression (OD) in two patients. Both recovered visual acuity, while proptosis fell under 25 mm Hertel. The control group had 29 persons (aged 51.86 +/-10.52, M:F = 16:13). A significant difference was found in the serum levels of IL-1alpha between the groups of controls (0.74+/-0.55 pg/ml) and patients before treatment (1.85 +/- 1.85 pg/ml; p TED treatment its concentration raised to 2.07 +/- 1.82 pg/ml (higher than the pretreatment; NS). For patients with low Clinical Activity Score (CAS) scores (1-5) there was no change in IL-6 concentrations before (1.03+/-o.64 pg/ml) and after treatment (1.07 +/- 0.63 pg/ml). Patients with higher CAS scores (6-10) had a change in IL-6 levels (from 1.32+/-1.00 to 2.67 +/- 4.84; p > 0.05). In addition, patients with pathological VEP had no changes in IL-6 (from 0.93 +/- 0.53 to 0.97 +/- 0.32 pg/ml), while patients with normal VEP had

  15. The application of alpha-card measurement to search underground water resources in bedrock

    International Nuclear Information System (INIS)

    Xiong Fulin; Liang Jinhua

    1986-01-01

    The alpha-card measurement is a new kind of nuclear technique to evaluate the concentration of the radium and thorium emanations with a short-time accumulation. It has shown the wonderful characteristics of higher sensitivity, greater detective depth, less interference and shorter work period when applied to search underground water resources in bedrock. The satisfactory result of its application in Pingyin limestone area is described. The principle of the alpha-card measurement and the foundation of its application are briefly discussed

  16. Energy dependence of event shapes and of $\\alpha_s$ at LEP 2

    CERN Document Server

    Abreu, P; Adye, T; Adzic, P; Albrecht, Z; Alderweireld, T; Alekseev, G D; Alemany, R; Allmendinger, T; Allport, P P; Almehed, S; Amaldi, Ugo; Amapane, N; Amato, S; Anassontzis, E G; Andersson, P; Andreazza, A; Andringa, S; Antilogus, P; Apel, W D; Arnoud, Y; Åsman, B; Augustin, J E; Augustinus, A; Baillon, Paul; Bambade, P; Barão, F; Barbiellini, Guido; Barbier, R; Bardin, Dimitri Yuri; Barker, G; Baroncelli, A; Battaglia, Marco; Baubillier, M; Becks, K H; Begalli, M; Behrmann, A; Beillière, P; Belokopytov, Yu A; Belous, K S; Benekos, N C; Benvenuti, Alberto C; Bérat, C; Berggren, M; Bertini, D; Bertrand, D; Besançon, M; Bianchi, F; Bigi, M; Bilenky, S M; Bizouard, M A; Bloch, D; Blom, H M; Bonesini, M; Bonivento, W; Boonekamp, M; Booth, P S L; Borgland, A W; Borisov, G; Bosio, C; Botner, O; Boudinov, E; Bouquet, B; Bourdarios, C; Bowcock, T J V; Boyko, I; Bozovic, I; Bozzo, M; Branchini, P; Brenke, T; Brenner, R A; Brückman, P; Brunet, J M; Bugge, L; Buran, T; Burgsmüller, T; Buschbeck, Brigitte; Buschmann, P; Cabrera, S; Caccia, M; Calvi, M; Camporesi, T; Canale, V; Carena, F; Carroll, L; Caso, Carlo; Castillo-Gimenez, M V; Cattai, A; Cavallo, F R; Chabaud, V; Chapkin, M M; Charpentier, P; Chaussard, L; Checchia, P; Chelkov, G A; Chierici, R; Chliapnikov, P V; Chochula, P; Chorowicz, V; Chudoba, J; Cieslik, K; Collins, P; Contri, R; Cortina, E; Cosme, G; Cossutti, F; Cowell, J H; Crawley, H B; Crennell, D J; Crépé, S; Crosetti, G; Cuevas-Maestro, J; Czellar, S; Davenport, Martyn; Da Silva, W; Deghorain, A; Della Ricca, G; Delpierre, P A; Demaria, N; De Angelis, A; de Boer, Wim; De Clercq, C; De Lotto, B; De Min, A; De Paula, L S; Dijkstra, H; Di Ciaccio, Lucia; Dolbeau, J; Doroba, K; Dracos, M; Drees, J; Dris, M; Duperrin, A; Durand, J D; Eigen, G; Ekelöf, T J C; Ekspong, Gösta; Ellert, M; Elsing, M; Engel, J P; Erzen, B; Espirito-Santo, M C; Falk, E; Fanourakis, G K; Fassouliotis, D; Fayot, J; Feindt, Michael; Fenyuk, A; Ferrari, P; Ferrer, A; Ferrer-Ribas, E; Ferro, F; Fichet, S; Firestone, A; Flagmeyer, U; Föth, H; Fokitis, E; Fontanelli, F; Franek, B J; Frodesen, A G; Frühwirth, R; Fulda-Quenzer, F; Fuster, J A; Galloni, A; Gamba, D; Gamblin, S; Gandelman, M; García, C; Gaspar, C; Gaspar, M; Gasparini, U; Gavillet, P; Gazis, E N; Gelé, D; Ghodbane, N; Gil, I; Glege, F; Gokieli, R; Golob, B; Gómez-Ceballos, G; Gonçalves, P; González-Caballero, I; Gopal, Gian P; Gorn, L; Górski, M; Guz, Yu; Gracco, Valerio; Grahl, J; Graziani, E; Green, C; Grimm, H J; Gris, P; Grosdidier, G; Grzelak, K; Günther, M; Guy, J; Hahn, F; Hahn, S; Haider, S; Hallgren, A; Hamacher, K; Hansen, J; Harris, F J; Hedberg, V; Heising, S; Hernández, J J; Herquet, P; Herr, H; Hessing, T L; Heuser, J M; Higón, E; Holmgren, S O; Holt, P J; Hoorelbeke, S; Houlden, M A; Hrubec, Josef; Huet, K; Hughes, G J; Hultqvist, K; Jackson, J N; Jacobsson, R; Jalocha, P; Janik, R; Jarlskog, C; Jarlskog, G; Jarry, P; Jean-Marie, B; Johansson, E K; Jönsson, P E; Joram, C; Juillot, P; Kapusta, F; Karafasoulis, K; Katsanevas, S; Katsoufis, E C; Keränen, R; Kersevan, Borut P; Khomenko, B A; Khovanskii, N N; Kiiskinen, A P; King, B J; Kinvig, A; Kjaer, N J; Klapp, O; Klein, H; Kluit, P M; Kokkinias, P; Koratzinos, M; Kostyukhin, V; Kourkoumelis, C; Kuznetsov, O; Krammer, Manfred; Kriznic, E; Krstic, J; Krumshtein, Z; Kubinec, P; Kurowska, J; Kurvinen, K L; Lamsa, J; Lane, D W; Langefeld, P; Lapin, V; Laugier, J P; Lauhakangas, R; Leder, Gerhard; Ledroit, F; Lefébure, V; Leinonen, L; Leisos, A; Leitner, R; Lemonne, J; Lenzen, Georg; Lepeltier, V; Lesiak, T; Lethuillier, M; Libby, J; Liko, D; Lipniacka, A; Lippi, I; Lörstad, B; Loken, J G; Lopes, J H; López, J M; López-Fernandez, R; Loukas, D; Lutz, P; Lyons, L; MacNaughton, J N; Mahon, J R; Maio, A; Malek, A; Malmgren, T G M; Maltezos, S; Malychev, V; Mandl, F; Marco, J; Marco, R P; Maréchal, B; Margoni, M; Marin, J C; Mariotti, C; Markou, A; Martínez-Rivero, C; Martínez-Vidal, F; Martí i García, S; Mastroyiannopoulos, N; Matorras, F; Matteuzzi, C; Matthiae, Giorgio; Masik, J; Mazzucato, F; Mazzucato, M; McCubbin, M L; McKay, R; McNulty, R; McPherson, G; Meroni, C; Meyer, W T; Migliore, E; Mirabito, L; Mitaroff, Winfried A; Mjörnmark, U; Moa, T; Moch, M; Møller, R; Mönig, K; Monge, M R; Moreau, X; Morettini, P; Morton, G A; Müller, U; Münich, K; Mulders, M; Mulet-Marquis, C; Muresan, R; Murray, W J; Muryn, B; Myatt, Gerald; Myklebust, T; Naraghi, F; Nassiakou, M; Navarria, Francesco Luigi; Navas, S; Nawrocki, K; Negri, P; Némécek, S; Neufeld, N; Neumeister, N; Nicolaidou, R; Nielsen, B S; Nikolenko, M; Nomokonov, V P; Normand, Ainsley; Nygren, A; Obraztsov, V F; Olshevskii, A G; Onofre, A; Orava, Risto; Orazi, G; Österberg, K; Ouraou, A; Paganoni, M; Paiano, S; Pain, R; Paiva, R; Palacios, J; Palka, H; Papadopoulou, T D; Papageorgiou, K; Pape, L; Parkes, C; Parodi, F; Parzefall, U; Passeri, A; Passon, O; Pegoraro, M; Peralta, L; Pernicka, Manfred; Perrotta, A; Petridou, C; Petrolini, A; Phillips, H T; Pierre, F; Pimenta, M; Piotto, E; Podobnik, T; Pol, M E; Polok, G; Poropat, P; Pozdnyakov, V; Privitera, P; Pukhaeva, N; Pullia, Antonio; Radojicic, D; Ragazzi, S; Rahmani, H; Ratoff, P N; Read, A L; Rebecchi, P; Redaelli, N G; Regler, Meinhard; Reid, D; Reinhardt, R; Renton, P B; Resvanis, L K; Richard, F; Rídky, J; Rinaudo, G; Røhne, O M; Romero, A; Ronchese, P; Rosenberg, E I; Rosinsky, P; Roudeau, Patrick; Rovelli, T; Royon, C; Ruhlmann-Kleider, V; Ruiz, A; Saarikko, H; Sacquin, Yu; Sadovskii, A; Sajot, G; Salt, J; Sampsonidis, D; Sannino, M; Schneider, H; Schwemling, P; Schwering, B; Schwickerath, U; Schyns, M A E; Scuri, F; Seager, P; Sedykh, Yu; Segar, A M; Sekulin, R L; Shellard, R C; Sheridan, A; Siebel, M; Simard, L C; Simonetto, F; Sissakian, A N; Smadja, G; Smirnov, N; Smirnova, O G; Smith, G R; Sopczak, André; Sosnowski, R; Spassoff, Tz; Spiriti, E; Sponholz, P; Squarcia, S; Stanescu, C; Stanic, S; Stevenson, K; Stocchi, A; Strub, R; Stugu, B; Szczekowski, M; Szeptycka, M; Tabarelli de Fatis, T; Tegenfeldt, F; Terranova, F; Thomas, J; Timmermans, J; Tinti, N; Tkatchev, L G; Todorova-Nová, S; Tomaradze, A G; Tomé, B; Tonazzo, A; Tortora, L; Tranströmer, G; Treille, D; Tristram, G; Trochimczuk, M; Troncon, C; Tsirou, A L; Turluer, M L; Tyapkin, I A; Tzamarias, S; Ullaland, O; Uvarov, V; Valenti, G; Vallazza, E; Van der Velde, C; van Apeldoorn, G W; van Dam, P; Van Doninck, W K; Van Eldik, J; Van Lysebetten, A; Van Vulpen, I B; Vassilopoulos, N; Vegni, G; Ventura, L; Venus, W A; Verbeure, F; Verlato, M; Vertogradov, L S; Verzi, V; Vilanova, D; Vitale, L; Vlasov, E; Vodopyanov, A S; Vollmer, C F; Voulgaris, G; Vrba, V; Wahlen, H; Walck, C; Weiser, C; Wicke, D; Wickens, J H; Wilkinson, G R; Winter, M; Witek, M; Wolf, G; Yi, J; Yushchenko, O P; Zaitsev, A; Zalewska-Bak, A; Zalewski, Piotr; Zavrtanik, D; Zevgolatakos, E; Zimin, N I; Zucchelli, G C; Zumerle, G

    1999-01-01

    Infrared and collinear safe event shape distributions and their mean values are determined using the data taken at ve di erent centre of mass energies above $M_Z$ with the DELPHI detector at LEP. From the event shapes, the strong coupling $\\alpha_s$ is extracted in $O(\\alpha^2_s)$, NLLA and a combined scheme using hadronisation corrections evaluated with fragmentation model generators as well as using an analytical power ansatz. Comparing these measurements to those obtained at MZ, the energy dependence (running) of $\\alpha_s$ is accessible. The logarithmic energy slope of the inverse strong coupling is measured to be $d\\alpha_{s}^{-1}/d log(E_{cm}) = 1.39 \\pm 0.34(stat) \\pm 0.17(syst)$, in good agreement with the QCD expectation of 1.27.

  17. Cell-Free DNA, High-Mobility Group Box-1, and Procalcitonin Concentrations in Dogs With Gastric Dilatation–Volvulus Syndrome

    Directory of Open Access Journals (Sweden)

    Roberta Troia

    2018-04-01

    Full Text Available Canine gastric dilatation–volvulus (GDV is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA, high-mobility group box-1 (HMGB1, and procalcitonin (PCT in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLEfast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann–Whitney U and correlations between continuous variables were assessed by calculation of Spearman’s correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls (P = 0.0009, P = 0.004, and P = 0.009, respectively. PCT concentrations were significantly higher in non-survivors compared to survivors (P = 0.008. Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis (P = 0.0005. The APPLEfast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated (rs 0.51, P = 0.0005. Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively.

  18. Cell-Free DNA, High-Mobility Group Box-1, and Procalcitonin Concentrations in Dogs With Gastric Dilatation-Volvulus Syndrome.

    Science.gov (United States)

    Troia, Roberta; Giunti, Massimo; Calipa, Stefano; Goggs, Robert

    2018-01-01

    Canine gastric dilatation-volvulus (GDV) is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA), high-mobility group box-1 (HMGB1), and procalcitonin (PCT) in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLE fast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann-Whitney U and correlations between continuous variables were assessed by calculation of Spearman's correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls ( P  = 0.0009, P  = 0.004, and P  = 0.009, respectively). PCT concentrations were significantly higher in non-survivors compared to survivors ( P  = 0.008). Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis ( P  = 0.0005). The APPLE fast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated ( r s 0.51, P  = 0.0005). Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively.

  19. Cell-Free DNA, High-Mobility Group Box-1, and Procalcitonin Concentrations in Dogs With Gastric Dilatation–Volvulus Syndrome

    Science.gov (United States)

    Troia, Roberta; Giunti, Massimo; Calipa, Stefano; Goggs, Robert

    2018-01-01

    Canine gastric dilatation–volvulus (GDV) is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA), high-mobility group box-1 (HMGB1), and procalcitonin (PCT) in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLEfast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann–Whitney U and correlations between continuous variables were assessed by calculation of Spearman’s correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls (P = 0.0009, P = 0.004, and P = 0.009, respectively). PCT concentrations were significantly higher in non-survivors compared to survivors (P = 0.008). Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis (P = 0.0005). The APPLEfast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated (rs 0.51, P = 0.0005). Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively. PMID:29686994

  20. Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14, and 21: implications for recombination between nonhomologues and Robertsonian translocations

    Energy Technology Data Exchange (ETDEWEB)

    Choo, K H; Vissel, B; Brown, R; Filby, R G; Earle, E

    1988-02-25

    The authors report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a consensus in situ hybridization profile derived from 13 normal individuals revealed the localization of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocation involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.

  1. [Prevalence survey and molecular characterization of alpha and beta thalassemia in Liuzhou city of Guangxi].

    Science.gov (United States)

    Cai, Ren; Li, Liyan; Liang, Xin; Liu, Zhongying; Su, Liu; Li, Wenjun; Zhu, Qiangui; Mo, Qiuhua; Pan, Lizhen; Ouyang, Hong; Huang, Lihua; Xu, Xiangmin

    2002-08-01

    To investigate the gene frequencies and mutation patterns of alpha thalassemia (alpha-thal) and beta thalassemia (beta-thal) in Liuzhou city of Guangxi Zhuang Autonomous Region. Cluster sampling was used. A total of 1 028 of umbilical blood samples were collected for a prevalence study of alpha-thal and a total of 1 312 healthy young people when receiving pre-marriage consultation were recruited for a beta-thal prevalence survey. Individuals live in city or town area of Liuzhou. A complete blood count as well as hemoglobin electrophoresis analysis were done in all of samples for phenotyping of alpha and beta-thals. Those with Hb Bart's for alpha-thal indicator and those with both microcytosis (MCV /=4.0%) for beta-thal were further studied by DNA analysis. PCR-based methodologies were used to characterize the mutation contributions of alpha and beta-thals. All the subjects were tested for the state of carrying beta-thala alleles for evaluating the situation of the compound heterozygotes of alpha-thal with beta-thal. Of 1 028 random samples of umbilical blood screened, 112 of subjects were defined to be the gene carriers of alpha-thal. The alpha-thal carrier rate was as high as 11.19% including 3 compound heterozygotes. Five well-known types of alpha-thal alleles were detected with gene contributions of 37.4% (--(SEA) deletion), 31.3% (-alpha(3.7) deletion), 17.4% (-alpha(4.2) deletion), 12.1% (alpha(CS)alpha mutation), and 0.9% (alpha(QS)alpha mutation), successively. Of the 1 312 adult specimens studied, 89 with beta-thal including 14 of the compound higher Hb F subjects were detected. All of the 89 phenotypic beta-thal carriers had the mutations in the beta-globin gene, making the overall prevalence 6.78%. The commonly seen three mutations, beta CD41 - 42 (-CTTT) frameshift, beta CD17 (T-A) nonsense mutation and beta-28 (A-G) promoter variation were accounted for 90% of the beta-thal alleles in Liuzhou. Of these beta-thal subjects, 16 (accounting for 18%) were

  2. Identification and characterization of alpha 1 adrenergic receptors in the canine prostate using [125I]-Heat

    International Nuclear Information System (INIS)

    Lepor, H.; Baumann, M.; Shapiro, E.

    1987-01-01

    We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, [ 125 I]-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using [ 125 I]-Heat. The Scatchard plots were linear indicating homogeneity of [ 125 I]-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of [ 125 I]-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of [ 125 I]-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific [ 125 I]-Heat binding at a single ligand concentration. [ 125 I]-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate

  3. ALPHA/AMPU, Radionuclide Radioactivity from Alpha Spectrometer Measurements

    International Nuclear Information System (INIS)

    Sill, D.S.

    1990-01-01

    1 - Description of program or function: The two computer programs, ALPHA and AMPU, take raw data obtained from alpha spectrometry and from these calculate activities and uncertainties of the radionuclides present in the sample. ALPHA determines activities of any alpha emitter in a sample that has been directly precipitated with NdF 3 . AMPU determines the Pu-239, Pu-238,and Am-241 activities using Pu-236 and Am-243 tracers. 2 - Method of solution: These programs propagate all random and systematic uncertainties, found anywhere in the experimental process, to the final result. The result is rounded and is in decimal agreement with the uncertainty. 3 - Restrictions on the complexity of the problem: In ALPHA, a chemical yield of 98% is assumed

  4. Core nucleosomes by digestion of reconstructed histone-DNA complexes

    Energy Technology Data Exchange (ETDEWEB)

    Bryan, P N; Wright, E B; Olins, D E

    1979-04-01

    Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (..nu../sub 1/). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(dA-dT).poly(dA-dT) have been examined by circular dichroism, thermal denaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260 to 280 nm and a prominent ..cap alpha..-helix signal at 222 nm. All particles show biphasic melting except ..nu../sub 1/(dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of ..nu../sub 1/ (dA-dT) produces a ladder of DNA fragments differing in length by one base residue. ..nu../sub 1/ (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4 to 10.5 bases per turn. There appear to be two regions of different DNA pitch within ..nu../sub 1/ (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

  5. T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

    Energy Technology Data Exchange (ETDEWEB)

    Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L. (Stanford Univ., CA (USA)); Sherritt, M.; Bernard, C.C. (LaTrobe Univ., Victoria (Australia)); Begovich, A.B.; Erlich, H.A. (Cetus Corporation, Emeryville, CA (USA))

    1989-02-01

    Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.

  6. [Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

    Science.gov (United States)

    Liu, Fang; Wuren, Tana; Ma, Lan; Yang, Ying-Zhong; Ge, Ri-Li

    2011-12-25

    In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

  7. Deficiency of a alpha-1-antitrypsin influences systemic iron homeostasis

    Science.gov (United States)

    Abstract Background: There is evidence that proteases and anti-proteases participate in the iron homeostasis of cells and living systems. We tested the postulate that alpha-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this anti-protease in humans are asso...

  8. hnRNP L regulates differences in expression of mouse integrin alpha2beta1.

    Science.gov (United States)

    Cheli, Yann; Kunicki, Thomas J

    2006-06-01

    There is a 2-fold variation in platelet integrin alpha2beta1 levels among inbred mouse strains. Decreased alpha2beta1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet alpha2beta1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L-specific siRNA. Thus, decreased surface alpha2beta1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1.

  9. Involvement of prostaglandins F/sub 2. cap alpha. / and E/sub 1/ with rabbit endometrium

    Energy Technology Data Exchange (ETDEWEB)

    Orlicky, D.J.

    1985-01-01

    Several growth factors and hormones are thought to play a role in the growth control of endometrial cells. The authors have shown that prostaglandin F/sub 2..-->../ (PGF/sub 2..cap alpha../) is a growth factor for primary cultures of rabbit endometrium cultured in chemically-defined serum-free medium and that prostaglandin E/sub 1/ (PGE/sub 1/) antagonizes the PGF/sub 2..-->../ induction of growth. Both (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ bind in a time and temperature dependent, dissociable, saturable and specific manner. The binding of (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ can be both down and up regulated and is enzyme sensitive. PGE /sub 1/ stimulates intracellular cAMP synthesis and accumulation in a time and concentration dependent manner. PGF/sub 2..cap alpha../ probably exerts its effects through an amiloride-sensitive intermediate. Both PGF/sub 2..cap alpha../ and PGE/sub 1/ are constitutively synthesized by these primary cultures, and they have shown this synthesis to be both drug and hormone sensitive. They hypothesize that it is the ratio, rather than the absolute quantities, of PGF/sub 2..cap alpha../ and PGE/sub 1/ which is of more importance in the regulation of endometrial cell growth. Furthermore, they believe this regulation of endometrial growth plays a role in control of proliferation during the decidual response and that a derangement in the ratio of these prostaglandins may lead to either infertility or hyperplasia. The ability of these cultures to synthesize prostaglandins in a hormonally regulatable manner may be of importance in the study of dysmenorrhea and uterine cramping as caused by the myometrial contracting prostaglandin, PGF/sub 2..cap alpha../.

  10. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  11. Alpha-defensin DEFA1A3 gene copy number elevation in Danish Crohn's disease patients

    DEFF Research Database (Denmark)

    Jespersgaard, Cathrine; Fode, Peder; Dybdahl, Marianne

    2011-01-01

    BACKGROUND AND PURPOSE OF STUDY: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association...... of DEFA1A3 with CD. METHODS: Two-hundred and forty ethnic Danish CD patients were included in the study. Reverse transcriptase PCR assays determined DEFA1A3 expression in colonic tissue from a subset of patients. Immunohistochemical analysis identified alpha-defensin peptides in colonic tissue. Copy...

  12. Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

    Science.gov (United States)

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

    2014-10-01

    DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Urinary alpha 1-microglobulin as an indicator protein of renal tubular dysfunction caused by environmental cadmium exposure

    Energy Technology Data Exchange (ETDEWEB)

    Tohyama, C.; Kobayashi, E.; Saito, H.; Sugihara, N.; Nakano, A.; Mitane, Y.

    1986-06-01

    An epidemiologic investigation was carried out to clarify the significance of the urinary excretion of alpha 1-microglobulin (alpha 1-MG) in people aged 50 years and over living in a Cd-polluted area in Japan. Approximately 80% of the population participated in the health examination. The urinary and serum levels and the relative clearance of alpha 1-MG to creatinine clearance were compared with various parameters (age, urinary beta 2-microglobulin (beta 2-MG), total protein, Cd, Cu and Zn, serum beta 2-MG, creatinine and blood urea nitrogen and relative clearances of alpha 1-MG, beta 2-MG, inorganic phosphate and uric acid). It was found that the urinary excretion of alpha 1-MG is closely associated with the urinary Cd and Cu and with the indices of renal dysfunction listed above. These results suggest that the urinary alpha 1-MG level markedly reflects a degree of proximal tubular dysfunction and that it may be useful as one of the screening measures for proximal tubular dysfunction caused by environmental Cd exposure.

  14. Conformation of glycomimetics in the free and protein-bound state: structural and binding features of the C-glycosyl analogue of the core trisaccharide alpha-D-Man-(1 --> 3)-[alpha-D-Man-(1 --> 6)]-D-Man.

    Science.gov (United States)

    Mikkelsen, Lise Munch; Hernáiz, María José; Martín-Pastor, M; Skrydstrup, Troels; Jiménez-Barbero, Jesús

    2002-12-18

    The conformational properties of the C-glycosyl analogue of the core trisaccharide alpha-D-Man-(1 --> 3)-[alpha-D-Man-(1 --> 6)]-D-Man in solution have been carefully analyzed by a combination of NMR spectroscopy and time-averaged restrained molecular dynamics. It has been found that both the alpha-1,3- and the alpha-1,6-glycosidic linkages show a major conformational averaging. Unusual Phi ca. 60 degrees orientations for both Phi torsion angles are found. Moreover, a major conformational distinction between the natural compound and the glycomimetic affects to the behavior of the omega(16) torsion angle around the alpha-1 --> 6-linkage. Despite this increased flexibility, the C-glycosyl analogue is recognized by three mannose binding lectins, as shown by NMR (line broadening, TR-NOE, and STD) and surface plasmon resonance (SPR) methods. Moreover, a process of conformational selection takes place, so that these lectins probably bind the glycomimetic similarly to the way they recognize the natural analogue. Depending upon the architecture and extension of the binding site of the lectin, loss or gain of binding affinity with respect to the natural analogue is found.

  15. Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

    OpenAIRE

    Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.

    2003-01-01

    Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fu...

  16. [O III] EMISSION AND GAS KINEMATICS IN A LYMAN-ALPHA BLOB AT z {approx} 3.1

    Energy Technology Data Exchange (ETDEWEB)

    McLinden, Emily M. [McDonald Observatory, University of Texas at Austin, Austin, TX 78712 (United States); Malhotra, Sangeeta; Rhoads, James E. [School of Earth and Space Exploration, Arizona State University, Tempe, AZ 85287 (United States); Hibon, Pascale [Gemini Observatory, La Serena (Chile); Weijmans, Anne-Marie [Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 50 St. George Street, Toronto, ON M5S 3H4 (Canada); Tilvi, Vithal [George P. and Cynthia Woods Mitchell Institute for Fundamental Physics and Astronomy, and Department of Physics and Astronomy, Texas A and M University, College Station, TX 77843 (United States)

    2013-04-10

    We present spectroscopic measurements of the [O III] emission line from two subregions of strong Ly{alpha} emission in a radio-quiet Lyman-alpha blob (LAB). The blob under study is LAB1 at z {approx} 3.1, and the [O III] detections are from the two Lyman break galaxies (LBGs) embedded in the blob halo. The [O III] measurements were made with LUCIFER on the 8.4 m Large Binocular Telescope and NIRSPEC on 10 m Keck Telescope. Comparing the redshift of the [O III] measurements to Ly{alpha} redshifts from SAURON allows us to take a step toward understanding the kinematics of the gas in the blob. Using both LUCIFER and NIRSPEC we find velocity offsets between the [O III] and Ly{alpha} redshifts that are modestly negative or consistent with 0 km s{sup -1} in both subregions studied (ranging from -72 {+-} 42- + 6 {+-} 33 km s{sup -1}). A negative offset means Ly{alpha} is blueshifted with respect to [O III] a positive offset then implies Ly{alpha} is redshifted with respect to [O III]. These results may imply that outflows are not primarily responsible for Lyman alpha escape in this LAB, since outflows are generally expected to produce a positive velocity offset. In addition, we present an [O III] line flux upper limit on a third region of LAB1, a region that is unassociated with any underlying galaxy. We find that the [O III] upper limit from the galaxy-unassociated region of the blob is at least 1.4-2.5 times fainter than the [O III] flux from one of the LBG-associated regions and has an [O III] to Ly{alpha} ratio measured at least 1.9-3.4 times smaller than the same ratio measured from one of the LBGs.

  17. Initial experience with the Sophono Alpha 1 osseointegrated implant.

    Science.gov (United States)

    Escorihuela-García, Vicente; Llópez-Carratalá, Ignacio; Pitarch-Ribas, Ignacia; Latorre-Monteagudo, Emilia; Marco-Algarra, Jaime

    2014-01-01

    In the last several years, bone anchored hearing aids have proven to be useful in treating conductive and mixed unilateral or bilateral hearing loss, as well as for sensorineural unilateral hearing loss. The Sophono Alpha 1 model has the advantage of not requiring an abutment, with it being coupled by magnetism instead. We report the cases of 3 infants with congenital malformations of external and middle ear. Audiometry showed conductive hearing loss. All 3 patients were implanted with Alpha 1 model (Sophono). Patients evolved satisfactorily. After 30 days we applied the processor and the control audiometry showed a marked improvement of hearing thresholds, although without a complete closure of the gap. With minimal care, the skin over the implant remained in excellent condition, with a very satisfactory cosmetic outcome. Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Otorrinolaringología y Patología Cérvico-Facial. All rights reserved.

  18. Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study

    Directory of Open Access Journals (Sweden)

    Nava-Salazar Sonia

    2011-03-01

    Full Text Available Abstract Background Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S and G1790A (A588T polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. Results Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing. In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. Conclusion The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.

  19. Isothermal and aniso-thermal creep in the {alpha} phase domain, {beta} phase domain and {alpha}+{beta} two phase domain in a Zr-1%NbO alloy; Fluage isotherme et anisotherme dans les domaines monophases ({alpha} et {beta}) et biphases ({alpha} et {beta}) d'un alliage Zr-1%NbO

    Energy Technology Data Exchange (ETDEWEB)

    Kaddour, D

    2004-12-15

    The coupling between phase transformation and mechanical behaviour of a Zr-1%NbO alloy was studied using an original experimental device already used in a previous study devoted to the Zy-4 alloy. The Zr-1%NbO alloy undergoes a phase transformation {alpha} (hc) {r_reversible} (cc) typically between 750 and 1000 C. The transformation temperatures were measured in situ by using the resistivity and dilatometry techniques. The isothermal creep behaviour of fuel cladding tubes was studied, first after heating, in the {alpha} phase domain between 650 and 760 C, in the {beta} phase domain between 960 and 1100 C, as well as in the ({alpha} + {beta}) two phase domain between 800 and 900 C. The results are summarized in Ashby deformation mechanism maps. It is confirmed that the {beta} phase is much more sensitive to creep flow than the {alpha} phase. The effect of microstructure on the isothermal creep flow behaviour was then investigated by first applying a thermal cycle involving either a full or a partial transformation from {alpha} to {beta}. It was investigated both in the {alpha} phase domain, and after direct cooling into the ({alpha} + {beta}) phase domain. The behaviour in aniso-thermal conditions was finally studied at heating and cooling rates of 10 and 200 C/min. In both cases, we showed that there is no significant transformation plasticity in the stress range under investigation ({<=} 5 MPa). A finite element model using Voronoi polyhedra and eventually meshing a film of intergranular {beta} phase was used to describe the behaviour of material in the ({alpha} + {beta}) domain in various microstructural states. The model predictions are in good agreement with the experimental results for the microstructure obtained after cooling, but the model underestimates creep deformation in the as-received state. This difference is probably related to the fact that interface sliding is not taken into account in the model. (author)

  20. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

  1. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  2. Three-Dimensional Conformal Radiotherapy in Prostate Cancer Patients: Rise in Interleukin 6 (IL-6) but not IL-2, IL-4, IL-5, Tumor Necrosis Factor-{alpha}, MIP-1-{alpha}, and LIF Levels

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Lopes, Carlos [Universidade do Vale do Paraiba, Centro de Oncologia Radioterapica do Vale do Paraiba, Universidade do Vale do Paraiba Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraiba, Sao Jose dos Campos, Sao Paulo (Brazil); Callera, Fernando, E-mail: fcallera@gmail.com [Centro de Hematologia Onco-hematologia e Transplantes de Medula Ossea do Vale do Paraiba, Sao Paulo (Brazil)

    2012-03-15

    Purpose: To investigate the effect of radiotherapy (RT) on serum levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-{alpha}), macrophage inflammatory protein-1-alpha (MIP-1-{alpha}) and leukemia inhibitory factor (LIF) in patients with prostate cancer. Methods and Materials: Forty eight patients with prostate cancer received three-dimensional conformal blocking radiation therapy with a linear accelerator. IL-2, IL-4, IL-5, IL-6, TNF-{alpha}, MIP-1-{alpha}, and LIF levels were measured by the related immunoassay kit 1 day before the beginning of RT and during RT at days 15 and 30. Results: The mean IL-2 values were elevated before and during the RT in contrast with those of IL-4, IL-5, IL-6, TNF-{alpha}, MIP-1-{alpha}, and LIF, which were within the normal range under the same conditions. Regarding markers IL-2, IL-4, IL-5, TNF-{alpha}, MIP-1-{alpha}, and LIF, comparisons among the three groups (before treatment and 15 and 30 days during RT) did not show significant differences. Although values were within the normal range, there was a significant rise in IL-6 levels at day 15 of RT (p = 0.0049) and a decline at day 30 to levels that were similar to those observed before RT. Conclusions: IL-6 appeared to peak after 15 days of RT before returning to pre-RT levels. In contrast, IL-2, IL-4, IL-5, TNF-{alpha}, MIP-1-{alpha}, and LIF levels were not sensitive to irradiation. The increased levels of IL-6 following RT without the concurrent elevation of other cytokines involved in the acute phase reaction did not suggest a classical inflammatory response to radiation exposure. Further studies should be designed to elucidate the role of IL-6 levels in patients with prostate cancer treated with RT.

  3. Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

    DEFF Research Database (Denmark)

    Mundy, John; Brandt, Anders; Fincher, Geoffrey B

    1985-01-01

    Polyclonal antibodies raised against barley (1-->3,1-->4)-beta-d-glucanase, alpha-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley....... In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding alpha-amylase or (1-->3,1-->4)-beta-d-glucanase, while in the aleurone alpha-amylase and (1-->3,1-->4)-beta-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1......-->3,1-->4)-beta-d-glucanase, alpha-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation...

  4. Contribution to the study of the diffusion {alpha}-proton for {alpha} particles of 3,1 and 5,3 MeV; Contribution a l'etude de la diffusion {alpha}-proton pour des particules {alpha} d'energie comprise entre 3,1 et 5,3 MeV

    Energy Technology Data Exchange (ETDEWEB)

    Ruhla, C [Commissariat a l' Energie Atomique, Saclay(France). Centre d' Etudes Nucleaires

    1953-07-01

    The diffusion of the particles has by the light cores that present a weak gate of potential, must permit the survey of the nuclear strengths. Some authors, studying the distribution in energy of the protons given out by a hydrogenated target submitted to a bombardment has variable energy, signal that this distribution has a structure of groups. We tried to reproduce experiences of diffusion {alpha}-proton, in order to verify the existence of the groups of signaled protons in the previous works. However in spite of finer experimental conditions, we had recovered any group structures in the distribution of the protons. This work permits to conclude that there is not a resonance in the {alpha}-proton diffusion for included energies between 3,1 and 5,3 MeV. The absence of resonances confirms the existence of the fundamental level of {sup 5}Li above in the neighborhood of 1,8 MeV {sup 4}He + {sup 1}H. (M.B.) [French] La diffusion des particules a par les noyaux legers qui presentent une faible barriere de potentiel, doit permettre l'etude des forces nucleaires. certains auteurs, etudiant la distribution en energie des protons emis par une cible hydrogenee soumise a un bombardement a d'energie variable, signalent que cette distribution a une structure de groupes. Nous avons essaye de reproduire les experiences du type diffusion {alpha}-proton, afin de verifier l'existence des groupes de protons signales dans les travaux anterieurs. Cependant malgre des conditions experimentales plus fines, nous n'avons retrouve aucunce structure de groupe dans la distribution des protons. Ce travail permet de conclure qu'il n'y a pas de resonance dans la diffusion {alpha}-proton pour des energies comprises entre 3,1 et 5,3 MeV. L'absence de resonances confirme l'existence du niveau fondamental de {sup 5}Li au voisinage de 1,8 MeV au-dessus de {sup 4}He + {sup 1}H. (M.B.)

  5. Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

    Science.gov (United States)

    Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

    2004-12-01

    The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

  6. DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by {sup 32}P-postlabeling

    Energy Technology Data Exchange (ETDEWEB)

    Randerath, E. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States); Zhou, G.D. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States); Donnelly, K.C. [Department of Veterinary Anatomy and Public Health, Texas A and M University, College Station, TX (United States); Safe, S.H. [Department of Veterinary Physiology/Pharmacology, Texas A and M University, College Station, TX (United States); Randerath, K. [Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX (United States)

    1996-09-01

    In the present study, a mouse bioassay was used in combination with {sup 32}P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6 x 10{sup 6} DNA nucleotides in skin (both extracts) and one adduct in 3.2 x 10{sup 7} or 1.2 x 10{sup 7} DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the {sup 32}P-labeled derivative of the reaction product of 7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) with N{sup 2} of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. (orig.) (orig.). With 5 figs., 6 tabs.

  7. Association of polymorphism in the alpha-1-antitrypsin gene with ...

    African Journals Online (AJOL)

    Alpha-1-antitrypsin (A1AT) as a strong protease inhibitor plays a major role in the protection of tissues against proteolytic destruction by neutrophil elastase. Existence of this protein in the mammary gland may increase the survival of milk proteins such as lactoferrin and lysozyme. The biological role of A1AT in tissues such ...

  8. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  9. Quantitative immuno-electron microscopic analysis of depolarization-induced expression of PGC-1alpha in cultured rat visual cortical neurons.

    Science.gov (United States)

    Meng, Hui; Liang, Huan Ling; Wong-Riley, Margaret

    2007-10-17

    Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC- 1alpha) is a coactivator of nuclear receptors and other transcription factors that regulate several metabolic processes, including mitochondrial biogenesis, energy homeostasis, respiration, and gluconeogenesis. PGC-1alpha plays a vital role in stimulating genes that are important to oxidative metabolism and other mitochondrial functions in brown adipose tissue and skeleton muscles, but the significance of PGC-1alpha in the brain remains elusive. The goal of our present study was to determine by means of quantitative immuno-electron microscopy the expression of PGC-1alpha in cultured rat visual cortical neurons under normal conditions as well as after depolarizing stimulation for varying periods of time. Our results showed that: (a) PGC-1alpha was normally located in both the nucleus and the cytoplasm. In the nucleus, PGC-1alpha was associated mainly with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, it was associated mainly with free ribosomes. (b) Neuronal depolarization by KCl for 0.5 h induced a significant increase in PGC-1alpha labeling density in both the nucleus and the cytoplasm (Pneuronal activity by synthesizing more proteins in the cytoplasm and translocating them to the nucleus for gene activation. PGC-1alpha level in neurons is, therefore, tightly regulated by neuronal activity.

  10. Differential involvement of 5-HT(1A) and 5-HT(1B/1D) receptors in human interferon-alpha-induced immobility in the mouse forced swimming test.

    Science.gov (United States)

    Zhang, Hongmei; Wang, Wei; Jiang, Zhenzhou; Shang, Jing; Zhang, Luyong

    2010-01-01

    Although Interferon-alpha (IFN-alpha, CAS 9008-11-1) is a powerful drug in treating several viral infections and certain tumors, a considerable amount of neuropsychiatric side-effects such as depression and anxiety are an unavoidable consequence. Combination with the selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine (CAS 56296-78-7) significantly improved the situation. However, the potential 5-HT(1A) receptor- and 5-HT(1B) receptor-signals involved in the antidepressant effects are still unclear. The effects of 5-HT(1A) receptor- and 5-HT(1B) receptor signals were analyzed by using the mouse forced swimming test (FST), a predictive test of antidepressant-like action. The present results indicated that (1) fluoxetine (administrated intragastrically, 30 mg/kg; not subactive dose: 15 mg/kg) significantly reduced IFN-alpha-induced increase of the immobility time in the forced swimming test; (2) 5-HT(1A) receptor- and 5-HT(1B) receptor ligands alone or in combination had no effects on IFN-alpha-induced increase of the immobility time in the FST; (3) surprisingly, WAY 100635 (5-HT(1A) receptor antagonist, 634908-75-1) and 8-OH-DPAT(5-HT(1A) receptor agonist, CAS 78950-78-4) markedly enhanced the antidepressant effect of fluoxetine at the subactive dose (15 mg/kg, i. g.) on the IFN-alpha-treated mice in the FST. Further investigations showed that fluoxetine combined with WAY 100635 and 8-OH-DPAT failed to produce antidepressant effects in the FST. (4) Co-application of CGS 12066A (5-HT(1B) receptor agonist, CAS 109028-09-3) or GR 127935 (5-HT(1B/1D) receptor antagonist, CAS 148642-42-6) with fluoxetine had no synergistic effects on the IFN-alpha-induced increase of immobility time in FST. (5) Interestingly, co-administration of GR 127935, WAY 100635 and fluoxetine significantly reduced the IFN-alpha-induced increase in immobility time of FST, being more effective than co-administration of WAY 100635 and fluoxetine. All results suggest that (1) compared to

  11. The effect of the Z mutation on the ability of alpha 1-antitrypsin to prevent neutrophil mediated tissue damage.

    Science.gov (United States)

    Llewellyn-Jones, C G; Lomas, D A; Carrell, R W; Stockley, R A

    1994-11-29

    Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.

  12. Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA

    DEFF Research Database (Denmark)

    Christiansen, G; Pedersen, Lotte Bang; Koehler, J E

    1993-01-01

    maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA. Hc1 alone is thus able to package DNA into condensed spherical...

  13. HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

    Science.gov (United States)

    Niehof, Monika; Borlak, Jürgen

    2011-01-27

    Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS). In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII) remain elusive. Notably, angiotensinogen (AGT) is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1), angiotensin I converting enzyme (ACE), and angiotensin I converting enzyme 2 (ACE2) as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition of nuclear

  14. HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

    Directory of Open Access Journals (Sweden)

    Monika Niehof

    Full Text Available Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS. In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII remain elusive. Notably, angiotensinogen (AGT is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1, angiotensin I converting enzyme (ACE, and angiotensin I converting enzyme 2 (ACE2 as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition

  15. The tree-alpha Faddeev calculation on 12C bound states with a Pauli correct alpha-alpha potential

    International Nuclear Information System (INIS)

    Kamada, Hiroyuki; Oryu, Shinsho

    1986-01-01

    The three-alpha model of 12 C is investigated by the Faddeev formalism with the UIM alpha-alpha potential, in which the Pauli effect between two-alpha system was taken into account adequately. The potential can reproduce the on- and off-shell effects of the alpha-alpha interaction by the rank-4 separable type for the S-wave, the rank-3 one for the D-wave, and the rank-2 one for the G-wave, in which two of the ranks in the S-wave, and one in the D-wave are prepared to eliminate the Pauli forbidden states. We obtained three even states J π = 0 + , 2 + , 4 + , and two odd states 1 - , 3 - , below the alpha- 8 Be(0 + g.s) threshold energy. The even parity states gain larger binding energies than those which have been obtained by former Faddeev calculation with the rank-1 Kukulin and Neudatchin (KN) potential. On the other hand, for the odd parity states, we obtained smaller binding energies than the former one. It is found that our Faddeev calculation with the UIM potential does not miss any important low-lying levels of 12 C, in which any spurious states do not appear. (author)

  16. Modification of certain pharmacological effects of ethanol by lipophilic alpha-1 adrenergic agonists

    Energy Technology Data Exchange (ETDEWEB)

    Menon, M.K.; Dinovo, E.C.; Haddox, V.G.

    1987-09-28

    The influence of four centrally-acting alpha-1 adrenoceptor agonists, namely, 2(2-chloro-5-trifluoromethylphenylimino) imidazolidine (St 587), cirazoline, (-) 1,2,3,4-tetrahydro-8-methoxy-5-methylthio-2-naphthalenamine ((-)SKF 89748A) and 2-(2-methylindazol-4-imino)imidazolidine (Sgd 101/75) on the pharmacological effects of ethanol was investigated. All four drugs reduced the duration of ethanol-induced hypnosis in C57B1/6 mice, this effect being proportional to their relative potencies to exert central alpha-1 agonism. In prazosin-pretreated mice, St 587 failed to reduce the hypnotic effect of ethanol, which provided strong evidence for the role of alpha-1 agonism for the hypnosis reducing effect of St 587. Hyperactivity induced in C57B1/6 mice by a subhypnotic dose of ethanol and St 587 was reported earlier. In the present study, St 587, cirazoline and (-)SKF 89748A produced similar response, but no correlation between this effect and ethanol hypnosis blockade could be established. 19 references, 8 figures, 2 tables.

  17. Relative workload determines exercise-induced increases in PGC-1alpha mRNA

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte

    2010-01-01

    INTRODUCTION:: The hypothesis that brief intermittent exercise induced increases in human skeletal muscle metabolic mRNA is dependent on relative workload was investigated. METHODS:: Trained (n=10) and untrained (n=8) subjects performed exhaustive intermittent cycling exercise (4x4 min @ 85% of VO2...... peak, interspersed by 3 min). Trained subjects also performed the intermittent exercise at the same absolute workload as untrained, corresponding to 70% of VO2 peak (n=6). RESULTS:: Exercise at 85% of VO2 peak elevated (P... and untrained, respectively. PGC-1alpha mRNA expression was increased (Pelevated (3.1+/-0.7 mM) and PGC-1alpha mRNA content was less (P

  18. Mechanism of action of the endo-(1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum

    NARCIS (Netherlands)

    Grün, Christian H.; Dekker, Nick; Nieuwland, Alexander A.; Klis, Frans M.; Kamerling, Johannis P.; Vliegenthart, Johannes F. G.; Hochstenbach, Frans

    2006-01-01

    (1-->3)-alpha-glucanases catalyze the hydrolysis of fungal cell wall (1-->3)-alpha-glucan, and function during cell division of yeasts containing this cell wall component or act in mycoparasitic processes. Here, we characterize the mechanism of action of the (1-->3)-alpha-glucanase MutAp from the

  19. The Ar{sup 17+} Ly{sub {alpha}2}/Ly{sub {alpha}1} ratio in Alcator C-Mod tokamak plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Rice, J E; Reinke, M L; Ince-Cushman, A C; Podpaly, Y A [Plasma Science and Fusion Center, MIT, Cambridge, MA (United States); Ashbourn, J M A [Mathematical Institute, University of Oxford, Oxford (United Kingdom); Gu, M F [SSL, University of California Berkeley, CA (United States); Bitter, M; Hill, K [Princeton Plasma Physics Laboratory, Princeton, NJ (United States); Rachlew, E, E-mail: rice@psfc.mit.edu [KTH, Stockholm (Sweden)

    2011-08-28

    High-quality spectra of hydrogen-like Ar{sup 17+} have been obtained from Alcator C-Mod tokamak plasmas using a spatially imaging high-resolution x-ray spectrometer system in an extensive study of the underlying high-n satellite lines. The ratio of Ly{sub {alpha}2} (1S{sub 1/2}-2P{sub 1/2}) to Ly{sub {alpha}1} (1S{sub 1/2}-2P{sub 3/2}) was found to be {approx}0.52 regardless of plasma parameters, which is somewhat greater than the ratio of the statistical weights of the upper n = 2 levels, 0.5. This difference is mainly due to the effects of collisional excitation of fine-structure sub-levels. For the observations presented here, electron densities were in an extended range from 3x10{sup 19} to 4x10{sup 20} m{sup -3} with electron and ion temperatures between 1 and 4 keV. Experimental results are compared to calculations from COLRAD, a collisional-radiative modelling code, and good agreement is shown.

  20. Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

    Science.gov (United States)

    Takeshita, Kohei; Suetake, Isao; Yamashita, Eiki; Suga, Michihiro; Narita, Hirotaka; Nakagawa, Atsushi; Tajima, Shoji

    2011-01-01

    Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes. PMID:21518897

  1. Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

    Science.gov (United States)

    Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

    2013-11-01

    Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

  2. Loss of Smyhc1 or Hsp90alpha1 function results in different effects on myofibril organization in skeletal muscles of zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Marta Codina

    Full Text Available BACKGROUND: Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90alpha1 (Hsp90alpha1 has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90alpha1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90alpha1 function or indirectly through the disorganization of myosin thick filaments. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1 resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90alpha1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. CONCLUSION/SIGNIFICANCE: Together, these studies indicate that the hsp90alpha1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90alpha1 may play a role in the assembly and organization of other sarcomeric structures.

  3. Expression and characterization of a recombinant maize CK-2 alpha subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Dobrowolska, G

    1993-01-01

    to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its......CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order...... molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39,228 Da (332 amino acids). The recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties as the recombinant human CK-2 alpha (rhCK-2...

  4. The lower genital tract microbiota in relation to cytokine-, SLPI- and endotoxin levels: application of checkerboard DNA-DNA hybridization (CDH).

    Science.gov (United States)

    Nikolaitchouk, Natalia; Andersch, Björn; Falsen, Enevold; Strömbeck, Louise; Mattsby-Baltzer, Inger

    2008-04-01

    In the present study the lower genital tract microbiota in asymptomatic fertile women (n=34) was identified and quantified by culturing vaginal secretions. Also, vaginal and cervical samples were analyzed by a semiquantitative checkerboard DNA-DNA hybridization technique (CDH) based on genomic probes prepared from 13 bacterial species (Bacteroides ureolyticus, Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Mobiluncus curtisii ss curtisii, Prevotella bivia, Prevotella disiens, Prevotella melaninogenica, Atopobium vaginae, Lactobacillus iners, Staphylococcus aureus ss aureus, Streptococcus anginosus, and Streptococcus agalactiae). The bacterial species found by either culture or CDH were correlated with proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8), secretory leukocyte protease inhibitor (SLPI), and endotoxin in the cervicovaginal samples. Grading the women into healthy, intermediate, or bacterial vaginosis (BV) as based on Gram staining of vaginal smears, the viable counts of lactobacilli (L. gasseri) and of streptococci-staphylococci combined were highest in the intermediate group. In BV, particularly the high concentrations of Actinomyces urogenitalis, Atopobium vaginae, and Peptoniphilus harei were noted (>or=10(11) per ml). The total viable counts correlated with both cervical IL-1 alpha and IL-1 beta. A strong negative correlation was observed between L. iners and total viable counts, G. vaginalis, or cervical IL-1 alpha, while it correlated positively with SLPI. Analysis of vaginal and cervical samples from 26 out of the 34 women by CDH showed that anaerobic bacteria were more frequently detected by CDH compared to culture. By this method, A. vaginae correlated with G. vaginalis, and L. iners with S. aureus. With regard to cytokines, B. ureolyticus correlated with both cervical and vaginal IL-1 alpha as well as with cervical IL-8, while F. nucleatum, S. agalactiae, S. anginosus, or S. aureus correlated with vaginal IL-1 alpha

  5. GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-01-01

    Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140

  6. Influence of fast alpha diffusion and thermal alpha buildup on tokamak reactor performance

    International Nuclear Information System (INIS)

    Uckan, N.A.; Tolliver, J.S.; Houlberg, W.A.; Attenberger, S.E.

    1988-01-01

    The effect of fast alpha diffusion and thermal alpha accumulation on the confinement capability of a candidate Engineering Test Reactor plasma (Tokamak Ignition/Burn Experimental Reactor) in achieving ignition and steady-state driven operation has been assessed using both global and 1-1/2-dimensional transport models. Estimates are made of the threshold for radial diffusion of fast alphas and thermal alpha buildup. It is shown that a relatively low level of radial transport, when combined with large gradients in the fast alpha density, leads to a significant radial flow with a deleterious effect on plasma performance. Similarly, modest levels of thermal alpha concentration significantly influence the ignition and steady-state burn capability

  7. Acute effect of oral, intraperitoneal, and intravenous 1 alpha-hydroxycholecalciferol on markers of bone metabolism

    DEFF Research Database (Denmark)

    Joffe, P; Ladefoged, S D; Cintin, C

    1994-01-01

    ,25-(OH)2D3 was measured. DESIGN: Single doses of 1 alpha-OHD3 (80 ng/kg body wt) were given in randomized cross-over fashion, orally, intraperitoneally (i.p.) and intravenously (i.v.) on three occasions. Blood was sampled at 0, 1, 6, 12, and 24 h after administration of 1 alpha-OHD3. MAIN RESULTS...

  8. Alpha1A-adrenergic receptor-directed autoimmunity induces left ventricular damage and diastolic dysfunction in rats.

    Directory of Open Access Journals (Sweden)

    Katrin Wenzel

    Full Text Available BACKGROUND: Agonistic autoantibodies to the alpha(1-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. METHODS/PRINCIPAL FINDINGS: We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A-adrenergic receptor and maintained them for one year. Alpha(1A-adrenergic antibodies (alpha(1A-AR-AB were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min. Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. CONCLUSIONS/SIGNIFICANCE: We show that alpha(1A-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A-AR-AB could contribute to cardiovascular endorgan damage.

  9. Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA

    Directory of Open Access Journals (Sweden)

    Yubin Li

    2018-01-01

    Full Text Available A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA was developed on the surface of silver-coated glass slide (SCGS. Oligonucleotide-1 (Oligo-1 was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2 was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4, signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

  10. Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

    Science.gov (United States)

    Pietrowski, D; Durante, M J; Liebstein, A; Schmitt-John, T; Werner, T; Graw, J

    1994-07-08

    The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

  11. PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Leick, Lotte; Wojtaszewski, Jørgen F P; Johansen, Sune T.

    2008-01-01

    The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor-gamma coactivator (PGC) 1alpha is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1alpha knockout (KO) and littermate wild-type (WT) mice performed....... Resting muscles of the PGC-1alpha KO mice had lower ( approximately 20%) cytochrome c (cyt c), cytochrome oxidase (COX) I, and aminolevulinate synthase (ALAS) 1 mRNA and protein levels than WT, but similar levels of AMP-activated protein kinase (AMPK) alpha1, AMPKalpha2, and hexokinase (HK) II compared...

  12. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  13. PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPAR{gamma} activation in the liver

    Energy Technology Data Exchange (ETDEWEB)

    Oishi, Katsutaka, E-mail: k-ooishi@aist.go.jp [Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki (Japan); Uchida, Daisuke [Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki (Japan); Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki (Japan); Ohkura, Naoki [Department of Clinical Molecular Biology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa (Japan); Horie, Shuichi [Department of Clinical Biochemistry, Kagawa Nutrition University, Sakado, Saitama (Japan)

    2010-10-15

    Research highlights: {yields} PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression. {yields} Hepatic expressions of PPAR{gamma} and PCG-1{alpha} are induced by a ketogenic diet. {yields} PPAR{gamma} antagonist attenuates a ketogenic diet-induced PAI-1 expression. {yields} Ketogenic diet advances the phase of circadian clock in a PPAR{alpha}-independent manner. -- Abstract: An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPAR{alpha}-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPAR{alpha}-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPAR{alpha} target genes such as Cyp4A10 and FGF21 was damped in PPAR{alpha}-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPAR{alpha}-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPAR{alpha} activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPAR{gamma} and its coactivator PCG-1{alpha} were more effectively induced in PPAR{alpha}-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPAR{gamma} antagonist, in both WT and PPAR{alpha

  14. 17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

    Science.gov (United States)

    Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

    2015-01-05

    Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

    International Nuclear Information System (INIS)

    Sebastian, J.; Sancar, G.B.

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

  16. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-05-01

    Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

  17. Antagonism of Lateral Amygdala Alpha1-Adrenergic Receptors Facilitates Fear Conditioning and Long-Term Potentiation

    Science.gov (United States)

    Lazzaro, Stephanie C.; Hou, Mian; Cunha, Catarina; LeDoux, Joseph E.; Cain, Christopher K.

    2010-01-01

    Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala…

  18. An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1.

    Science.gov (United States)

    Calvo, K R; Knoepfler, P; McGrath, S; Kamps, M P

    1999-12-23

    The Pbx/Exd family of homeodomain (HD) proteins contribute to the transcriptional and developmental roles of other Hox and Meis/Prep1/Hth HD proteins through heterodimer formation. E2a-Pbx1 is an oncogenic derrivative of Pbx1 produced by the t(1;19) translocation in pediatric pre-B cell acute lymphoblastic leukemia. E2a-Pbx1 heterodimerizes with Hox but not with Meis/Prep1 proteins, produces acute myeloid leukemia in mice, and blocks differentiation of cultured murine myeloid progenitors. Here, we characterize negative and positive regulatory sequences that flank the Pbx1 HD and determine their importance for myeloid immortalization by E2a-Pbx1. A 25 residue predicted alpha helix preceding the Pbx1 HD bound the HD and prevented both its binding to DNA and its ability to heterodimerize with Hox proteins. Addition of 39 residues N-terminal to this inhibitory helix exposed a Pbx dimerization interface that orchestrated cooperative DNA-binding of E2a-Pbx1 and all Pbx proteins as homodimers and heterdimers. Sequences inhibiting DNA-binding and mediating Pbx dimerization coincided with those reported to have nuclear export function. An additional 103 residues N-terminal to the Pbx dimerization interface restored heterodimerization with Hox and Meis1/Prep1 proteins. This negative switch domain - comprised of the inhibitory helix and N-terminal regions required for its partner-mediated derepression - was dispensable for myeloid immortalization by E2a-Pbx1. While stabilizing the heterodimer, the 310 helix C-terminal to the Pbx1 HD was also dispensable for the ability of E2a-Pbx1 to heterodimerize with Hox proteins and immortalize myeloblasts. Retention of myeloid immortalization by E2a-Pbx1 proteins lacking all Pbx1 sequences N- or C-terminal to the HD indicates that Hox proteins, or a yet undefined factor that binds the Pbx1 HD and derepresses DNA-binding by the HD, cooperate with E2a-Pbx1 in myeloid immortalization.

  19. Improving adherence to alpha-1 antitrypsin deficiency screening guidelines using the pulmonary function laboratory

    Directory of Open Access Journals (Sweden)

    Luna Diaz LV

    2017-07-01

    Full Text Available Landy V Luna Diaz,1 Isabella Iupe,1 Bruno Zavala,1 Kira C Balestrini,1 Andrea Guerrero,1 Gregory Holt,1,2 Rafael Calderon-Candelario,1,2 Mehdi Mirsaeidi,1,2 Michael Campos1,21Miami Veterans Administration Medical Center, Miami, FL, 2Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Miami School of Medicine, Miami, FL, USAAlpha-1 antitrypsin deficiency (AATD is the only well-recognized genetic disorder associated with an increased risk of emphysema and COPD.1 Identifying AATD allows genetic counseling and the chance to offer specific augmentation therapy to slow emphysema progression. Despite specific recommendations from the World Health Organization, American Thoracic Society and European Respiratory Society to screen all patients with COPD and other at-risk conditions,2–4 testing rates are low (<15%.5We conducted a project to improve AATD screening at the Miami VA Medical Center using the pulmonary function test (PFT laboratory. We instructed the PFT personnel to perform reflex testing on all patients with pre-bronchodilator airflow obstruction (forced expiratory volume in 1 second/forced vital capacity <70% and then evaluated if the screening was appropriate according to guidelines. Trained PFT personnel explained AATD disease to patients and provided them with an informational brochure. After obtaining verbal consent, AATD screening was performed using dried blood spot kits provided by the Alpha-1 Foundation as part of the Florida Screening Program (noncommercial.6 The PFT lab director was the responsible physician of record, in charge of discussing positive results to patients and documenting results in the electronic medical record. The Miami Veterans Affairs Medical Center Institutional Review Board approved the protocol as a quality improvement project.

  20. Gastric clearance of alpha-1-antitrypsin under cimetidine perfusion. New test to detect protein-losing gastropathy

    International Nuclear Information System (INIS)

    Florent, C.; Vidon, N.; Flourie, B.; Carmantrand, A.; Zerbani, A.; Maurel, M.; Bernier, J.J.

    1986-01-01

    Gastric losses of plasma are usually measured with radiolabeled macromolecules. This method is expensive and cumbersome. Direct measurement of exudated plasma proteins are ineffective since proteins are denaturated by acidic gastric juice and pepsin. It was recently shown that albumin measurement after immediate neutralization allowed detection of gastric protein losses, but this method is quite complex and time consuming. We studied alpha 1-antitrypsin and 51Cr-labeled protein clearance in gastric juice during normal saline and cimetidine (1.5 mg/kg/hr) infusion in six healthy volunteers and six patients with exudative gastropathy. alpha 1-Antitrypsin was measurable in all samples during cimetidine infusion: alpha 1-AT and 51Cr losses were significantly correlated (P less than 0.001). The upper limit of gastric alpha 1-AT clearance in controls was 0.86 ml/hr (mean + 2 SD). Using this value, there was no overlapping between patients and controls. The upper limit of 51Cr test was 1.87 ml/hr (mean + 2 SD) in controls but gastric clearance of 51Cr was below this value in one patient. This suggests that the measurement of alpha 1-AT gastric clearance during cimetidine perfusion is a good test to detect an exudative gastropathy. This test is inexpensive and lasts only 3 hr

  1. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  2. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  3. Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage

    Czech Academy of Sciences Publication Activity Database

    Kanagaraj, R.; Parasuraman, P.; Mihaljevic, B.; van Loon, B.; Burdová, Kamila; König, C.; Furrer, A.; Bohr, V.A.; Hübscher, U.; Janscak, P.

    2012-01-01

    Roč. 40, č. 17 (2012), s. 8449-8459 ISSN 0305-1048 Grant - others:Swiss National Science Foundation(CH) 31003A-129747/1; Swiss National Science Foundation(CH) 3100-109312/2; Oncosuisse(CH) KLS-02344-02-2009; NIH(US) Z01-AG000726-17 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : DNA repair * oxidative stress * MUTYH * WRN * Pol lambda Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  4. Neutron Diffraction Study On Gamma To Alpha Phase Transition In Ce0.9th0.1 Alloy

    Energy Technology Data Exchange (ETDEWEB)

    Lashley, Jason C1 [Los Alamos National Laboratory; Heffner, Robert H [Los Alamos National Laboratory; Llobet, A [Los Alamos National Laboratory; Darling, T W [U OF NEVADA; Jeong, I K [PUSAN NATL UNIV

    2008-01-01

    Comprehensive neutron diffraction measurements were performed to study the isostructural {gamma} {leftrightarrow} {alpha} phase transition in Ce{sub 0.9}Th{sub 0.1} alloy. Using Rietveld refinements, we obtained lattice and thermal parameters as a function of temperature. From the temperature slope of the thermal parameters, we determined Debye temperatures {Theta}{sup {gamma}}{sub D} = 133(1) K and {Theta}{sup {alpha}}{sub D} = 140(1) K for the {gamma} phase and the {alpha} phase, respectively. This result implies that the vibrational entropy change is not significant at the {gamma} {leftrightarrow} {alpha} transition, contrary to that from elemental Cerium [Phys. Rev. Lett. 92, 105702, 2004].

  5. Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

    Science.gov (United States)

    Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

    2010-01-01

    The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

  6. Reciprocal Regulation between DNA-PKcs and Snail1 Conferring Genomic Instability

    International Nuclear Information System (INIS)

    Seo, Haeng Ran; Lee, Hae June; Jin, Yeung Bae; Bae, Sang Woo; Lee, Yun Sil; Kim, Nam Hee; Kim, Hyun Sil; Nam, Hyung Wook; Yook, Jong In

    2010-01-01

    Although the roles of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) involving non-homologous end joining (NHEJ) of DNA repair are well recognized, the biological mechanisms and regulators by which DNA-PKcs regulate genomic instability are not clearly defined. We show herein that DNA-PKcs activity resulting from DNA damage caused by ionizing radiation (IR) phosphorylates Snail1 at serine 100, which results in increased Snail1 expression and its function by inhibition of GSK-3-mediated phosphorylation. Furthermore, Snail1 phosphorylated at serine 100 can reciprocally inhibit kinase activity of DNA-PKcs, resulting in an inhibition to recruit DNA-PKcs or Ku70/80 to a DNA double-strand break site, and ultimately inhibition of DNA repair activity. The impairment of repair activity by a direct interaction between Snail1 and DNA-PKcs increases the resistance to DNA damaging agents, such as IR, and genomic instability. Our findings provide a novel cellular mechanism for induction of genomic instability by reciprocal regulation of DNA-PKcs and Snail1

  7. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  8. [A compound heterozygosity mutation in the interleukin-7 receptor-alpha gene resulted in severe combined immunodeficiency in a Chinese patient].

    Science.gov (United States)

    Zhang, Zhi-yong; Zhao, Xiao-dong; Wang, Mo; Yu, Jie; An, Yun-fei; Yang, Xi-qiang

    2009-09-01

    Mutation in the interleukin-7 receptor-alpha (IL-7R alpha) chain causes a rare type of severe combined immunodeficiency (SCID) with presence of NK cells in the peripheral blood. Here we report the molecular and clinical characterization of a compound heterozygosity mutation in the interleukin-7 receptor-alpha gene that resulted in SCID in a patient firstly from China. A 5 month-old male patient and his parents were enrolled in this study. Since 15 days of age, the patient had had recurrent fever, persistent cough and diarrhea. He was in poor general condition with pyorrhea and ulceration of the BCG scar. His brother died of severe infection at 4 months of age. He was initially diagnosed as SCID according to clinical manifestation and immunological analysis. A panel of SCID candidate genes including IL-2RG, RAG1/RAG2 and IL-7R alpha of patient and his parents were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the IL-7R alpha transcripts. Sequencing was performed directly on the PCR products forward and reversely. The serum immunoglobulin (Ig) profile was IgG 6867 mg/L (normal range, 3050 - 8870 mg/L); IgM 206 mg/L and IgA 249 mg/L, IgE 2.3 IU/ml (normal range microscope and by culture. The patient had a compound heterozygosity mutation in the IL-7R alpha gene:on one allele, there was a splice-junction mutation in intron 4 (intron 4(+1)G > A), for which his father was a carrier; whereas on the other allele, a nonsense mutation at position 638 in exon 5 with a premature stop codon (638 C > T, R206X) was identified, for which his mother was a carrier. The splice-junction mutation in intron 4 of IL-7R alpha was firstly reported. The IL-7R alpha mRNA expression of the patient was remarkably reduced whereas the parents had relatively normal IL-7R alpha mRNA expression. IL-7R alpha cDNA of the patient was amplified by nested PCR. The PCR products were purified, cloned with a TA

  9. 1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines.

    Science.gov (United States)

    Lagishetty, Venu; Chun, Rene F; Liu, Nancy Q; Lisse, Thomas S; Adams, John S; Hewison, Martin

    2010-07-01

    Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  10. Problem solving in foundation engineering using foundationPro

    CERN Document Server

    Yamin, Mohammad

    2016-01-01

    This book is at once a supplement to traditional foundation engineering textbooks and an independent problem-solving learning tool. The book is written primarily for university students majoring in civil or construction engineering taking foundation analysis and design courses to encourage them to solve design problems. Its main aim is to stimulate problem solving capability and foster self-directed learning. It also explains the use of the foundationPro software, available at no cost, and includes a set of foundation engineering applications. Taking a unique approach, Dr. Yamin summarizes the general step-by-step procedure to solve various foundation engineering problems, illustrates traditional applications of these steps with longhand solutions, and presents the foundationPro solutions. The special structure of the book allows it to be used in undergraduate and graduate foundation design and analysis courses in civil and construction engineering. The book stands as valuable resource for students, faculty, ...

  11. Bovine serum albumin-catalyzed deprotonation of [1-(13)C]glycolaldehyde: protein reactivity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon.

    Science.gov (United States)

    Go, Maybelle K; Malabanan, M Merced; Amyes, Tina L; Richard, John P

    2010-09-07

    Bovine serum albumin (BSA) in D(2)O at 25 degrees C and pD 7.0 was found to catalyze the deuterium exchange reactions of [1-(13)C]glycolaldehyde ([1-(13)C]GA) to form [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA. The formation of [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA in a total yield of 51 +/- 3% was observed at early reaction times, and at later times, [1-(13)C,2-(2)H]GA was found to undergo BSA-catalyzed conversion to [1-(13)C,2,2-di-(2)H]GA. The overall second-order rate constant for these deuterium exchange reactions [(k(E))(P)] equals 0.25 M(-1) s(-1). By comparison, (k(E))(P) values of 0.04 M(-1) s(-1) [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778] and 0.06 M(-1) s(-1) [Go, M. K., Koudelka, A., Amyes, T. L., and Richard, J. P. (2010) Biochemistry 49, 5377-5389] have been determined for the wild-type- and K12G mutant TIM-catalyzed deuterium exchange reactions of [1-(13)C]GA, respectively, to form [1-(13)C,2,2-di-(2)H]GA. These data show that TIM and BSA exhibit a modest catalytic activity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon. We suggest that this activity is intrinsic to many globular proteins, and that it must be enhanced to demonstrate meaningful de novo design of protein catalysts of proton transfer at alpha-carbonyl carbon.

  12. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    International Nuclear Information System (INIS)

    Kitagawa, Yukiko; Kameoka, Masanori; Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-01-01

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2α) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2α. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2α. Confocal fluorescence microscopy revealed that a subpopulation of AP2α was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin β and Nup153, implying that AP2α negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2α may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle

  13. Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1[alpha

    Energy Technology Data Exchange (ETDEWEB)

    Rho, H.; Jones, C.N.; Rose, R.B. (NCSU)

    2010-12-07

    The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1{alpha} (hepatocyte nuclear factor-1{alpha}), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1{alpha} complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1{alpha} through simple mixing. To further investigate regulation of DCoH/HNF-1{alpha} complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1{alpha}. The 1.8 {angstrom} resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1{alpha} complex formation are discussed.

  14. Tonic pain and continuous EEG: prediction of subjective pain perception by alpha-1 power during stimulation and at rest.

    Science.gov (United States)

    Nir, Rony-Reuven; Sinai, Alon; Moont, Ruth; Harari, Eyal; Yarnitsky, David

    2012-03-01

    Pain neurophysiology has been chiefly characterized via event-related potentials (ERPs), which are exerted using brief, phase-locked noxious stimuli. Striving for objectively characterizing clinical pain states using more natural, prolonged stimuli, tonic pain has been recently associated with the individual peak frequency of alpha oscillations. This finding encouraged us to explore whether alpha power, reflecting the magnitude of the synchronized activity within this frequency range, will demonstrate a corresponding relationship with subjective perception of tonic pain. Five-minute-long continuous EEG was recorded in 18 healthy volunteers under: (i) resting-state; (ii) innocuous temperature; and (iii) psychophysically-anchored noxious temperature. Numerical pain scores (NPSs) collected during the application of tonic noxious stimuli were tested for correlation with alpha-1 and alpha-2 power. NPSs and alpha power remained stable throughout the recording conditions (Ps⩾0.381). In the noxious condition, alpha-1 power obtained at the bilateral temporal scalp was negatively correlated with NPSs (Ps⩽0.04). Additionally, resting-state alpha-1 power recorded at the bilateral temporal scalp was negatively correlated with NPSs reported during the noxious condition (Ps⩽0.038). Current findings suggest alpha-1 power may serve as a direct, objective and experimentally stable measure of subjective perception of tonic pain. Furthermore, resting-state alpha-1 power might reflect individuals' inherent tonic pain responsiveness. The relevance of alpha-1 power to tonic pain perception may deepen the understanding of the mechanisms underlying the processing of prolonged noxious stimulation. Copyright © 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  15. Lower lid entropion secondary to treatment with alpha-1a receptor antagonist: a case report

    Directory of Open Access Journals (Sweden)

    Simcock Peter

    2010-03-01

    Full Text Available Abstract Introduction The use of alpha-1a receptor antagonists (tamsulosin is widely accepted in the treatment of benign prostatic hypertrophy (BPH. It has previously been implicated as a causative agent in intra-operative floppy iris syndrome due to its effects on the smooth muscle. We report a case of lower lid entropion that may be related to a patient commencing treatment of tamsulosin. Case presentation A 74-year-old Caucasian man was started on alpha 1-a receptor antagonist (Tamsulosin treatment for benign prostatic hypertrophy. Eight days later, he presented to the ophthalmology unit with a right lower lid entropion which was successfully treated surgically with a Weiss procedure. Conclusion We report a case of lower lid entropion that may be secondary to the recent use of an alpha-1a blocker (tamsulosin. This can be explained by considering the effect of autonomic blockade on alpha-1 receptors in the Muller's muscle on a patient that may already have an anatomical predisposition to entropion formation due to a further reduction in muscle tone.

  16. Synthesis and antimicrobial evaluation of new 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinones.

    Science.gov (United States)

    Güzeldemirci, Nuray Ulusoy; Ilhan, Eser; Küçükbasmaci, Omer; Satana, Dilek

    2010-01-01

    New 4-thiazolidinone derivatives of benzilic acid (alpha,alpha-diphenyl-alpha-hydroxyacetic acid) have been synthesized and evaluated for antibacterial and antifungal activities. The reaction of 1- (alpha,alpha-diphenyl-alpha-hydroxy)acetyl-4-alkyl/arylthiosemicarbazides with ethyl 2-bromopropionate gave 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinone derivatives. Their antibacterial and antifungal activities were evaluated against S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. coli ATCC 25922, C. albicans ATCC 10231, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, T. mentagrophytes var. erinacei NCPF 375, M. gypseum NCPF 580 and T. tonsurans NCPF 245. 3e, 3f, 3g and 3h showed the highest antibacterial activity. Particularly 3a and 3e showed the highest antifungal activities against C. parapsilosis ATCC 22019, T. tonsurans NCPF 245 and M. gypseum NCPF 580.

  17. Downregulation of MIP-1alpha/CCL3 with praziquantel treatment in Schistosoma haematobium and HIV-1 co-infected individuals in a rural community in Zimbabwe

    DEFF Research Database (Denmark)

    Zinyama-Gutsire, Rbl; Gomo, E.; Kallestrup, P

    2009-01-01

    influence. METHODS: To determine levels of MIP-1alpha/CCL3 chemokine in plasma of S. haematobium and HIV-1 co-infected and uninfected individuals in a rural black Zimbabwean community.A cohort was established of HIV-1 and schistosomiasis infection and co-infection comprising 379 participants. Outcome...... measures consisted of HIV-1 and schistosomiasis status and levels of MIP-1alpha/CCL3 in plasma at baseline and three months post treatment. An association was established between MIP-1alpha/CCL3 plasma levels with HIV-1 and S. haematobium infections. RESULTS: A total of 379 adults formed the established...... cohort comprising 76 (20%) men and 303 (80%) women. Mean age was 33.25, range 17 - 62 years. The median MIP-1alpha/CCL3 plasma concentration was significantly higher in S. haematobium infected compared with uninfected individuals (p = 0.029). In contrast, there was no difference in the median MIP-1alpha...

  18. Crystal structure of the Msx-1 homeodomain/DNA complex.

    Science.gov (United States)

    Hovde, S; Abate-Shen, C; Geiger, J H

    2001-10-09

    The Msx-1 homeodomain protein plays a crucial role in craniofacial, limb, and nervous system development. Homeodomain DNA-binding domains are comprised of 60 amino acids that show a high degree of evolutionary conservation. We have determined the structure of the Msx-1 homeodomain complexed to DNA at 2.2 A resolution. The structure has an unusually well-ordered N-terminal arm with a unique trajectory across the minor groove of the DNA. DNA specificity conferred by bases flanking the core TAAT sequence is explained by well ordered water-mediated interactions at Q50. Most interactions seen at the TAAT sequence are typical of the interactions seen in other homeodomain structures. Comparison of the Msx-1-HD structure to all other high resolution HD-DNA complex structures indicate a remarkably well-conserved sphere of hydration between the DNA and protein in these complexes.

  19. Main metabolites of 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea and 1-(2-chloroethyl)-3-(2',3', 4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea in rats

    International Nuclear Information System (INIS)

    Madelmont, J.C.; Moreau, M.F.; Godeneche, D.; Duprat, J.; Plagne, R.; Meyniel, G.

    1982-01-01

    The metabolism of two glycosylnitrosoureas, 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) and 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea (RPCNU), has been investigated in the rat. With the label on the carboxyl moiety of RFCNU, we have shown that hydrolysis of the 4-nitrobenzoyl ester occurred to a large extent in vivo; 4-nitrobenzoic acid and its glucuronide were the major urinary metabolites. Two other minor metabolites and their glucuronides were identified as 4-aminobenzoic acid and 4-acetamidobenzoic acid. With the label on the chloroethyl moieties of RFCNU and RPCNU, we have shown that chloroethanol was a major degradation product of this alkylating part of the molecule. The concentration of chloroethanol in plasma vs. time has been determined. In urine, four metabolites derived from alkylated glutathione, namely thiodiacetic acid and its sulfoxide, N-acetylcarboxymethylcysteine, and N-acetylhydroxyethylcysteine, have been identified

  20. Safety and efficacy outcome of mentor alpha-1 inflatable penile prosthesis implantation for impotence treatment.

    Science.gov (United States)

    Goldstein, I; Newman, L; Baum, N; Brooks, M; Chaikin, L; Goldberg, K; McBride, A; Krane, R J

    1997-03-01

    We investigated safety and efficacy outcome pertaining to the Mentor Alpha-1, 3-piece inflatable penile prosthesis for impotence treatment. A 2-phase, multi-institutional, large scale retrospective study, with independently analyzed medical record (phase I) and questionnaire (phase II) data from consecutive eligible patients of 7 physician investigators was performed from March to October 1993. In phase I there were no morbidities of any type in 394 of the 434 patients (90.8%) (mean age 61 years, range 24 to 88) who underwent Alpha-1 implantation (mean followup 22.2 months, range 0.67 to 44.5). The risk of prosthesis malfunction (fluid leak and auto-inflation) was 2.5%. No cylinder aneurysms were reported. A total of 93.1% of Alpha-1 devices was free from explantation (4.4%) or revision surgery (2.5%) for approximately 2 years from the original implant date. Kaplan-Meier actuarial analyses revealed that cumulative survival of the Alpha-1 prostheses at 12, 24 and 36 months was 98 +/- 1%, 93 +/- 2% and 85 +/- 7% until device malfunction, and 91 +/- 2% 83 +/- 4% and 75 +/- 7% until surgical intervention (revision or explantation). In phase II 89% of the men claimed fulfilled expectations with the Alpha-1 prosthesis as impotence treatment. Satisfaction responses 80% or greater were noted with regard to intercourse ability and confidence, and device rigidity and function. Implantation did not result in greater than 80% satisfaction in partner relationships or feelings (as judged by the patient), social or work confidence, or intercourse frequency. Factors adversely affecting satisfaction included partner feelings of dissatisfaction (as judged by the patient), specific physician investigators and need for explantation/revision surgery. In 1 of the largest multi-institutional implant outcome studies thus far performed, safety and efficacy data concerning the Alpha-1 contemporary inflatable device were found markedly improved over earlier inflatable prostheses and now

  1. Drugs interacting with alpha adrenoceptors

    NARCIS (Netherlands)

    van Zwieten, P. A.

    1989-01-01

    Alpha adrenoceptors should be divided into various subtypes, comprising pre/postsynaptic and alpha 1/alpha 2-subpopulations, respectively. This classification implicates important functional differences between the various alpha-receptor subtypes, including certain differences in signal transduction

  2. DNA probe for lactobacillus delbrueckii

    Energy Technology Data Exchange (ETDEWEB)

    Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  3. New production processes for alpha hemihydrate open up new marketing opportunities

    International Nuclear Information System (INIS)

    Engert, W.; Lehmkaemper, O.; Bunte, H.P.

    1991-01-01

    New production processes and markets for alpha hemihydrate are discussed. Utility studies concluded that lignite gypsum is harmless in terms of public and occupational health, and is technically comparable to or superior to natural gypsum by virtue of greater purity. Semi-commercial and pilot-scale studies were carried out on the use of flue gas desulfurization (FGD) gypsum for producing alpha hemihydrate, with successful results. The process enabled pure alpha hemihydrate to be produced without dihydrate or dihydrate impurities, and of a constant, uniform quality. The treatment consists of forming pressed mouldings of FGD gypsum followed by steam autoclaving, drying and milling. Agents are used to stabilize the stackable moldings, and to act as growth inhibitors during transformation of dihydrite to alpha-hemihydrate. Markets for the product are found in mining, tunneling and road building, foundation work, floor systems, as hard plaster for dental and moulding applications, for construction industry use, and as structural and non-structural material. Details are presented of the production process and marketing concepts. 12 figs

  4. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    DEFF Research Database (Denmark)

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels

    2010-01-01

    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

  5. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    Directory of Open Access Journals (Sweden)

    Sanjeev Gupta

    2010-07-01

    Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  6. Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-05-05

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  7. Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-06-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  8. Radioprotection of the intestinal crypts of mice by recombinant human interleukin-1 alpha

    International Nuclear Information System (INIS)

    Wu, S.G.; Miyamoto, T.

    1990-01-01

    Recombinant human interleukin-1 alpha (rHIL-1 alpha or IL-1) protected the intestinal crypt cells of mice against X-ray-induced damage. The survival of crypt cells measured in terms of their ability to form colonies of regenerating duodenal epithelium in situ was increased when IL-1 was given either before or after irradiation. The maximum degree of radioprotection was seen when the drug was given between 13 and 25 h before irradiation. The IL-1 dose producing maximum protection was about 6.3 micrograms/kg. This is the first report indicating that the cytokine IL-1 has a radioprotective effect in the intestine. The finding suggests that IL-1 may be of potential value in preventing radiation injury to the gut in the clinic

  9. Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    Xingxing Kong

    2010-07-01

    Full Text Available Sirtuin 3 (SIRT3 is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR binding element (ERRE (-407/-399 mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2C(12 myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2C(12 myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2C(12 myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy

  10. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  11. Auger radiation targeted into DNA: a therapy perspective

    Energy Technology Data Exchange (ETDEWEB)

    Buchegger, Franz [University Hospital of Lausanne CHUV, Service of Nuclear Medicine, Lausanne (Switzerland); University Hospital of Lausanne, Service of Nuclear Medicine, Lausanne (Switzerland); Perillo-Adamer, Florence; Bischof Delaloye, Angelika [University Hospital of Lausanne CHUV, Service of Nuclear Medicine, Lausanne (Switzerland); Dupertuis, Yves M. [University Hospital of Geneva, Service of Nutrition, Geneva (Switzerland)

    2006-11-15

    Auger electron emitters that can be targeted into DNA of tumour cells represent an attractive systemic radiation therapy goal. In the situation of DNA-associated decay, the high linear energy transfer (LET) of Auger electrons gives a high relative biological efficacy similar to that of {alpha} particles. In contrast to {alpha} radiation, however, Auger radiation is of low toxicity when decaying outside the cell nucleus, as in cytoplasm or outside cells during blood transport. The challenge for such therapies is the requirement to target a high percentage of all cancer cells. An overview of Auger radiation therapy approaches of the past decade shows several research directions and various targeting vehicles. The latter include hormones, peptides, halogenated nucleotides, oligonucleotides and internalising antibodies. Here, we will discuss the basic principles of Auger electron therapy as compared with vector-guided {alpha} and {beta} radiation. We also review some radioprotection issues and briefly present the main advantages and disadvantages of the different targeting modalities that are under investigation. (orig.)

  12. Immunohistochemical and radioimmunological demonstration of alpha/sub 1/-fetoprotein in nonmalignant changes of human gastric mucosa

    Energy Technology Data Exchange (ETDEWEB)

    Falser, N; Lederer, B; Reissigl, H [Innsbruck Univ. (Austria). Pathologisch-Anatomisches Lab.; Innsbruck Univ. (Austria). Gastroenterologisches Lab.)

    1977-07-01

    The occurence of ..cap alpha../sub 1/ fetoprotein in nonmalignant changes of the gastric mucosa was investigated by means of immunohistochemistry and radioimmunonoassay. The investigations were performed in tissue sections, cytological imprint preparations as well as in homogenized tissue samples (obtained by gastroscopy). ..cap alpha../sub 1/ fetoprotein could be demonstrated by immuno-histochemistry in about 90% of the samples originating from the surroundings of gastric ulcer and the region of gastrojejunostomy after B II-resection. The RIA was positive in about 75% of the tissue samples, whereas from gastric juice only 40% of positive results could be obtained. No ..cap alpha../sub 1/ fetoprotein-activity could be demonstrated in serum samples. These investigations indicate that ..cap alpha../sub 1/ fetoprotein is not exclusively synthesized by embryonic or neoplastic tissues and also can be synthesized also by regenerating cell-systems. It may be supposed that this synthesis represents an unspecific answer to growth-stimulation.

  13. Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy.

    Science.gov (United States)

    Jung, Yeonjoo; Park, Jinah; Kim, Tai Young; Park, Jung-Hyun; Jong, Hyun-Soon; Im, Seock-Ah; Robertson, Keith D; Bang, Yung-Jue; Kim, Tae-You

    2007-10-01

    The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

  14. Association between Insulin Like Growth Factor-1 (IGF-1) gene ...

    African Journals Online (AJOL)

    The insulin-like growth factor-1 (IGF1) is a key regulator of muscle development and metabolism in birds and other vertebrate. Our objective was to determine the association between IGF1 gene polymorphism and carcass traits in FUNAAB Alpha chicken. Genomic DNA was extracted from the blood of 50 normal feathered ...

  15. Sterol synthesis. A novel reductive rearrangement of an alpha,beta-unsaturated steroidal epoxide; a new chemical synthesis of 5alpha-cholest-8(14)-en-3beta, 15alpha-diol.

    Science.gov (United States)

    Parish, E J; Schroepfer, G J

    1977-04-01

    Reduction of 3beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with either lithium triethylboro-hydride or lithium aluminum hydride (4 molar excess) gave 5-alpha-cholest-8(14)-en-3beta,15alpha-diol in high yield. Reduction of the epoxy ester with lithium triethylborodeuteride or lithium aluminum deuteride (4 molar excess) gave [7alpha-2-H]-5alpha-cholest-8(14)-en-3beta,15alpha-diol. Reduction of 2beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with a large excess (24 molar excess) of lithium aluminum hydride gave, in addition to the expected 5alpha-cholest-8(14)-en-3beta,15alpha-diol, a significant yield (33%) of 5alpha-cholest-8(14)-en-3beta-o1. Reduction of the epoxy ester with a large excess (24 molar excess) of lithium aluminum deuteride gave [7alpha-2H]-5alpha-cholest-8(14)-en-3beta,15alpha-diol and 5alpha-cholest-8(14)-en-3beta-o1 which contained two atoms of stably bound deuterium.

  16. Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

    Science.gov (United States)

    Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

    2007-12-01

    An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

  17. Interferon alpha as antiviral therapy in chronic active Epstein-Barr virus disease with interstitial pneumonia - case report.

    Science.gov (United States)

    Roliński, Jacek; Grywalska, Ewelina; Pyzik, Aleksandra; Dzik, Michał; Opoka-Winiarska, Violetta; Surdacka, Agata; Maj, Maciej; Burdan, Franciszek; Pirożyński, Michał; Grabarczyk, Piotr; Starosławska, Elżbieta

    2018-04-20

    Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is defined as a severe, progressive lymphoproliferative disorder associated with active EBV infection persisting longer than 6 months and developing in patients without recognised immunodeficiency. Rarely, interstitial pneumonitis (IP) occurs as a serious complication in CAEBV patients. The standard therapeutic regimen for IP in CAEBV has not yet been defined. Although interferon alpha (IFN-alpha) is known to suppress viral DNA replication by affecting its basal promoter activation process, it is rarely used in CAEBV patients. A 22-year-old Caucasian woman, diagnosed with CAEBV 1.5 years earlier, was admitted to the Immunology Clinic due to a 4-week history of productive cough, fever and general weakness. Cultures of blood, urine and sputum were negative, but EBV DNA copies were found in the sputum, whole blood, isolated peripheral blood lymphocytes as well as in the blood plasma. Cytokine assessment in peripheral blood revealed the lack of IFN-alpha synthesis. Disseminated maculate infiltrative areas in both lungs were observed on a computed tomography (CT) chest scan. The patient was not qualified for the allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the risk of immunosuppression-related complications of infectious IP. Inhaled (1.5 million units 3 times a day) and subcutaneous (6 million units 3 times a week) IFN-alpha was implemented. To the best of our knowledge, this was the first documented use of inhaled IFN-alpha in a patient with CAEBV and concomitant IP. Patient's status has improved, and she was eventually qualified to allo-HSCT with reduced conditioning. Currently, the patient feels well, no EBV was detected and further regression of pulmonary changes was documented. CAEBV should be considered in patients who present with interstitial lung infiltration and involvement of other organs. Although more promising results have been obtained with allo-HSCT, inhaled IFN-alpha may

  18. Immunostimulatory effects of natural human interferon-alpha (huIFN-alpha) on carps Cyprinus carpio L.

    Science.gov (United States)

    Watanuki, Hironobu; Chakraborty, Gunimala; Korenaga, Hiroki; Kono, Tomoya; Shivappa, R B; Sakai, Masahiro

    2009-10-15

    Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.

  19. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

    Science.gov (United States)

    Taherzadeh, S; Sharma, S; Chhajlani, V; Gantz, I; Rajora, N; Demitri, M T; Kelly, L; Zhao, H; Ichiyama, T; Catania, A; Lipton, J M

    1999-05-01

    The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

  20. TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

    DEFF Research Database (Denmark)

    Le Douarin, B; You, J; Nielsen, Anders Lade

    1998-01-01

    Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new...... family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

  1. Different patterns of HIV-1 DNA after therapy discontinuation

    Directory of Open Access Journals (Sweden)

    Ghinelli Florio

    2005-09-01

    Full Text Available Abstract Background By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16 – 24 weeks to study the possible relationship between DNA and RNA viral load. Methods The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia (RNA viral load. Results Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA. Conclusion Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects.

  2. Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

    Science.gov (United States)

    Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2009-11-01

    Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

  3. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

    Energy Technology Data Exchange (ETDEWEB)

    Prior, Sara; Miousse, Isabelle R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nzabarushimana, Etienne [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Department of Bioinformatics, School of Informatics and Computing, Indiana University, Bloomington, IN 47405 (United States); Pathak, Rupak [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Skinner, Charles; Kutanzi, Kristy R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Allen, Antiño R. [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Raber, Jacob [Departments of Behavioral Neuroscience, Neurology, and Radiation Medicine, Division of Neuroscience, ONPRC, Oregon Health & Science University, Portland, OR 97239 (United States); Tackett, Alan J. [Department of Biochemistry, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Hauer-Jensen, Martin [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nelson, Gregory A. [Department of Basic Sciences, Division of Radiation Research, Loma Linda University, Loma Linda, CA 92350 (United States); and others

    2016-10-15

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

  4. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

    International Nuclear Information System (INIS)

    Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.

    2016-01-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

  5. The influence of nifedipine and pertussis toxin (PTX) on vascular responsiveness to alpha1- and alpha2-adrenergic stimulation of isolated femoral arteries

    Czech Academy of Sciences Publication Activity Database

    Líšková, Silvia; Kuneš, Jaroslav; Paulis, Ĺudovít; Zicha, Josef

    2006-01-01

    Roč. 48, č. 4 (2006), s. 769-769 ISSN 0194-911X. [Annual Meeting of the European Council for Cardiovascular Research (ECCR) /11./. 29.09.2006-01.10.2006, La Colle sur Loup] R&D Projects: GA MZd NR7786 Institutional research plan: CEZ:AV0Z50110509 Keywords : nifedipine * pertussis toxin * vascular responsiveness * alpha1- and alpha2-adrenergic stimulation * femorel arteria Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  6. Kinetics and mechanism of DNA repair; Evaluation of caged compounds for use in studies of u. v. -induced DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Meldrum, R.A.; Wharton, C.W. (Birmingham Univ. (UK). Dept. of Biochemistry); Shall, S. (Sussex Univ., Brighton (UK). School of Biological Sciences)

    1990-03-15

    Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the {gamma}-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside trisphosphate is isotopically labelled in the {alpha}-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair. (author).

  7. Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

    Science.gov (United States)

    Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

    2009-11-01

    Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

  8. Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt

    DEFF Research Database (Denmark)

    Frandsen, T P; Lok, F; Mirgorodskaya, E

    2000-01-01

    in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a c......High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4...

  9. Analysis of DNA strand break induction and repair in plants from the vicinity of Chernobyl

    International Nuclear Information System (INIS)

    Syomov, A.B.; Ptitsyna, S.N.; Sergeeva, S.A.

    1992-01-01

    For 3 years following the Chernobyl accident DNA repair efficiency was studied in irradiated and control populations of various plan species. Compared with the control populations, some irradiated populations exhibited increases in the yield of DNA single-strand breaks per unit dose of challenge radiation. The effect was registered in low-dose-rate alpha-irradiated populations, but was absent in plant populations growing in conditions of low-dose-rate beta-irradiation. The efficiency of single-strand DNA repair was identical in control and irradiated populations and approximated 100%. (author). 12 refs.; 1 fig.; 2 tabs

  10. Effects of anti-tumor necrosis factor-alpha and anti-intercellular adhesion molecule-1 antibodies on ischemia/reperfusion lung injury.

    Science.gov (United States)

    Chiang, Chi-Huei

    2006-10-31

    Inhibition of neutrophil activation and adherence to endothelium by antibodies to tumor necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecules (ICAM-1), respectively, might attenuate ischemia-reperfusion injury (I/R). I/R was conducted in an isolated rat lung model. Anti-TNF-alpha antibody and/or anti-ICAM-1 antibody were added before ischemia or after reperfusion. Hemodynamic changes, lung weight gain (LWG), capillary filtration coefficients (Kfc), and pathologic changes were assessed to evaluate the severity of I/R. The LWG, Kfc, pathological changes and lung injury score of treatment groups with anti-TNF-alpha antibody treatment, either pre-ischemia or during reperfusion, were less than those observed in control groups. Similar findings were found in group treated with anti-ICAM-1 antibody or combination therapy during reperfusion. In contrast, pre-I/R treatment with anti-ICAM-1 antibody induced severe lung edema and failure to complete the experimental procedure. No additional therapeutic effect was found in combination therapy. We conclude that TNF-alpha and ICAM-1 play important roles in I/R. Anti-TNF-alpha antibody has therapeutic and preventive effects on I/R. However, combined therapy with anti-TNF-alpha antibody and anti-ICAM-1 antibody may have no additive effect and need further investigation.

  11. Evaluation through comet assay of DNA damage induced in human lymphocytes by alpha particles. Comparison with protons and Co-60 gamma rays

    International Nuclear Information System (INIS)

    Di Giorgio, M.; Kreiner, A. J.; Schuff, J. A.; Vallerga, M. B.; Taja, M. R.; Lopez, F. O.; Alvarez, D. E.; Saint Martin, G.; Burton, A.; Debray, M. E.; Kesque, J. M.; Somacal, H.; Stoliar, P.; Valda, A.; Davidson, J.; Davidson, M.; Ozafran, M. J.; Vazquez, M. E.

    2004-01-01

    Several techniques with different sensitivity to single-strand breaks and/or double strand breaks were applied to detect DNA breaks generated by high LET particles. Tests that assess DNA damage in single cells might be the appropriate tool to estimate damage induced by particles, facilitating the assessment of heterogeneity of damage in a cell population. The microgel electrophoresis (comet) assay is a sensitive method for measuring DNA damage in single cells. The objective of this work was to evaluate the proficiency of comet assay to assess the effect of high LET radiation on peripheral blood lymphocytes, compared to protons and Co-60 gamma rays. Materials and methods: Irradiations of blood samples were performed at TANDAR laboratory (Argentina). Thin samples of human peripheral blood were irradiated with different doses (0-2.5 Gy) of 20.2 MeV helium-4 particles in the track segment mode, at nearly constant LET. Data obtained were compared with the effect induced by a MeV protons and Co-60 gamma rays. Alkaline comet assay was applied. Comets were quantified by the Olive tail moment. Distribution of the helium-4 particle and protons were evaluated considering Poisson distribution in lymphocyte nuclei. The mean dose per nucleus per particle result 0.053 Gy for protons and 0.178 Gy for helium-4 particles. When cells are exposed to a dose of 0.1 Gy, the hit probability model predicts that 43% of the nuclei should have experienced and alpha traversal while with protons, 85% of the nuclei should be hit. The experimental results show a biphasic response for helium-4 particles (0.1 Gy), indicating the existence of two subpopulations: unhit and hit. Distributions of tail moment as a function of fluence and experimental dose for comets induced by helium-4 particles, protons and Co-60 gamma rays were analyzed. With helium-4 irradiations, lymphocyte nuclei show an Olive tail moment distribution flattened to higher tail moments a dose increase. However, for irradiations with

  12. Function of BRCA1 at a DNA Replication Origin

    National Research Council Canada - National Science Library

    Lieberman, Paul

    2004-01-01

    ... and allow efficient repair of damaged DNA. In this proposal, we present preliminary data that BRCA1 functions in a DNA checkpoint response for the origin of Epstein-Barr Virus DNA replication (Ori P...

  13. Studying effects of Magnolol on alpha-particle induced bystander effects using PADC-film based dishes

    Energy Technology Data Exchange (ETDEWEB)

    Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Tse, A.K.W.; Fong, W.F. [Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University, Baptist University Road, Kowloon Tong (Hong Kong); Yu, K.N., E-mail: peter.yu@cityu.edu.h [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)

    2009-10-15

    Radiation-induced bystander effect refers to the biological response found in cells (called bystander cells) which are not irradiated directly by ionizing radiation but are next to cells irradiated directly by ionizing radiation. In the present paper, the effects of Magnolol, an extract from the bark of Magnolia officinalis which is used as a traditional Chinese medicine, were studied on alpha-particle induced bystander effects. In our experiments, Chinese hamster ovary (CHO) cells were cultured in PADC-film based dishes and were irradiated with low fluences of alpha particles passing through the PADC films. The precise number of cells traversed or missed by alpha particles could be determined by studying the alpha-particle tracks developed on the PADC films upon subsequent chemical etching. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed to analyze the biological response of bystander cells in terms of DNA strand breaks. With the pretreatment of Magnolol, the DNA strand breaks in bystander cells were reduced, which showed that the alpha-particle induced bystander effects were suppressed with the presence of Magnolol. Since Magnolol is an antioxidant which can scavenge reactive oxygen species (ROS), our results give support to that ROS play a role in the bystander signal transmission in our experiments.

  14. Studying effects of Magnolol on alpha-particle induced bystander effects using PADC-film based dishes

    International Nuclear Information System (INIS)

    Wong, T.P.W.; Tse, A.K.W.; Fong, W.F.; Yu, K.N.

    2009-01-01

    Radiation-induced bystander effect refers to the biological response found in cells (called bystander cells) which are not irradiated directly by ionizing radiation but are next to cells irradiated directly by ionizing radiation. In the present paper, the effects of Magnolol, an extract from the bark of Magnolia officinalis which is used as a traditional Chinese medicine, were studied on alpha-particle induced bystander effects. In our experiments, Chinese hamster ovary (CHO) cells were cultured in PADC-film based dishes and were irradiated with low fluences of alpha particles passing through the PADC films. The precise number of cells traversed or missed by alpha particles could be determined by studying the alpha-particle tracks developed on the PADC films upon subsequent chemical etching. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed to analyze the biological response of bystander cells in terms of DNA strand breaks. With the pretreatment of Magnolol, the DNA strand breaks in bystander cells were reduced, which showed that the alpha-particle induced bystander effects were suppressed with the presence of Magnolol. Since Magnolol is an antioxidant which can scavenge reactive oxygen species (ROS), our results give support to that ROS play a role in the bystander signal transmission in our experiments.

  15. Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo

    2010-12-01

    Full Text Available Abstract Background Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins. Results Using the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD, a glyceraldehyde-3-P dehydrogenase (GAPDH and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment. Conclusions This study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association

  16. Effect of Temperature on Topological States of Circular DNA

    Science.gov (United States)

    Fan, Yang-Tao; Li, Xiu-Yan; Liu, Yan-Hui; Chen, Hu

    2017-07-01

    The different topological states of circular double-stranded DNA can be defined by their linking number. The equilibrium distribution of linking number can be obtained by circularizing a linear DNA into a circle by ligase. Based on the recent experimental results that the DNA bending rigidity and twist rigidity strongly depend on temperature, the reduced bending rigidity can be approximated by g=(3.19× {10}-19-T\\cdot 4.14× {10}-22) {erg}\\cdot {cm} over the temperature interval (5 ∼ 53) °C, and the temperature dependence of twist rigidity can be fitted by C(T)=(4588.89{exp} (-T/117.04)-251.33) nm. The temperature dependence of the linking number distribution of circular DNAs can be predicted by using Monte Carlo simulation. The variance of linking number distribution on temperature is in accordance with the previous experimental results. Compared with the temperature dependence of bending rigidity, the temperature dependence of twist rigidity causes a noticeable fluctuation in linking number distribution and mainly contribute towards the variance change of linking number distribution of circular DNA. The variance of the writhe number and twist number in the equation = + depends on the length of circular DNA. When the length of circular DNA is less than 230 nm, the variance of twist number is dominant over the variance of writhe number ( ), whereas for the condition that the length of the circular DNA is larger than 370 nm. Supported by the National Natural Science Foundation of China under Grant Nos. 11047022, 11204045, and 11464004, Guizhou Provincial Tracking Key Program of Social Development (SY20123089, SZ20113069), the General Financial Grant from the China Postdoctoral Science Foundation (2014M562341), the Research Foundation for Young University Teachers from Guizhou University (201311), and College Innovation Talent Team of Guizhou Province (2014)32

  17. SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

    Science.gov (United States)

    Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

    2013-01-01

    Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

  18. INFLUENCE OF ALPHA-1-ACID GLYCOPROTEIN UPON PRODUCTION OF CYTOKINES BY PERIPHERAL BLOOD MONONUCLEARS

    Directory of Open Access Journals (Sweden)

    М. V. Osikov

    2007-01-01

    Full Text Available Abstract. Alpha-1-acid glycoprotein (orosomucoid is a multifunctional acute phase reactant belonging to the family of lipocalines from plasma alpha-2 globulin fraction. In present study, we investigated dosedependent effects of orosomucoid upon secretion of IL-1â, IL-2, IL-3, IL-4 by mononuclear cells from venous blood of healthy volunteers. Mononuclear cells were separated by means of gradient centrifugation, followed by incubation for 24 hours with 250, 500, or 1000 mcg of orosomucoid per ml RPMI-1640 medium (resp., low, medium and high dose. The levels of cytokine production were assayed by ELISA technique. Orosomucoid-induced secretion of IL-1â and IL-4 was increased, whereas IL-3 secretion was inhibited. IL-2 production was suppressed at low doses of orosomucoid, and stimulated at medium and high doses. The effect of alpha-1-acid glycoprotein upon production of IL-2, IL-3 and IL-4 was dose-dependent. Hence, these data indicate that orosomucoid is capable of modifying IL-1â, IL-2, IL-3, and IL-4 secretion by blood mononuclear cells.

  19. Individual variation in p53 and Cip1 expression profiles in normal human fibroblast strains following exposure to high-let radiation

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, T.R.; Johnson, N.F.; Gilliland, F.D. [Univ. of New Mexico, Albuquerque, NM (United States)] [and others

    1995-12-01

    Exposure to {alpha}-particles emitted by radon progeny appears to be the second-leading cause of lung cancer mortality. However, individual susceptibility to the carcinogenic effects of {alpha}-particles remains poorly characterized. Variation in susceptibility to cancer produced by certian classes of DNA-damaging chemicals is suspected to involve differences in metabolic activation and detoxication. Susceptibility to {alpha}-particle-induced cancer may involve variations in capacity or opportunity to repair DNA damage. Subtle variations in DNA repair capacity would more likely explain radon-related lung cancer susceptibility. The p53 tumor suppressor protein accumulates as a cellular response to DNA damage from ionizing radiation and regulates arrest in the G{sub 1} portion of the cell cycle. Arrest in G{sub 1} portion of the cell cycle. While upstream regulation of p53 protein stability is poorly understood, variations in the ability to accumulate p53 following DNA damage represent potential variations in lung cancer susceptibility related to radon progeny. Further, transcription of the cell-cycle regulatory gene Cip1 is regulated by p53 and increases following ionizing radiation. Therefore, variations in the expression of Cip1 following {alpha}-particle exposure may also be a susceptibility factor in radon-related lung cancers. The purpose of the present investigation was to measure p53 and Cip1 protein induction following {alpha}-particle exposure of fibroblast lines from nine individuals to determine if there were significant variations. The expression of Cip1 protein indicates the differences in response are biologically relevant.

  20. A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

    Energy Technology Data Exchange (ETDEWEB)

    Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others

    1994-09-01

    Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

  1. Intermittent intravenous followed by intermittent oral 1 alpha(OH)D3 treatment of secondary hyperparathyroidism in uraemia

    DEFF Research Database (Denmark)

    Brandi, L; Daugaard, H; Egsmose, C

    1996-01-01

    OBJECTIVES: To examine whether intermittent oral 1 alpha(OH)D3 treatment of patients on haemodialysis with secondary hyperparathyroidism (HPT) was able to maintain the marked suppression of PTH, which previously had been induced by an intermittent intravenous administration of 1 alpha(OH)D3....... Simultaneously, the effect of the different routes of administration of 1 alpha(OH)D3 on the circulating levels of N- and C-terminal PTH fragments was measured. DESIGN: An open study of patients on chronic haemodialysis. SETTING: Renal division, Rigshospitalet, Copenhagen, Denmark. SUBJECTS: A total of 26...

  2. Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances.

    Science.gov (United States)

    Ashley, Neil; Adams, Susan; Slama, Abdelhamid; Zeviani, Massimo; Suomalainen, Anu; Andreu, Antonio L; Naviaux, Robert K; Poulton, Joanna

    2007-06-15

    Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.

  3. PGC-1{alpha} is required for AICAR induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Leick, Lotte; Fentz, Joachim; Biensø, Rasmus S

    2010-01-01

    We tested the hypothesis that repeated activation of AMPK induces mitochondrial and glucose membrane transporter gene/protein expression via a peroxisome proliferator activated receptor Upsilon co-activator (PGC)-1alpha dependent mechanism. Whole body PGC-1alpha knockout (KO) and littermate wild...... GLUT4, cytochrome c oxidase (COX)I and cytochrome (cyt) c protein expression ~10-40% relative to saline in white muscles of the WT mice, but not of the PGC-1alpha KO mice. In line, GLUT4 and cyt c mRNA content increased 30-60% 4h after a single AICAR injection relative to saline only in WT mice. One...... and PGC-1alpha KO mice. In conclusion, we here provide genetic evidence for a major role of PGC-1alpha in AMPK mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle....

  4. Alpha thalassaemia-mental retardation, X linked

    Directory of Open Access Journals (Sweden)

    Gibbons Richard

    2006-05-01

    Full Text Available Abstract X-linked alpha thalassaemia mental retardation (ATR-X syndrome in males is associated with profound developmental delay, facial dysmorphism, genital abnormalities and alpha thalassaemia. Female carriers are usually physically and intellectually normal. So far, 168 patients have been reported. Language is usually very limited. Seizures occur in about one third of the cases. While many patients are affectionate with their caregivers, some exhibit autistic-like behaviour. Patients present with facial hypotonia and a characteristic mouth. Genital abnormalities are observed in 80% of children and range from undescended testes to ambiguous genitalia. Alpha-thalassaemia is not always present. This syndrome is X-linked recessive and results from mutations in the ATRX gene. This gene encodes the widely expressed ATRX protein. ATRX mutations cause diverse changes in the pattern of DNA methylation at heterochromatic loci but it is not yet known whether this is responsible for the clinical phenotype. The diagnosis can be established by detection of alpha thalassaemia, identification of ATRX gene mutations, ATRX protein studies and X-inactivation studies. Genetic counselling can be offered to families. Management is multidisciplinary: young children must be carefully monitored for gastro-oesophageal reflux as it may cause death. A number of individuals with ATR-X are fit and well in their 30s and 40s.

  5. Expression of HIF-1{alpha} in irradiated tissue is altered by topical negative-pressure therapy

    Energy Technology Data Exchange (ETDEWEB)

    Grimm, A.; Stange, S.; Labanaris, A.; Horch, R.E. [Erlangen-Nuernberg Univ., Erlangen (Germany). Dept. of Plastic and Hand Surgery; Dimmler, A. [Erlangen-Nuernberg Univ., Erlangen (Germany). Dept. of Pathology; Sauer, R.; Grabenbauer, G. [Erlangen-Nuernberg Univ. (Germany). Dept. of Radiation Oncology

    2007-03-15

    Background and Purpose: Despite the enormous therapeutic potential of modern radiotherapy, common side effects such as radiation-induced wound healing disorders remain a well-known clinical phenomenon. Topical negative pressure therapy (TNP) is a novel tool to alleviate intraoperative, percutaneous irradiation or brachytherapy. Since TNP has been shown to positively influence the perfusion of chronic, poorly vascularized wounds, the authors applied this therapeutic method to irradiated wounds and investigated the effect on tissue oxygenation in irradiated tissue in five patients. Material and Methods: With informed patients' consent, samples prior to and 4 and 8 days after continuous TNP with -125 mmHg were obtained during routine wound debridements. Granulation tissue was stained with hematoxylin-eosin, and additionally with CD31, HIF-1{alpha} (hypoxia-inducible factor-1{alpha}), and D2-40 to detect blood vessels, measure indirect signs of hypoxia, and lymph vessel distribution within the pre- and post-TNP samples. Results: In this first series of experiments, a positive influence of TNP onto tissue oxygenation in radiation-induced wounds could be demonstrated. TNP led to a significant decrease of 53% HIF-1{alpha}-positive cell nuclei. At the same time, a slight reduction of CD31-stained capillaries was seen in comparison to samples before TNP. Immunostaining with D2-40 revealed an increased number of lymphatic vessels with distended lumina and an alteration of the parallel orientation within the post-TNP samples. Conclusion: This study is, to the authors' knowledge, the first report on a novel previously not described histological marker to demonstrate the effects of TNP on HIF-1{alpha} expression as an indirect marker of tissue oxygenation in irradiated wounds, as demonstrated by a reduction of HIF-1{alpha} concentration after TNP. Since this observation may be of significant value to develop possible new strategies to treat radiation-induced tissue

  6. No-carrier-added (NCA) synthesis of 6-[{sup 18}F]fluoro-L-DOPA using 3,5,6,7,8,8a-hexahydro-7,7,8a-trimethyl-[6S-(6{alpha}, 8{alpha}, 8{alpha}{beta})]-6,8-methano-2H-1,4-benzoxazin-2-one

    Energy Technology Data Exchange (ETDEWEB)

    Horti, A. [Yale Univ., New Haven, CT (United States). School of Medicine]|[Yale Univ., West Haven, CT (United States). PET Center; Redmond, D.E. Jr. [Yale Univ., New Haven, CT (United States). School of Medicine; Soufer, R. [Yale Univ., West Haven, CT (United States). PET Center

    1995-12-31

    3,5,6,7,8,8a-Hexahydro-7,7,8a-trimethyl-[6S-(6{alpha},8{alpha} , 8{alpha}{beta})]-6,8-methano-2H-1,4-benzoxazino-2-one (2) was investigated as chiral auxiliary for asymmetric NCA nucleophilic synthesis of 6-[{sup 18}F]Fluoro-L-DOPA. Direct condensation of 3,4-dimethoxy-2-[{sup 18}F]fluorobenzaldehyde (1a) or 6-[{sup 18}F]fluoro-piperonal (1b) in the presence of NaH with 2 gave the corresponding [{sup 18}F]-3-[(2-fluorophenyl)methylene]-3,5,6,7,8,8a-hexahydro-7,7,8 a-trimethyl-[6S-(3Z,3{alpha},6{alpha},8{alpha},8{alpha}{beta})]-6, 8-methano-2H-1,4-benzoxazin-2-one derivative 3a or 3b as a single stereoisomer. L-Selectride promoted hydrogenation of the olefinic double bond of these derivatives, in presence of tertbutyl alcohol, afforded the corresponding [{sup 18}F]-3-[(2-fluorophenyl) methyl]-3,5,6,7,8,8a-hexahydro-7,7,8a-trimethyl-[3S-(3{alpha}, 6{alpha}, 8{alpha}8{alpha}{beta})]-6,8-methano-2H-1,4-benzoxazin-2-one derivatives (4a,b) without affecting the orientation of diasterofacial discrimination. Deprotection of the derivatives 4a,b yielded 6-[{sup 18}F]fluoro-L-DOPA (e.e. >90%, 3% radiochemical yield (EOB), total synthesis time 125 min, specific activity >2000 mCi/{mu}mol). Direct deprotection/reduction of the compounds 3a,b provides the enantiomeric mixture of 6-[{sup 18}F]fluoro-D,L-DOPA (10-12% radiochemical yield) and, after chiral separation, 6-[{sup 18}F]fluoro-L-DOPA (e.e. 98%, 4-5% radiochemical yield). (author).

  7. Prostaglandin E1 and prostaglandin F2 alpha in exudate in nickel allergy

    DEFF Research Database (Denmark)

    Lerche, A; Bisgaard, H; Kassis, V

    1989-01-01

    Ten nickel-allergic patients and 5 healthy control subjects participated in a study of the kinetics of the flux and concentration of migrated leukocytes and extracellular PGE1 and PGF2 alpha during a 48 h period, using a skin chamber technique. The patients were provided with two skin chambers, one...... with and one without nickel challenge. A higher flux of leukocytes, PGE1 and PGF2 alpha was observed during the second day of allergen exposure, while the concentrations probably due to dilution were unchanged or diminished, indicating an unspecific role of the prostaglandins during the contact allergic...

  8. Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles

    Science.gov (United States)

    2012-01-01

    Background Little is known about the cellular effects of exposure to mixed beams of high and low linear energy transfer radiation. So far, the effects of combined exposures have mainly been assessed with clonogenic survival or cytogenetic methods, and the results are contradictory. The gamma-H2AX assay has up to now not been applied in this context, and it is a promising tool for investigating the early cellular response to mixed beam irradiation. Purpose To determine the dose response and repair kinetics of gamma-H2AX ionizing radiation-induced foci in VH10 human fibroblasts exposed to mixed beams of 241Am alpha particles and X-rays. Results VH10 human fibroblasts were irradiated with each radiation type individually or both in combination at 37°C. Foci were scored for repair kinetics 0.5, 1, 3 and 24 h after irradiation (one dose per irradiation type), and for dose response at the 1 h time point. The dose response effect of mixed beam was additive, and the relative biological effectiveness for alpha particles (as compared to X-rays) was of 0.76 ± 0.52 for the total number of foci, and 2.54 ± 1.11 for large foci. The repair kinetics for total number of foci in cells exposed to mixed beam irradiation was intermediate to that of cells exposed to alpha particles and X-rays. However, for mixed beam-irradiated cells the frequency and area of large foci were initially lower than predicted and increased during the first 3 hours of repair (while the predicted number and area did not). Conclusions The repair kinetics of large foci after mixed beam exposure was significantly different from predicted based on the effect of the single dose components. The formation of large foci was delayed and they did not reach their maximum area until 1 h after irradiation. We hypothesize that the presence of low X-ray-induced damage engages the DNA repair machinery leading to a delayed DNA damage response to the more complex DNA damage induced by alpha particles. PMID:23121736

  9. Mapping of the serotonin 5-HT{sub 1D{alpha}} autoreceptor gene (HTR1D) on chromosome 1 using a silent polymorphism in the coding region

    Energy Technology Data Exchange (ETDEWEB)

    Ozaki, N.; Lappalainen, J.; Linnoila, M. [National Institute on Alcohol Abuse and Alcoholism, Rockville, MD (United States)] [and others

    1995-04-24

    Serotonin (5-HT){sub ID} receptors are 5-HT release-regulating autoreceptors in the human brain. Abnormalities in brain 5-HT function have been hypothesized in the pathophysiology of various psychiatric disorders, including obsessive-compulsive disorder, autism, mood disorders, eating disorders, impulsive violent behavior, and alcoholism. Thus, mutations occurring in 5-HT autoreceptors may cause or increase the vulnerability to any of these conditions. 5-HT{sub 1D{alpha}} and 5-HT{sub 1D{Beta}} subtypes have been previously localized to chromosomes 1p36.3-p34.3 and 6q13, respectively, using rodent-human hybrids and in situ localization. In this communication, we report the detection of a 5-HT{sub 1D{alpha}} receptor gene polymorphism by single strand conformation polymorphism (SSCP) analysis of the coding sequence. The polymorphism was used for fine scale linkage mapping of 5-HT{sub 1D{alpha}} on chromosome 1. This polymorphism should also be useful for linkage studies in populations and in families. Our analysis also demonstrates that functionally significant coding sequence variants of the 5-HT{sub 1D{alpha}} are probably not abundant either among alcoholics or in the general population. 14 refs., 1 fig., 1 tab.

  10. CC1, a novel crenarchaeal DNA binding protein.

    Science.gov (United States)

    Luo, Xiao; Schwarz-Linek, Uli; Botting, Catherine H; Hensel, Reinhard; Siebers, Bettina; White, Malcolm F

    2007-01-01

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

  11. The L444P Gba1 mutation enhances alpha-synuclein induced loss of nigral dopaminergic neurons in mice

    Science.gov (United States)

    Migdalska-Richards, Anna; Wegrzynowicz, Michal; Rusconi, Raffaella; Deangeli, Giulio; Di Monte, Donato A; Spillantini, Maria G; Schapira, Anthony H V

    2017-01-01

    Abstract Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson’s disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson’s disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression. PMID:28969384

  12. The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

    DEFF Research Database (Denmark)

    Lafuente-Barquero, Juan; Luke-Glaser, Sarah; Graf, Marco

    2017-01-01

    of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids...

  13. Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

    Directory of Open Access Journals (Sweden)

    Kihyuck Kwak

    Full Text Available Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV L1 and L2 DNA vaccination.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8 or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2 vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type

  14. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    International Nuclear Information System (INIS)

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

    2005-01-01

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

  15. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    Science.gov (United States)

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  16. Development of a socketed foundation for the Midwest Weak Post (MWP) v1.

    Science.gov (United States)

    2014-07-01

    A socketed foundation was designed and evaluated for use with the Midwest Weak Post (MWP), Version 1. : Dynamic component testing was conducted on five different design configurations with varying embedment : depths, steel reinforcement, and soil con...

  17. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE binds to alpha-actinin 1: novel pathways in skeletal muscle?

    Directory of Open Access Journals (Sweden)

    Shira Amsili

    Full Text Available BACKGROUND: Hereditary inclusion body myopathy (HIBM is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. METHODOLOGY/PRINCIPAL FINDINGS: We used a Surface Plasmon Resonance (SPR-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. CONCLUSIONS/SIGNIFICANCE: The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM.

  18. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

    International Nuclear Information System (INIS)

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

  19. THE LYMAN ALPHA REFERENCE SAMPLE: EXTENDED LYMAN ALPHA HALOS PRODUCED AT LOW DUST CONTENT

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, Matthew [Universite de Toulouse, UPS-OMP, IRAP, Toulouse (France); Oestlin, Goeran; Duval, Florent; Guaita, Lucia; Melinder, Jens; Sandberg, Andreas [Department of Astronomy, Oskar Klein Centre, Stockholm University, AlbaNova University Centre, SE-106 91 Stockholm (Sweden); Schaerer, Daniel [CNRS, IRAP, 14, avenue Edouard Belin, F-31400 Toulouse (France); Verhamme, Anne; Orlitova, Ivana [Geneva Observatory, University of Geneva, 51 Chemin des Maillettes, CH-1290 Versoix (Switzerland); Mas-Hesse, J. Miguel; Oti-Floranes, Hector [Centro de Astrobiologia (CSIC-INTA), Departamento de Astrofisica, POB 78, 28691 Villanueva de la Canada (Spain); Adamo, Angela [Max Planck Institute for Astronomy, Koenigstuhl 17, D-69117 Heidelberg (Germany); Atek, Hakim [Laboratoire d' Astrophysique, Ecole Polytechnique Federale de Lausanne (EPFL), Observatoire, CH-1290 Sauverny (Switzerland); Cannon, John M. [Department of Physics and Astronomy, Macalester College, 1600 Grand Avenue, Saint Paul, MN 55105 (United States); Herenz, E. Christian [Leibniz-Institut fuer Astrophysik (AIP), An der Sternwarte 16, D-14482 Potsdam (Germany); Kunth, Daniel [Institut d' Astrophysique de Paris, UMR 7095 CNRS and UPMC, 98 bis Bd Arago, F-75014 Paris (France); Laursen, Peter, E-mail: matthew@astro.su.se [Dark Cosmology Centre, Niels Bohr Institute, University of Copenhagen, Juliane Maries Vej 30, DK-2100 Copenhagen (Denmark)

    2013-03-10

    We report on new imaging observations of the Lyman alpha emission line (Ly{alpha}), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 < z < 0.18 in continuum-subtracted Ly{alpha}, H{alpha}, and the far ultraviolet continuum. We show that Ly{alpha} is emitted on scales that systematically exceed those of the massive stellar population and recombination nebulae: as measured by the Petrosian 20% radius, R{sub P20}, Ly{alpha} radii are larger than those of H{alpha} by factors ranging from 1 to 3.6, with an average of 2.4. The average ratio of Ly{alpha}-to-FUV radii is 2.9. This suggests that much of the Ly{alpha} light is pushed to large radii by resonance scattering. Defining the Relative Petrosian Extension of Ly{alpha} compared to H{alpha}, {xi}{sub Ly{alpha}} = R {sup Ly{alpha}}{sub P20}/R {sup H{alpha}}{sub P20}, we find {xi}{sub Ly{alpha}} to be uncorrelated with total Ly{alpha} luminosity. However, {xi}{sub Ly{alpha}} is strongly correlated with quantities that scale with dust content, in the sense that a low dust abundance is a necessary requirement (although not the only one) in order to spread Ly{alpha} photons throughout the interstellar medium and drive a large extended Ly{alpha} halo.

  20. sup. alpha. N-acetyl derivatives of. beta. -endorphin-(1-31) and -(1-27) regulate the supraspinal antinociceptive activity of different opioids in mice

    Energy Technology Data Exchange (ETDEWEB)

    Garzon, J.; Sanchez-Blazquez, P. (Cajal Institute, Madrid (Spain))

    1991-01-01

    {sup {alpha}}N-acetyl human {beta}-endorphin(1-31) injected icv to mice antagonized the analgesic activity of {beta}-endorphin-(1-31) and morphine whereas the analgesia evoked by DADLE and DAGO was enhanced by this treatment. The modulatory activity of {sup {alpha}}N-acetyl {beta}-endorphin-(1-31) was exhibited at remarkable low doses (fmols) reaching a maximum that persisted even though the dose was increased 100,000 times. The regulatory effect of a single dose of the acetylated neuropeptide lasted for 24h. The activity of {sup {alpha}}N-acetyl human {beta}-endorphin(1-31) was partially retained by the shorter peptide {sup {alpha}}N-acetyl human {beta}-endorphin-(1-27) and to a lesser extent by {beta}-endorphin-(1-27), {beta}-endorphin-(1-31) lacked this regulatory activity on opioid analgesia. Acetylated {beta}-endorphin-(1-31) displayed a biphasic curve when competing with 5 pM ({sup 125}I)-Tyr{sup 27} human {beta}-endorphin-(1-31) specific binding, the first step was abolished with an apparent IC{sub 50} of 0.35 nM, and the rest with an IC{sub 50} of 200 nM. It is suggested that {sup {alpha}}N-acetyl {beta}-endorphin-(1-31) changed the efficiency of the opioid analgesics by acting upon a specific substrate that is functionally coupled to the opioid receptor, presumably the guanine nucleotide binding regulatory proteins G{sub i}/G{sub 0}.

  1. Bucket Foundation Response Under Various Displacement Rates

    DEFF Research Database (Denmark)

    Vaitkunaite, Evelina; Nielsen, Benjaminn Nordahl; Ibsen, Lars Bo

    2016-01-01

    in a multi-bucket foundation system. The foundation model is at a scale of approximately 1:20 prototype foundation size. The tests are performed in a pressure tank with the foundation model installed in dense sand. Based on the data, the conclusion is that the bucket foundation design in a storm case should......The present testing program aims at showing the pore pressure response around a bucket foundation skirt as well as the load and displacement change due to ten different displacement rates. Research findings are useful for a numerical model calibration focusing on the design of the upwind foundation...

  2. Specificity of DNA repair in plants exposed at low dose-rate

    International Nuclear Information System (INIS)

    Semov, A.B.; Ptitsina, S.N.; Shevchenko, V.A.

    1997-01-01

    Intensity of gamma-ray induced unscheduled DNA synthesis (UDS) as well as yield and repair of single-strand DNA breaks (SSB) were investigated in control and exposed higher plant populations. Populations of V. cracca have been chronically irradiating by 90 Sr-beta-particles due to Kyshtym accident (South Ural) or have been growing on the uranium-miner tails (alpha-irradiation). In former case increased radioresistance was revealed (the phenomenon previously called radio-adaptation and that probably has something in common with adaptive response). This radioresistance correlates with higher intensity of UDS. On the basis of experiments with specific inhibitors of alpha- and beta- like DNA polymerases (aphidicolin, di-deoxy-thymidine) and protein synthesis (cycloheximide) it was assumed that the enhanced UDS in radioresistant population is an partially inducible process in which both DNA polymerases take part. In control population UDS is not inducible and totally inhibited by ddT. Differences in induction and repair of gamma-ray induced SSB between control and radioresistant populations were not registered. In case of chronic alpha-irradiation increased radiosensitivity and slightly decreased UDS were found. In this population and in some populations from Chernobyl vicinity, analyzed in 1986-1991, higher yield of SSB was registered but repair of SSB was not differ from control ones. (authors)

  3. Sequestering HMGB1 via DNA-Conjugated Beads Ameliorates Murine Colitis

    Science.gov (United States)

    Antoine, Daniel J.; Dancho, Meghan; Tsaava, Teá; Li, Jianhua; Lu, Ben; Levine, Yaakov A.; Stiegler, Andrew; Tamari, Yehuda; Al-Abed, Yousef; Roth, Jesse; Tracey, Kevin J.; Yang, Huan

    2014-01-01

    Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract that affects millions of people worldwide. Although the etiology of IBD is not clear, it is known that products from stressed cells and enteric microbes promote intestinal inflammation. High mobility group box 1 (HMGB1), originally identified as a nuclear DNA binding protein, is a cytokine-like protein mediator implicated in infection, sterile injury, autoimmune disease, and IBD. Elevated levels of HMGB1 have been detected in inflamed human intestinal tissues and in feces of IBD patients and mouse models of colitis. Neutralizing HMGB1 activity by administration of anti-HMGB1 antibodies or HMGB1-specific antagonist improves clinical outcomes in animal models of colitis. Since HMGB1 binds to DNA with high affinity, here we developed a novel strategy to sequester HMGB1 using DNA immobilized on sepharose beads. Screening of DNA-bead constructs revealed that B2 beads, one linear form of DNA conjugated beads, bind HMGB1 with high affinity, capture HMGB1 ex vivo from endotoxin-stimulated RAW 264.7 cell supernatant and from feces of mice with colitis. Oral administration of B2 DNA beads significantly improved body weight, reduced colon injury, and suppressed colonic and circulating cytokine levels in mice with spontaneous colitis (IL-10 knockout) and with dextran sulfate sodium-induced colitis. Thus, DNA beads reduce inflammation by sequestering HMGB1 and may have therapeutic potential for the treatment of IBD. PMID:25127031

  4. Are Ichthyosporea animals or fungi? Bayesian phylogenetic analysis of elongation factor 1alpha of Ichthyophonus irregularis.

    Science.gov (United States)

    Ragan, Mark A; Murphy, Colleen A; Rand, Thomas G

    2003-12-01

    Ichthyosporea is a recently recognized group of morphologically simple eukaryotes, many of which cause disease in aquatic organisms. Ribosomal RNA sequence analyses place Ichthyosporea near the divergence of the animal and fungal lineages, but do not allow resolution of its exact phylogenetic position. Some of the best evidence for a specific grouping of animals and fungi (Opisthokonta) has come from elongation factor 1alpha, not only phylogenetic analysis of sequences but also the presence or absence of short insertions and deletions. We sequenced the EF-1alpha gene from the ichthyosporean parasite Ichthyophonus irregularis and determined its phylogenetic position using neighbor-joining, parsimony and Bayesian methods. We also sequenced EF-1alpha genes from four chytrids to provide broader representation within fungi. Sequence analyses and the presence of a characteristic 12 amino acid insertion strongly indicate that I. irregularis is a member of Opisthokonta, but do not resolve whether I. irregularis is a specific relative of animals or of fungi. However, the EF-1alpha of I. irregularis exhibits a two amino acid deletion heretofore reported only among fungi.

  5. Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

    Science.gov (United States)

    Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni

    2005-10-01

    We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

  6. Life cycle assessment of the offshore wind farm alpha ventus

    International Nuclear Information System (INIS)

    Wagner, Hermann-Josef; Baack, Christoph; Eickelkamp, Timo; Epe, Alexa; Lohmann, Jessica; Troy, Stefanie

    2011-01-01

    Due to better wind conditions at sea, offshore wind farms have the advantage of higher electricity production compared to onshore and inland wind farms. In contrast, a greater material input, leading to increased energy consumptions and emissions during the production phase, is required to build offshore wind farms. These contrary effects are investigated for the first German offshore wind farm alpha ventus in the North Sea. In a life cycle assessment its environmental influence is compared to that of Germany's electricity mix. In comparison to the mix, alpha ventus had better indicators in nearly every investigated impact category. One kilowatt-hour electricity, generated by the wind farm, was burdened with 0.137 kWh Primary Energy-Equivalent and 32 g CO 2 -Equivalent, which represented only a small proportion of the accordant values for the mix. Furthermore, the offshore foundations as well as the submarine cable were the main energy intensive components. The energetic and greenhouse gas payback period was less than one year. Therefore, offshore wind power, even in deep water, is compatible with the switch to sustainable electricity production relying on renewable energies. Additional research, taking backup power plants as well as increasingly required energy storage systems into account, will allow further calculation. -- Highlights: → Offshore wind energy represents an environmentally friendly way of power generation. → The offshore foundations and the submarine cable are energy intensive components. → Alpha ventus emits 30 g CO 2 per kWh electricity over the entire life cycle. → Less specific emissions occur in comparison to the existing German electricity grid. → The energetic and greenhouse gas payback periods are less than one year.

  7. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    Science.gov (United States)

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  8. Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors.

    Science.gov (United States)

    Dreiling, Michelle; Bischoff, Sabine; Schiffner, Rene; Rupprecht, Sven; Kiehntopf, Michael; Schubert, Harald; Witte, Otto W; Nathanielsz, Peter W; Schwab, Matthias; Rakers, Florian

    2016-09-01

    Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.

  9. 17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

    Science.gov (United States)

    Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

    2010-03-01

    The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

  10. Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

    Directory of Open Access Journals (Sweden)

    Eva Polanská

    Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

  11. Synthesis and evaluation of phytotoxic activity of {alpha}-Santonin derivatives; Sintese e avaliacao da atividade fitotoxica de derivados da {alpha}-Santonina

    Energy Technology Data Exchange (ETDEWEB)

    Alvarenga, Elson S.; Barbosa, Luiz C.A.; Saliba, William A.; Arantes, Francisco F.P.; Demuner, Antonio J. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Quimica]. E-mail: elson@ufv.br; Silva, Antonio A. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Fitotecnia

    2009-07-01

    Mixtures of {alpha}-Santonin and various solvents were irradiated by either high or low pressure mercury lamps. The photochemical reactions afforded lumisantonin (11) (76% in acetonitrile), (3 S,3a S,9{beta}S)-3,6,6-trimethyl-3,3a,4,5-tetrahydronafto[1,2-b]furan-2,7({eta}6,9{beta}{eta}) dione (12) (100% in acetonitrile), 10{alpha}-acetoxy-3-oxo-1,7{alpha}H{eta},6,11{alpha}a{eta}-guaia-4-en-6,12-oli= de (8) (26% in acetic acid), 10{alpha}-hydroxy-3-oxo-1,7{alpha}a{eta},6,11{alpha}{eta}-guaia-4-en-6,12-olid= e (10) (32%) and (E)-3-((3 S,3a S,7{alpha}S)-3-methyl-2-oxo-6-(propan-2-ylidene)hexahydrobenzofuran- 7 - (7{alpha}{eta})-ylidene)propanoic acid (9) (44%) (in water/ acetic acid 1:1, v/v). Lactone 12 was also prepared by irradiation of lumisantonin in diethyl ether. Lactones 8 and 10 were converted, respectively, into the 10 {alpha}-acetoxy-3{alpha}-hydroxy-1,7{alpha}H,6,11{alpha}H-guaia-4-en-6,12-olid= e (13) (87%) and 3a,10a-dihydroxy-1,7{alpha}H,6,11{alpha}H-guaia-4-en-6,12-olide (14) (75%) by sodium borohydride reduction. The effects of the compounds on the development of radicle of Sorghum bicolor and Cucumis sativus were evaluated. (author)

  12. Chl1 DNA helicase regulates Scc2 deposition specifically during DNA-replication in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Soumya Rudra

    Full Text Available The conserved family of cohesin proteins that mediate sister chromatid cohesion requires Scc2, Scc4 for chromatin-association and Eco1/Ctf7 for conversion to a tethering competent state. A popular model, based on the notion that cohesins form huge ring-like structures, is that Scc2, Scc4 function is essential only during G1 such that sister chromatid cohesion results simply from DNA replisome passage through pre-loaded cohesin rings. In such a scenario, cohesin deposition during G1 is temporally uncoupled from Eco1-dependent establishment reactions that occur during S-phase. Chl1 DNA helicase (homolog of human ChlR1/DDX11 and BACH1/BRIP1/FANCJ helicases implicated in Fanconi anemia, breast and ovarian cancer and Warsaw Breakage Syndrome plays a critical role in sister chromatid cohesion, however, the mechanism through which Chl1 promotes cohesion remains poorly understood. Here, we report that Chl1 promotes Scc2 loading unto DNA such that both Scc2 and cohesin enrichment to chromatin are defective in chl1 mutant cells. The results further show that both Chl1 expression and chromatin-recruitment are tightly regulated through the cell cycle, peaking during S-phase. Importantly, kinetic ChIP studies reveals that Chl1 is required for Scc2 chromatin-association specifically during S-phase, but not during G1. Despite normal chromatin enrichment of both Scc2 and cohesin during G1, chl1 mutant cells exhibit severe chromosome segregation and cohesion defects--revealing that G1-loaded cohesins is insufficient to promote cohesion. Based on these findings, we propose a new model wherein S-phase cohesin loading occurs during DNA replication and in concert with both cohesion establishment and chromatin assembly reactions--challenging the notion that DNA replication fork navigates through or around pre-loaded cohesin rings.

  13. Antagonizing the alpha(4)beta(1) Integrin, but Not alpha(4)beta(7), Inhibits Leukocytic Infiltration of the Central Nervous System in Rhesus Monkey Experimental Autoimmune Encephalomyelitis

    NARCIS (Netherlands)

    Haanstra, Krista G.; Hofman, Sam O.; Estevao, Dave M. Lopes; Blezer, Erwin L. A.; Bauer, Jan; Yang, Li-Li; Wyant, Tim; Csizmadia, Vilmos; 't Hart, Bert A.; Fedyk, Eric R.

    2013-01-01

    The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the alpha(4) integrins expressed by T lymphocytes. The alpha(4)beta(1) integrin mediates migration of memory T lymphocytes into the

  14. SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

    International Nuclear Information System (INIS)

    Campos, D; Peeters, W; Nickel, K; Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M

    2015-01-01

    Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response

  15. SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

    Energy Technology Data Exchange (ETDEWEB)

    Campos, D [University of Wisconsin Madison, Madison, WI (United States); Peeters, W [Radboud University Medical Center, Nijmegen, GA (United States); Nickel, K [University of Wisconsin, Madison, WI (United States); Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M [University of Wisconsin, Madison, Wisconsin (United States)

    2015-06-15

    Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

  16. Mapping and understanding the diversity of insects in the tropics: past achievements and future directions

    Czech Academy of Sciences Publication Activity Database

    Novotný, Vojtěch; Miller, S. E.

    2014-01-01

    Roč. 53, č. 3 (2014), s. 259-267 ISSN 2052-1758 R&D Projects: GA ČR GA13-10486S Grant - others:European Social Fund(CZ) CZ.1.07/2.3.00/20.0064; US National Science Foundation(US) DEB 0515678 Institutional support: RVO:60077344 Keywords : alpha diversity * beta diversity * DNA barcording Subject RIV: EH - Ecology, Behaviour http://onlinelibrary.wiley.com/doi/10.1111/aen.12111/pdf

  17. Synthesis and evaluation of alpha-[[(2-haloethyl)amino]methyl]-2- nitro-1H-imidazole-1-ethanols as prodrugs of alpha-[(1-aziridinyl)methyl]-2- nitro-1H-imidazole-1-ethanol (RSU-1069) and its analogues which are radiosensitizers and bioreductively activated cytotoxins

    International Nuclear Information System (INIS)

    Jenkins, T.C.; Naylor, M.A.; O'Neill, P.; Threadgill, M.D.; Cole, S.; Stratford, I.J.; Adams, G.E.; Fielden, E.M.; Suto, M.J.; Stier, M.A.

    1990-01-01

    alpha-[(1-Aziridinyl)methyl]-2-nitro-1H-imidazole-1-ethanols, of general formula ImCH2CH(OH)CH2NCR1R2CR3R4, where Im = 2-nitroimidazole and R1, R2, R3, R4 = H, Me, are radiosensitizers and selective bioreductively activated cytotoxins toward hypoxic tumor cells in vitro and in vivo. Treatment of the aziridines with hydrogen halide in acetone or aqueous acetone gave the corresponding 2-haloethylamines of general formula ImCH2CH(OH)CH2(+)-NH2CR1R2CR3R4X X-, where R1, R2, R3, R4 = H, Me, and X = F, Cl, Br, I. These 2-haloethylamines were evaluated as prodrugs of the parent aziridines. The rates of ring closure in aqueous solution at pH approximately 6 were found to increase with increasing methyl substitution and to depend on the nature of the leaving group (I approximately Br greater than Cl much greater than F). A competing reaction of ImCH2CH(OH)CH2+NH2CH2CH2X X- (X = Cl, Br) with aqueous HCO3- ions gives 3-[2-hyroxy-3-(2-nitro-1H-imidazol-1-yl)propyl]-2-oxazolidinone. The activities of these prodrugs as radiosensitizers or as bioreductively activated cytotoxins were consistent with the proportion converted to the parent aziridine during the course of the experiment. alpha-[[(2-Bromoethyl)amino]methyl]-2-nitro-1H-imidazole-1- ethanol (RB 6145, 10), the prodrug of alpha-[(1-aziridinyl)methyl]-2-nitro-1H-imidazole-1-ethanol (RSU-1069, 3), is identified as the most useful compound in terms of biological activity and rate of ring closure under physiological conditions

  18. Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

    Science.gov (United States)

    Vyhlidal, C; Samudio, I; Kladde, M P; Safe, S

    2000-06-01

    17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

  19. Identification of a new defective SERPINA1 allele (PI*Zla palma) encoding an alpha-1-antitrypsin with altered glycosylation pattern.

    Science.gov (United States)

    Hernández-Pérez, José M; Ramos-Díaz, Ruth; Pérez, José A

    2017-10-01

    Alpha-1-antitrypsin (AAT) deficiency is a genetic condition that arises from mutations in the SERPINA1 gene and predisposes to develop pulmonary emphysema and, less frequently, liver disease. Occasionally, new defective SERPINA1 alleles are detected as an outcome of targeted-screening programs or case-findings. This study began with a female patient showing bronchial hyperreactivity. Serum level and phenotype for AAT was analysed by immunonephelometry and isoelectric focusing electrophoresis. The SERPINA1 gene of the proband was genotyped by PCR amplification and DNA sequencing. Analysis of AAT deficiency was extended to the proband's family. An abnormal AAT variant that migrated to a more cathodal position than PiZ AAT was detected in the proband's serum. Genetic analysis demonstrated that proband is heterozygous for a new defective SERPINA1 allele (PI*Z la palma ) characterized by the c.321C > A (p.Asn83Lys) mutation in the M1Val213 background. This mutation abolishes the N-glycosylation site in position 83 of the mature AAT. Eight relatives of the proband are carriers of the PI*Z la palma allele and four of them have shown symptoms of bronchial asthma or bronchial hyperreactivity. The mean α1AT level in the serum of PI*MZ la palma individuals was 87.1 mg/dl. The reduction in circulating AAT levels associated to the PI*Z la palma allele was similar to that of PI*Z allele, representing a risk of impairment in lung function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Anti-interleukin-1 alpha autoantibodies in humans: Characterization, isotype distribution, and receptor-binding inhibition--higher frequency in Schnitzler's syndrome (urticaria and macroglobulinemia)

    International Nuclear Information System (INIS)

    Saurat, J.H.; Schifferli, J.; Steiger, G.; Dayer, J.M.; Didierjean, L.

    1991-01-01

    Since autoantibodies (Abs) to cytokines may modify their biologic activities, high-affinity binding factors for interleukin-1 alpha (IL-1 alpha BF) were characterized in human sera. IL-1 alpha BF was identified as IgG (1) by sucrose density-gradient centrifugation followed by immunodiffusion autoradiography, (2) by ligand-blotting method, (3) by ligand binding to affinity-immobilized serum IgG, and (4) by IgG affinity purification followed by sucrose density-gradient centrifugation. IL-1 alpha binding activity resided in the F(ab)2 fragment. The apparent equilibrium constant was in the range of IgG found after immunization with conventional antigens (i.e., 10(-9) to 10(-10) mol/L). Anti-IL-1 alpha IgG auto-Abs represented only an extremely small fraction of total IgG (less than 1/10(-5)). Some sera with IL-1 alpha BF and purified IgG thereof were able to inhibit by 96% to 98% the binding of human recombinant IL-1 alpha to its receptor on murine thymoma EL4-6.1 cells, whereas other sera did not. When 125I-labeled anti-IL-1 alpha IgG complexes were injected into rats, they prolonged the plasma half-life of 125I-labeled IL-1 alpha several fold and altered its tissue distribution. The predominant class was IgG (12/19), mainly IgG4 (9/19), but in five of the sera, anti-IL-1 alpha IgA was also detected. In a screening of 271 sera, IL-1 alpha BF was detected in 17/98 normal subjects and was not more frequent in several control groups of patients, except in patients with Schnitzler's syndrome (fever, chronic urticaria, bone pain, and monoclonal IgM paraprotein) (6/9; p less than 0.005). The pathologic significance of these auto-Abs remains to be determined