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Sample records for acute wild-type canine

  1. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

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    Cui Shang-jin

    2010-05-01

    Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

  2. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

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    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  3. Experimental adaptation of wild-type canine distemper virus (CDV) to the human entry receptor CD150.

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    Bieringer, Maria; Han, Jung Woo; Kendl, Sabine; Khosravi, Mojtaba; Plattet, Philippe; Schneider-Schaulies, Jürgen

    2013-01-01

    Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red) adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2) pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5) pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

  4. Experimental adaptation of wild-type canine distemper virus (CDV to the human entry receptor CD150.

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    Maria Bieringer

    Full Text Available Canine distemper virus (CDV, a close relative of measles virus (MV, is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2 pfu/ml in Vero cells expressing human CD150 (Vero-hSLAM. After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5 pfu/ml. Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L and Gly to Glu (G71E, and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

  5. Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients

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    Mello Francisco C A

    2012-07-01

    Full Text Available Abstract Background Lamivudine (LAM is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods. Results Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012. As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20 had minor subpopulations (4-17% of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies. Conclusions YMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD

  6. Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia.

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    Serrano-López, Juana; Serrano, Josefina; Figueroa, Vianihuini; Torres-Gomez, Antonio; Tabares, Salvador; Casaño, Javier; Fernandez-Escalada, Noemi; Sánchez-Garcia, Joaquín

    2013-12-01

    Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia.

  7. Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

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    Christian Lehmann

    2016-06-01

    Full Text Available Abstract Background Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML. In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. Methods The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Results Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that

  8. Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR.

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    Gao, Keliang; Huang, Xiaomeng; Chiang, Chi-Ling; Wang, Xinmei; Chang, Lingqian; Boukany, Pouyan; Marcucci, Guido; Lee, Robert; Lee, Ly James

    2016-05-01

    Nanochannel electroporation (NEP) was applied to deliver precise dosages of myeloid cell leukemia-1 (Mcl-1)-specific siRNA and molecular beacons to two types of acute myeloid leukemia (AML) cells, FMS-like tyrosine kinase-3 wild-type (WT) and internal tandem duplications (ITD) type at the single-cell level. NEP, together with single-cell quantitative reverse transcription PCR, led to an observation showing nearly 20-folds more Mcl-1 siRNA than MCL1 mRNA were required to induce cell death for both cell lines and patient blasts, i.e., ~8,800 siRNAs for ~500 ± 50 mRNAs in ITD cells and ~6,000 siRNAs for ~300 ± 50 mRNAs in WT cells. A time-lapse study revealed that >75% MCL1 mRNA was downregulated within 1 hour after delivery of a small amount of siRNA. However, additional siRNA was required to inhibit the newly transcribed mRNA for >12 hours until the cell lost its ability of self-protection recovery. A multidelivery strategy of low doses and short delivery interval, which require 77% less siRNA and has the potential of lower side effects and clinical cost, was as effective as a single high-dose siRNA delivery. Our method provides a viable analytical tool to investigate gene silencing at the single-cell level for oligonucleotide-based therapy.

  9. Double CEBPA mutations are prognostically favorable in non-M3 acute myeloid leukemia patients with wild-type NPM1 and FLT3-ITD.

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    Wen, Xiang-Mei; Lin, Jiang; Yang, Jing; Yao, Dong-Ming; Deng, Zhao-Qun; Tang, Chun-Yan; Xiao, Gao-Fei; Yang, Lei; Ma, Ji-Chun; Hu, Jia-Bo; Qian, Wei; Qian, Jun

    2014-01-01

    This study is aimed to investigate the pattern of CEBPA mutations and its clinical significance in Chinese non-M3 acute myeloid leukemia (AML) patients. The entire coding region of CEBPA gene was amplified by PCR and then sequenced in samples from 233 non-M3 AML patients. Fifty mutations were identified in 37 (15.8%) patients with eleven (4.7%) double mutated CEBPA (dmCEBPA) and twenty-six (11.1%) single mutated CEBPA (smCEBPA). dmCEBPA was exclusively observed in M1 and M2 subtypes of FAB classification (P = 0.008), whereas smCEBPA occurred in almost all subtypes (P = 0.401). Patients with dmCEBPA had significantly younger age and higher WBC counts than those with wtCEBPA (P = 0.016 and 0.043, respectively). Both dmCEBPA and smCEBPA were mainly present in cytogenetically normal patients. Patients with dmCEBPA achieved higher rate of complete (CR) than wtCEBPA patients (88% vs. 51%, P = 0.037), whereas smCEBPA and wtCEBPA groups are similar (47% vs. 51%, P = 0.810). Patients with dmCEBPA had a superior overall survival (OS) compared with patients with wtCEBPA (P = 0.033), whereas patients with smCEBPA had a similar OS as patients with wtCEBPA (P = 0.976). dmCEBPA but not smCEBPA was also associated with favorable outcome in patients with wild-type NPM1 and FLT3-ITD (NPM1(wt)FLT3-ITD(wt) ). Our data confirm that dmCEBPA but not smCEBPA is prognostically favorable in NPM1(wt)FLT3-ITD(wt) AML, and suggest that the entity AML with mutated CEBPA should be definitely designated as AML with dmCEBPA in WHO classification and smCEBPA should be excluded from the favorable risk of molecular abnormalities.

  10. A canine model of normovolaemic acute anaemia

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    T.C. Spotswood

    2005-09-01

    Full Text Available The objective was to develop a non-terminal, acute normovolaemic anaemia model in dogs that has minimal effects on patient well-being. Eleven normal Beagle dogs were used. About 20 % of the circulating blood volume was removed from the jugular vein 1-3 times per day over a 3-4 day period until a haematocrit (Ht of 13-17 % was obtained. Normovolaemia was maintained by replacing the volume deficit of the red blood cells with Ringer's lactate and re-infusing the plasma. Full blood count and Ht were monitored twice daily. The 13-17 % Ht was reached within 3-4 days with the number of phlebotomies ranging from four to seven. The model was primarily developed to determine echocardiographic values as well as Doppler abdominal splanchnic blood flow parameters in anaemic dogs as part of a study that will compare these results to similar studies in babesiosis-induced anaemia. The model may also be useful in the evaluation of the pathophysiology of anaemia in dogs or as a model for anaemia in humans.

  11. Acute pancreatitis : a newly recognised potential complication of canine babesiosis

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    A.J. Möhr

    2000-07-01

    Full Text Available This retrospective study describes 4 cases of canine babesiosis with histologically confirmed acute pancreatitis. In addition, 16 dogs with babesiosis are reported with serum amylase (>3500 U/l and/or lipase (>650 U/l activity elevations of a magnitude that would support a diagnosis of probable acute pancreatitis, although extra-pancreatic sources of the enzymes could not be excluded in these cases. Median time of pancreatitis diagnosis was 2.5 days post-admission, with primarily young (median age 3 years, sexually intact dogs affected. The development of pancreatitis was unrelated to the degree of anaemia at time of admission. In addition to pancreatitis, 80 % of cases suffered from other babesial complications, namely icterus (13, acute respiratory distress syndrome (6, immune-mediated haemolytic anaemia (6, renal failure (3, haemoconcentration (2 and cerebral syndrome (2. Acute respiratory distress syndrome, renal failure and cerebral syndrome were associated with a poor prognosis, with 4 of the 5 dogs included in the overall 26 % mortality rate having at least 1 of these complications. Haemolytic anaemia with ischaemia-reperfusion injury to the pancreas is proposed as a possible primary pathophysiological mechanism in babesial pancreatitis. Hypotensive shock, immune-mediated haemolytic anaemia, haemoconcentration and possibly altered lipid metabolism in babesiosis may also be involved. The previously postulated pro-inflammatory cytokine milieu of complicated babesiosis may underlie the progression, if not the primary initiation, of pancreatic pathology. Acute pancreatitis may represent the previously reported 'gut' form of babesiosis.

  12. Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice

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    Kasprzak, Kazimierz S. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Diwan, Bhalchandra A. [Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Kaczmarek, Monika Z. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Logsdon, Daniel L. [Laboratory Animal Sciences Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Fivash, Mathew J. [Data Management Services, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Salnikow, Konstantin, E-mail: salnikok@mail.nih.gov [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States)

    2011-11-15

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono- < gamma > -lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni{sub 3}S{sub 2}), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni{sub 3}S{sub 2} carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni{sub 3}S{sub 2}. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni{sub 3}S{sub 2} in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni{sub 3}S{sub 2} carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni{sub 3}S{sub 2} and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies. -- Highlights: Black-Right-Pointing-Pointer Ascorbate depletion enhances carcinogenicity and acute toxicity of nickel. Black-Right-Pointing-Pointer Gulo-/- mice unable to synthesize ascorbate were used in this study. Black

  13. PLZF-RAR[alpha] fusion proteins generated from the variant t(11; 17)(q23; q21) translocation in acute promyelocytic leukemia inhibit ligand-dependent transactivation of wild-type retinoic acid receptors

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    Chen, Zhu; Chen, Sai-Juan; Wang, Zhen-Yi (Shanghai Second Medical Univ. (China)); Guidez, F.; Rousselot, P.; Agadir, A.; Degos, L.; Chomienne, C. (Laboratoire de Biologie Cellulaire Hematopoietique, Paris (France)); Zelent, A. (Institute for Cancer Research, London (United Kingdom)); Waxman, S. (Mount Sinai Medical Center, New York, NY (United States))

    1994-02-01

    Recently, the authors described a recurrent variant translocation, t(11;17)(q23;q21), in acute promyelocytic leukemia (APL) which juxtaposes PLZF, a gene encoding a zinc finger protein, to RARA, encoding retinoic acid receptor [alpha] (RAR[alpha]). They have now cloned cDNAs encoding PLZF-RAR[alpha] chimeric proteins and studied their transactivating activities. In transient-expression assays, both the PLZF(A)-RAR[alpha] and PLZF(B)-RAR[alpha] fusion proteins like the PML-RAR[alpha] protein resulting from the well-known t(15;17) translocation in APL, antagonized endogenous and transfected wild-type RAR[alpha] in the presence of retinoic acid. Cotransfection assays showed that a significant repression of RAR[alpha] transactivation activity was obtained even with a very low PLZF-RAR[alpha]-expressing plasmid concentration. A [open quotes]dominant negative[close quotes] effect was observed with vectors expressing RAR[alpha] and retinoid X receptor [alpha] (RXR[alpha]). These abnormal transactivation properties observed in retinoic acid-sensitive myeloid cells strongly implicate the PLZF-RAR[alpha] fusion proteins in the molecular pathogenesis of APL.

  14. Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen α1(I-promoter-GFP reporter mice.

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    Shengli Ding

    Full Text Available BACKGROUND: Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. METHODS: FVB-N wild type mice and C57BL6 procollagen α1(I-GFP reporter mice were given one (DSS1 or two (DSS2 cycles of 3% DSS (5 days/cycle followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3, and DSS2+7. Procollagen α1(I-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I mRNA. RESULTS: Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin(+ cells and much fewer α-SMA(+ cells. GFP mRNA strongly correlated with collagen α1(I mRNA. CONCLUSIONS: One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I-GFP transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic

  15. Uncommon acute neurologic presentation of canine distemper in 4 adult dogs.

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    Galán, Alba; Gamito, Araceli; Carletti, Beatrice E; Guisado, Alicia; de las Mulas, Juana Martín; Pérez, José; Martín, Eva M

    2014-04-01

    Four uncommon cases of canine distemper (CD) were diagnosed in vaccinated adult dogs. All dogs had acute onset of neurologic signs, including seizures, abnormal mentation, ataxia, and proprioceptive deficits. Polymerase chain reaction for CD virus was positive on cerebrospinal fluid in 2 cases. Due to rapid deterioration the dogs were euthanized and CD was confirmed by postmortem examination.

  16. Urine neutrophil gelatinase-associated lipocalin (NGAL as a biomarker for acute canine kidney injury

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    Lee Ya-Jane

    2012-12-01

    Full Text Available Abstract Background Biomarkers for the early prediction of canine acute kidney injury (AKI are clinically important. Recently, neutrophil gelatinase-associated lipocalin (NGAL was found to be a sensitive biomarker for the prediction of human AKI at a very early stage and the development of AKI after surgery. However, NGAL has not yet been studied with respect to dog kidney diseases. The application of NGAL canine AKI was investigated in this study. Results The canine NGAL gene was successfully cloned and expressed. Polyclonal antibodies against canine NGAL were generated and used to develop an ELISA for measuring NGAL protein in serum and urine samples that were collected from 39 dogs at different time points after surgery. AKI was defined by the standard method, namely a serum creatinine increase of greater than or equal to 26.5 μmol/L from baseline within 48 h. At 12 h after surgery, compared to the group without AKI (12 dogs, the NGAL level in the urine of seven dogs with AKI was significantly increased (median 178.4 pg/mL vs. 88.0 pg/mL, and this difference was sustained to 72 h. Conclusion As the increase in NGAL occurred much earlier than the increase in serum creatinine, urine NGAL seems to be able to serve as a sensitive and specific biomarker for the prediction of AKI in dogs.

  17. Peritoneal dialysis in acute renal failure in canines: A review

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    R. H. Bhatt

    Full Text Available Peritoneal dialysis is a technique whereby infusion of dialysis solution into the peritoneal cavity is followed by a variable dwell time and subsequent drainage. During peritoneal dialysis, solutes and fluids are exchanged between the capillary blood and the intraperitoneal fluid through a biologic membrane, the peritoneum. Inadequate renal function leads to disturbance in the removal of the extra fluid and waste products. It removes the waste product and extra fluid from the body in renal failure in small animal practice. Peritoneal dialysis is more accessible, more affordable and easier to administer to the small animal patient. The most common indication for peritoneal dialysis in dogs is acute renal failure (ARF. Peritoneal dialysis is an important therapeutic tool for mitigating clinical signs of uremia and giving the kidneys time to recover in cats with acute kidney injury when conventional therapy is no longer effective. [Vet. World 2011; 4(11.000: 517-521

  18. 甘肃犬瘟热病毒流行株N蛋白和H蛋白基因的序列分析%Sequence analysis of nucleoprotein and hemagglutinin genes of wild type canine distemper virus isolated from dogs in Gansu Province

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    王小辉; 王旭荣; 郭庆勇; 李建喜; 李世宏

    2011-01-01

    采用RT-PCR方法扩增了3株犬瘟热病毒(CDV)分离株的N、H蛋白基因,将其克隆入pGEMT Easy载体并进行序列分析.结果表明,获得的N蛋白基因序列与GenBank上登录的其他国家和地区的CDV分离株相应序列的同源性为92.5%~99.9%,获得的H蛋白基因与其他国家和地区的CDV分离株相应序列的同源性为81.7%~100%.由N蛋白基因和H蛋白基因推导的氨基酸序列构建的遗传进化树可知,这3株CDV流行株均处于野毒株的分支上,在N蛋白基因的遗传进化树上,CDV流行株呈现明显的地域性差异,而在H蛋白基因的遗传进化树上,不同国家和地区的CDV分离株之间虽有一定的地域联系,但与N蛋白基因相比,其地域性联系并不明显.说明这3株CDV流行株为野毒株,H蛋白基因的变异水平明显高于N蛋白基因,而N蛋白基因更能显示毒株与地域的关系.%The nucleoprotein(N) gene and hemagglutinin(H) gene were amplified from 3 strains of canine distemper virus(CDV) isolated from dogs in Gansu Province by RT-PCR. Subsequently, the nucleotides of these two genes were sequenced and analyzed by Clustal V of DNAstar. The N gene shared from 92.5 % to 99.9 % homology with those of the CDV strains available in GenBank. The H gene shared from 81.7% to 100% homology with those of the CDV strains from other regions. The phylogenetic tree based on the amino acid sequences deduced from the nucleotides sequences of the N gene indicated that the strains of CDV had remarkable difference and characteristics among different regions, but the phylogenetic tree based on the deduced H protein's amino acid sequences showed that the CDV strains were not different among the different regions. The above results showed that the 3 CDV strains from Gansu were classified as wild type. The mutations in H gene were more than that in N gene and N gene were able to display the relationship between CDV strain and region.

  19. Controversial results of therapy with mesenchymal stem cells in the acute phase of canine distemper disease.

    Science.gov (United States)

    Pinheiro, A O; Cardoso, M T; Vidane, A S; Casals, J B; Passarelli, D; Alencar, A L F; Sousa, R L M; Fantinato-Neto, P; Oliveira, V C; Lara, V M; Ambrósio, C E

    2016-05-23

    Distemper disease is an infectious disease reported in several species of domestic and wild carnivores. The high mortality rate of animals infected with canine distemper virus (CDV) treated with currently available therapies has driven the study of new efficacious treatments. Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic option for many degenerative, hereditary, and inflammatory diseases. Therefore, the aim of this study was to characterize stem cells derived from the canine fetal olfactory epithelium and to assess the systemic response of animals infected with CDV to symptomatic therapy and treatment with MSCs. Eight domestic mongrel dogs (N = 8) were divided into two groups: support group (SG) (N = 5) and support group + cell therapy (SGCT) (N = 3), which were monitored over 15 days. Blood samples were collected on days 0, 6, 9, 12, and 15 to assess blood count and serum biochemistry (urea, creatinine, alanine transferase, alkaline phosphatase, gamma-glutamyl transferase, total protein, albumin, and globulin), and urine samples were obtained on days 0 and 15 for urinary evaluation (urine I). The results showed a high mortality rate (SG = 4 and SGCT = 2), providing inadequate data on the clinical course of CDV infection. MSC therapy resulted in no significant improvement when administered during the acute phase of canine distemper disease, and a prevalence of animals with high mortality rate was found in both groups due to the severity of symptoms.

  20. Accumulation of xenotransplanted canine bone marrow cells in NOD/SCID/γc(null) mice with acute hepatitis induced by CCl4.

    Science.gov (United States)

    Kato, Takashi; Hisasue, Masaharu; Segawa, Kazuhito; Fujimoto, Ayumi; Makiishi, Eri; Neo, Sakurako; Yasuno, Kyohei; Kobayashi, Ryosuke; Tsuchiya, Ryo

    2013-07-31

    Bone marrow cell infusion (BMI) has recently been suggested as an effective therapy for refractory liver disease; however, the efficiency of BMI using canine bone marrow cells (cBMCs) has not been reported. We evaluated the accumulation potential of cBMCs in a mouse model of acute liver failure. Acute hepatitis was induced by carbon tetrachloride (CCl4) treatment in NOD/SCID/γc(null)(NOG) mice and wild-type (WT) C57BL mice, and the characteristics of liver dysfunction and the degree of hepatic injury and regeneration were compared between the two mouse models. Next, female CCl4-treated NOG mice were xenotransplanted with male PKH26-labeled cBMCs, and the potential of cBMCs to accumulate in injured liver tissue compartments was examined. Fluorescence microscopy was performed to histologically detect the infused cBMCs, and DNA polymerase chain reaction was performed for detection of the male Y chromosome (SRY gene) in the recipient female NOG mice. The number of PKH26-positive cBMCs transplanted in the liver tissue gradually increased in the NOG mice. The infused cBMCs were located in the necrotic area of the liver at an early stage after transplantation, and most had accumulated a week after transplantation. However, the therapeutic efficacy of the xenotransplantation remained unclear, because no significant differences were observed concerning the extent liver injury and regeneration between the cBMC-transplanted and saline control mice. These results suggest that cBMCs will specifically accumulate in injured liver tissue and that BMC transplantation may have the potential to repair liver deficiency.

  1. Effect of hepatocyte growth factor on left ventricular remodeling after acute myocardial infarction in canine

    Institute of Scientific and Technical Information of China (English)

    Ping LI; Tingshu YANG; Liling LIANG

    2006-01-01

    Background and objectives To investigate the effect of hepatocyte growth factor (HGF) on left ventricular (LV) remodeling after acute myocardial infarction (AMI). Methods AMI was produced by ligation of proximal left anterior descending coronary artery(LAD) in 12 mongrel canines. These animals were randomized into 2 groups. In HGF group (n=6), canines were injected with pcDNA3-HGF lml (about 300ug) at the margin of infarcted myocardium; in control group (n=6) canines were injected with equal volume of normal saline. Cardiac function and left ventricular remodeling were evaluated with echocardiography at 1, 4, 8 weeks after MI. LV myocardium specimens were obtained at 8 weeks and stained with hematoxylin and eosin for histological examination or with sirius red to assess the collagen content. Results Compared with control group, LVEF in HGF group was significantly higher at 4 weeks (49.61+6.66 vs 39.84+6.39; P<0.05) and at 8 weeks (51.57+8.53 vs 40.61+7.67; P<0.05) after AMI, while LVESV was significantly lower in HGF group than that in control group at 8 weeks after AMI (18.98+3.47 vs 25.66+5.86; P<0.05). Posterior left ventricular wall thickness decreased significantly from 1 wk to 8 wks after AMI in control group, while remained unchanged in HGF group. Compared with control group, histological examination showed more neovascularization and less scar, and sirius red staining indicated higher volume of type Ⅲ collagen (7.10±4.06% vs 3.77±1.09%; P<0.05) and lower collagen Ⅰ/Ⅲ ratio value (1.11±0.52 vs 2.94±2.48; P<0.05)in HGF group. Conclusion HGF gene transfer might improve cardiac function and LV remodeling after acute myocardial infarction by stimulating angiogenesis, reducing fibrosis, and reducing myocardial scarring.

  2. Diagnostic features in 10 naturally occurring cases of acute fatal canine leptospirosis.

    Science.gov (United States)

    Rissi, Daniel R; Brown, Cathy A

    2014-11-01

    The current report describes the diagnostic features in 10 cases of acute fatal canine leptospirosis with minimal renal and hepatic changes that may present a diagnostic challenge for the pathologist. Most affected dogs were less than 6 months of age and had a biochemical profile consistent with hepatorenal dysfunction. Clinical signs consisted of vomiting, depression, icterus, dehydration, diarrhea, and anorexia. All dogs died or were humanely euthanized within 3-7 days after the onset of clinical disease. Necropsy findings included pulmonary edema with hemorrhages, icterus, renal and hepatic pallor and swelling, and gastric edema with hemorrhage. Despite severe azotemia, histological changes in the kidneys were subtle in all dogs, and included mild renal tubular simplification, with single-cell necrosis and attenuation, along with minimal interstitial lymphoplasmacytic inflammation, edema, and hemorrhage. Hepatic lesions included scattered hepatocellular single-cell necrosis and hepatocellular dissociation. Prominent extrarenal lesions typically associated with uremia including vascular fibrinoid necrosis in multiple organs, pulmonary mineralization with occasional fibrinosuppurative exudation, and gastric mineralization were also present. Postmortem diagnostic confirmation was based on the detection of leptospiral antigen on fresh renal samples by fluorescent antibody test and on the demonstration of intact spirochetes in sections of kidneys using immunohistochemical staining. Acute fatal canine leptospirosis occurred as a fulminant hepatorenal disease affecting mainly young dogs, and the diagnosis was dependent on the recognition of the subtle renal changes with confirmation via fluorescent antibody testing or immunohistochemical staining.

  3. Reduced Pancreatic Exocrine Function and Organellar Disarray in a Canine Model of Acute Pancreatitis.

    Directory of Open Access Journals (Sweden)

    Yuepeng Jin

    Full Text Available The aim of the present study was to investigate the pancreatic exocrine function in a canine model and to analyze the changes in organelles of pancreatic acinar cells during the early stage of acute pancreatitis (AP. AP was induced by retrograde injection of 5% sodium taurocholate (0.5 ml/kg into the main pancreatic duct of dogs. The induction of AP resulted in serum hyperamylasemia and a marked reduction of amylase activity in the pancreatic fluid (PF. The pancreatic exocrine function was markedly decreased in subjects with AP compared with the control group. After the induction of AP, histological examination showed acinar cell edema, cytoplasmic vacuolization, fibroblasts infiltration, and inflammatory cell infiltration in the interstitium. Electron micrographs after the induction of AP revealed that most of the rough endoplasmic reticulum (RER were dilated and that some of the ribosomes were no longer located on the RER. The mitochondria were swollen, with shortened and broken cristae. The present study demonstrated, in a canine model, a reduced volume of PF secretion with decreased enzyme secretion during the early stage of AP. Injury of mitochondria and dilatation and degranulation of RER may be responsible for the reduced exocrine function in AP. Furthermore, the present model and results may be useful for researching novel therapeutic measures in AP.

  4. Synovial fluid cytology in experimental acute canine monocytic ehrlichiosis (Ehrlichia canis).

    Science.gov (United States)

    Theodorou, Konstantina; Leontides, Leonidas; Siarkou, Victoria I; Petanides, Theodoros; Tsafas, Konstantinos; Harrus, Shimon; Mylonakis, Mathios E

    2015-05-15

    Evidence-based information of a cause-and-effect relationship between Ehrlichia canis infection and polyarthritis in naturally- or experimentally-infected dogs is currently lacking. The aim of this prospective study was to investigate whether synovial fluid cytological evidence of arthritis could be documented in dogs with acute monocytic ehrlichiosis. Direct synovial fluid cytology smears from eight Beagle dogs experimentally infected with E. canis were examined prior to, and on 21, 35 and 63 days post-inoculation. The cytological variables assessed included cellularity, percentages of mononuclear cells and neutrophils, macrophage reactivity and evidence of E. canis morulae. The median cellularity and percentages of mononuclear cells and neutrophils prior to inoculation did not differ when compared to post-inoculation cytological evaluation. Increased cellularity, E. canis morulae or cytological evidence of arthritis or macrophage reactivity were not observed throughout the course of the study. In the present study, no cytological evidence of arthritis was found in dogs with experimental acute canine monocytic ehrlichiosis, suggesting that E. canis infection should be considered a rather uncommon cause of arthritis in dogs.

  5. Effect of nifekalant on acute electrical remodelling in rapid atrial pacing canine model

    Institute of Scientific and Technical Information of China (English)

    TANG Min; ZHANG Shu; SUN Qi; HUA Wei; HUANG Cong-xin

    2006-01-01

    Background Nifekalant may prevent atrial fibrillation (AF) and possibly be useful in treatment of atrial tachyarrhythmia in patients with severe heart failure. This study investigated the electophysiologic effect of nifekalant on the acute atrial remodeling in rapid atrial pacing (RAP) model of canine.Methods Twelve mongrel dogs subjected to rapid stimulation (400 beats/min) at left atrial appendage (LAA)for 24 hours, were randomized into the control group (rapid pacing only, n=6) and the nifekalant group (intravenous nifekalant therapy immediately after RAP, n=6). Atrial electrophysiological parameters were measured in right atrium, coronary sinus, LAA, posterior wall of left atrium (PWLA) and left superior pulmonary vein (LSPV), before and after the RAP.Results In the control group, the effective refractory periods (ERP) were shortened greatly at all sites, paced dogs had substantially shorter ERPs in the high right atrium, LAA, and LSPV, but fewer changes in the PWLA,the coefficient variation of ERP (COV ERP) was increased significantly. After rapid atrial stimulation, the inducibility of AF increased significantly [induction number: pre-RAP vs post-RAP, 1.00± 0.89 vs 8.17 ± 2.79,P<0.01; duration of AF: pre-RAP vs post-RAP, (450.34± 362.59) ms vs (9975.77 ±4376.99) ms, P<0.01]. In the nifekalant group, although the ERPs were prolonged at all sites compared with those in pre-RAP state, only the value at LSPV differed significantly from that in pre-RAP state [pre-RAP vs post-RAP, (102.50±5.24) ms vs (132.51 ±5.20) ms, P<0.01]; the COV ERP did not change statistically in this group. The inducibility of AF slightly increased but insignificantly after pacing [induction number: pre-RAP vs post-RAP, 0.83 ±0.75 vs 1.67±0.82, P=0.19; duration of AF: pre-RAP vs post-RAP, (378.67±317.88) ms vs (1124.08±1109.77) ms,P=0.06]. Conduction time values did not alter significantly in either of the two groups after RAP.Conclusions In canine RAP model, nifekalant

  6. The role of wild type RAS isoforms in cancer.

    Science.gov (United States)

    Zhou, Bingying; Der, Channing J; Cox, Adrienne D

    2016-10-01

    Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.

  7. Transcutaneous Intraluminal Impedance Measurement for Minimally Invasive Monitoring of Gastric Motility: Validation in Acute Canine Models

    Directory of Open Access Journals (Sweden)

    Michael D. Poscente

    2014-01-01

    Full Text Available Transcutaneous intraluminal impedance measurement (TIIM is a new method to cutaneously measure gastric contractions by assessing the attenuation dynamics of a small oscillating voltage emitted by a battery-powered ingestible capsule retained in the stomach. In the present study, we investigated whether TIIM can reliably assess gastric motility in acute canine models. Methods. Eight mongrel dogs were randomly divided into 2 groups: half received an active TIIM pill and half received an identically sized sham capsule. After 24-hour fasting and transoral administration of the pill (active or sham, two force transducers (FT were sutured onto the antral serosa at laparotomy. After closure, three standard cutaneous electrodes were placed on the abdomen, registering the transluminally emitted voltage. Thirty-minute baseline recordings were followed by pharmacological induction of gastric contractions using neostigmine IV and another 30-minute recording. Normalized one-minute baseline and post-neostigmine gastric motility indices (GMIs were calculated and Pearson correlation coefficients (PCCs between cutaneous and FT GMIs were obtained. Statistically significant GMI PCCs were seen in both baseline and post-neostigmine states. There were no significant GMI PCCs in the sham capsule test. Further chronic animal studies of this novel long-term gastric motility measurement technique are needed before testing it on humans.

  8. Canine gastritis.

    Science.gov (United States)

    Webb, Craig; Twedt, David C

    2003-09-01

    Gastritis--inflammation of the stomach--is a frequently cited differential yet rarely characterized diagnosis in cases of canine anorexia and vomiting. Although the list of rule-outs for acute or chronic gastritis is extensive, a review of the veterinary literature reveals fewer than 15 articles that have focused on clinical cases of canine gastritis over the last 25 years. The dog frequently appears in the human literature as an experimentally manipulated model for the study of endoscopic techniques or the effect of medications on gastric mucosa. In the veterinary patient, cases of acute gastritis are rarely pursued with the complete diagnostic armamentarium, and cases of chronic gastritis are rarely found to occur as an entity isolated from the rest of the gastrointestinal tract. This article focuses on those findings most clinically relevant to cases of canine gastritis in veterinary medicine.

  9. Automatic Detection of Wild-type Mouse Cranial Sutures

    DEFF Research Database (Denmark)

    Ólafsdóttir, Hildur; Darvann, Tron Andre; Hermann, Nuno V.;

    , automatic detection of the cranial sutures becomes important. We have previously built a craniofacial, wild-type mouse atlas from a set of 10 Micro CT scans using a B-spline-based nonrigid registration method by Rueckert et al. Subsequently, all volumes were registered nonrigidly to the atlas. Using...

  10. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey, E-mail: carey.pope@okstate.edu

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  11. Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

    Directory of Open Access Journals (Sweden)

    Angez Mehar

    2012-02-01

    Full Text Available Abstract Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas

  12. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Gloria C.; Franco, Ricardo; Lu, Yi; Ma, Jian-Guo; Shelnutt, John A.

    1999-05-19

    Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has

  13. Innate immune responses to obesity in cloned and wild-type domestic pig

    DEFF Research Database (Denmark)

    Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Stagsted, Jan

    as a refined pig model for obesity-induced innate host responses by reducing pig-to-pig biological variation compared to wild-type (WT) pigs (n=19). Pigs were fed ad libitum with a high fat/high sucrose diet to induce obesity or kept lean on a restricted diet (60% of ad libitum intake) beginning at three...... months of age. mRNA expression levels were determined for 39 innate immune factors on a high-throughput qPCR system in samples from liver, abdominal fat, mesenteric fat and subcutaneous fat. Previous findings have suggested that cloning may affect certain phenotypic traits of pigs including basic......, mRNA expression profiles of certain acute phase proteins were significantly affected by cloning, being expressed at higher levels in the liver of both cloned groups compared to both WT groups but at lower levels in adipose tissues of cloned lean pigs as opposed to WT lean pigs. Also there were...

  14. MAP kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma.

    Science.gov (United States)

    Orouji, Elias; Orouji, Azadeh; Gaiser, Timo; Larribère, Lionel; Gebhardt, Christoffer; Utikal, Jochen

    2016-05-01

    Recent therapeutic advances have improved melanoma patientś clinical outcome. Novel therapeutics targeting BRAF, NRAS and cKit mutant melanomas are widely used in clinical practice. However therapeutic options in NRAS(wild-type) /BRAF(wild-type) /cKit(wild-type) melanoma patients are limited. Our study shows that gene copy numbers of members of the MAPK signaling pathway vary in different melanoma subgroups. NRAS(wild-type) /BRAF(wild-type) melanoma metastases are characterized by significant gains of MAP2K1 (MEK1) and MAPK3 (ERK1) gene loci. These additional gene copies could lead to an activation of the MAPK signaling pathway via a gene-dosage effect. Our results suggest that downstream analyses of the pMEK and pERK expression status in NRAS(wild-type) /BRAF(wild-type) melanoma patients identify patients that could benefit from targeted therapies with MEK and ERK inhibitors.

  15. Three-dimensional structure of the wild-type RHDV

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The three-dimensional (3D) structure of the wild-type rabbit hemorrhagic disease virus (RHDV) has been determined to a resolution of 3.2 nm by electron cryomicroscopy and computer image reconstruction techniques. The 3D density map exhibits characteristic structural features of a calicivirus: a T=3 icosahedral capsid with 90 arch-like capsomeres at the icosahedral and local 2-fold axes and 32 large surface hollows at the icosahedral 5- and 3-fold axes. This result confirms that the RHDV isolated in China is a member of the Caliciviridae family. A rather continuous capsid shell was found without channels. However, our RHDV structure also reveals some distinct structural charac-teristics not observed in other caliciviruses, including inter-connected capsomeres and the lack of protuberance on the base of each of the surface hollows. Two types of particles were identified with similar outer capsid structure but dif-ferent density distributions inside the capsid shells, which could not be distinguished by conventional negative staining electron microscopy. As the genomic and subgenomic RNAs are both packaged into particles for RHDV, we suggest that the two types of particles identified correspond to those con-taining either the genomic or subgenomic RNAs, respec-tively.

  16. Wild type measles virus attenuation independent of type I IFN

    Directory of Open Access Journals (Sweden)

    Horvat Branka

    2008-02-01

    Full Text Available Abstract Background Measles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt. Results The adaptation of a measles virus isolate (G954-PBL by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13 differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene. While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain. Conclusion Thus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system.

  17. Transcriptional profiling of gastric epithelial cells infected with wild type or arginase-deficient Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Kim Songhee H

    2012-08-01

    Full Text Available Abstract Background Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators. Results Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type Helicobacter pylori strain 26695, an arginase mutant (rocF- or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction. Conclusions We conclude that H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.

  18. Biosafety of Recombinant and Wild Type Nucleopolyhedroviruses as Bioinsecticides

    Directory of Open Access Journals (Sweden)

    Bruce D. Hammock

    2007-06-01

    Full Text Available The entomopathogenic Autographa californica (Speyer nucleopolyhedrovirus (AcMNPV has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 1012 PIBs/feddan, feddan = 4,200 m2 and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field, AcMNPV, and SlNPV using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 μg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%, AcAaIT-field (1.2%, and SlNPV (4.0%. Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal

  19. Wild-type measles virus is intrinsically dual-tropic

    Directory of Open Access Journals (Sweden)

    Makoto eTakeda

    2012-01-01

    Full Text Available Measles is a highly contagious disease that causes temporary and severe immunosuppression in patients. Signaling lymphocyte activation molecule (SLAM expressed on cells of the immune system functions as a receptor for measles virus (MV. In addition to SLAM, vaccine strains of MV also use a ubiquitously expressed complement regulatory protein, CD46, as a receptor, whereas wild-type (wt MV strains do not use this receptor. However, recent studies have indicated that SLAM is not the sole receptor for wt MV strains. These strains have an intrinsic ability to enter both immune and epithelial cells using distinct receptor binding sites in their hemagglutinin (H protein. Recently, a clear answer was obtained through the identification of an epithelial MV receptor, nectin4, expressed at adherens junctions, thereby greatly improving our knowledge of MV receptors. It is now clear that MV specifically targets two cell types, immune cells and epithelial cells, using SLAM and nectin4, respectively. MV loses the ability to use either SLAM or nectin4 when it possesses specific mutations in the H protein. However, nectin4-blind MV still infects SLAM-positive immune cells efficiently (SLAM-tropic, and conversely, SLAM-blind MV infects nectin4-positive epithelial cells efficiently (nectin4-tropic. In this regard, MV is intrinsically dual-tropic to immune cells and epithelial cells. Although many aspects and molecular mechanisms underlying immunosuppressive effects and a highly contagious nature of MV still remain to be elucidated, analyses of physiological functions of these two receptors would provide deep insights into MV pathogenesis.

  20. Systemic phosphatidylcholine pretreatment protects canine esophageal mucosa during acute experimental biliary reflux

    Institute of Scientific and Technical Information of China (English)

    Gabor Eros; Jozsef Kaszaki; Miklos Czobel; Mihaly Boros

    2006-01-01

    AIM: To characterize the consequences of short-term exposure to luminal bile on mucosal mast cell reactions in a canine model, and to determine the effects of systemic phosphatidylcholine pretreatment in this condition.METHODS: Twenty mongrel dogs were used for experiments. Group 1 (n = 5) served as a salinetreated control, while in group 2 (n=5) the esophagus was exposed to bile for 3 h. In group 3 (n=5) the animals were pretreated with 7-nitroindazole to inhibit the neuronal isoform of nitric oxide synthase.In group 4 (n = 5) phosphatidylcholine solution (50 mg/kg) was administered iv before the biliary challenge. Mucosal microcirculation was observed by intravital videomicroscopy. Myeloperoxidase and nitric oxide synthase activities, the degrees of mast cell degranulation and mucosal damage were evaluated via tissue biopsies.RESULTS: Exposure to bile evoked significant mast cell degranulation and leukocyte accumulation. The red blood cell velocity and the diameter of the postcapillary venules increased significantly. The tissue ATP content and constitutive nitric oxide synthase activity decreased,while the inducible nitric oxide synthase activity increased significantly as compared to the control values.7-nitroindazole treatment significantly exacerbated the mucosal mast cell degranulation and tissue damage. In contrast, phosphatidylcholine pretreatment prevented the bile-induced ATP depletion, the inducible nitric oxide synthase and myeloperoxidase activity and the mast cell degranulation increased.CONCLUSION: The neuronal nitric oxide synthase -mast cell axis plays an important role in the esophageal mucosal defense system. Systemic phosphatidylcholine pretreatment affords effective protection through ameliorating the bile-induced ATP depletion and secondary inflammatory reaction.

  1. Wild-Type Measles Virus with the Hemagglutinin Protein of the Edmonston Vaccine Strain Retains Wild-Type Tropism in Macaques

    Science.gov (United States)

    Nagata, Noriyo; Kato, Sei-ich; Ami, Yasushi; Suzaki, Yuriko; Suzuki, Tadaki; Sato, Yuko; Tsunetsugu-Yokota, Yasuko; Mori, Kazuyasu; Van Nguyen, Nguyen; Kimura, Hideki; Nagata, Kyosuke

    2012-01-01

    A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM+ as well as CD46+ cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM+ cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM+ lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo. PMID:22238320

  2. IGF-1,bFGF EXPRESSION AND VASCULAR REGENERATION IN ACUTE INFARCTED CANINE MYOCARDIUM AFTER AUTOLOGUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

    Institute of Scientific and Technical Information of China (English)

    朱洪生; 钟竑; 张臻

    2003-01-01

    Objective To study the cell growth factor secretion and vascular regeneration in acute infarcted myocardium after autologous skeletal muscle satellite cell implantation.MethodsAutologous skeletal muscle satellite cells from adult mongrel canine were implanted into the acute myocardial infarct site via the ligated left anterior descending (LAD) artery. Specimens were harvested at 2, 4, 8 weeks after implantation for the expression of insulin like growth factor-1 (IGF-1), basic fibroblast growth factor (Bfgf) and the vascular density.ResultsThe expression of IGF-1, Bfgf and the vascular density in skeletal muscle satellite cell implant group were higher than that in the control group.ConclusionThe skeletal muscle satellite cells, after being implanted into the acute myocardial infarction, not only showed myocardial regeneration, but also showed the ability to secrete the cell factors, hence representing a positive effect on the regeneration of the infarcted myocardium.

  3. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants

    DEFF Research Database (Denmark)

    Klausen, M.; Heydorn, Arne; Ragas, Paula Cornelia;

    2003-01-01

    Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary...... for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type...... and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type...

  4. ADENOVIRUS-MEDIATED WILD-TYPE P53 EXPRESSION SUPPRESSES GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji

    1998-01-01

    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  5. TREATMENT OF CANINE ACUTE LIVER FAILURE WITH MODIFIED EXTRACORPOREAL PIGLIVER PERFUSION

    Institute of Scientific and Technical Information of China (English)

    王博; 吕毅; 刘昌; 仵正; 潘承恩

    2003-01-01

    Objective To study the theraputic effect of extracorporeal liver perfusion on the treatment of acute liver failure. Methods Mongrel dogs weighing 12-14*!kg were selected. Hepatic failure was induced by an end-to-side portacaval shunt. The common hepatic and gastroduodenal arteries were occluded for 2 hours. To the control group (n=7), the dogs received standard medical therapy . To the treating group (n=10), the dogs received extracorporeal kidney and liver perfusion at the onset of the occlusion of the hepatic artery. During the liver support, the animals were frequently monitored regarding their clinical state, liver function, biochemical and hematological parameters. Results After the occlusion of the liver blood flow, all dogs died within 3-7.5 hours. The average survival time was (5.7±1.2) hours. Serum levels of ALT, AST, LDH and ammonia increased significantly. In the treating group, the dogs died within 7-10.5 hours. The average survival time was 8.6±1.1 hours. There were no significant diferences in serum levels of ALT, AST, LDH between the two groups(P>0.05). There were dramatic diferences in blood Ammonia level, PT, FIB between the two groups(P<0.05). The survival time was longer in treating group. The animals' blood pressure were more stable in the treating group than that in the control group. Conclusion The modified xenogenic liver perfusion can provide necessary hepatic function for the acute liver failure dogs.

  6. Canine Distemper

    Science.gov (United States)

    Although this brochure provides basic information about canine distemper, your veterinarian is always your best source of ... Consult your veterinarian for more information about canine distemper and its prevention. And Now A Note On ...

  7. Estimation of the wild-type minimum inhibitory concentration value distribution.

    Science.gov (United States)

    Jaspers, Stijn; Aerts, Marc; Verbeke, Geert; Beloeil, Pierre-Alexandre

    2014-01-30

    Antimicrobial resistance has become one of the main public health burdens of the last decades, and monitoring the development and spread of non-wild-type isolates has therefore gained increased interest. Monitoring is performed based on the minimum inhibitory concentration (MIC) values, which are collected through the application of dilution experiments. In order to account for the unobserved population heterogeneity of wild-type and non-wild-type isolates, mixture models are extremely useful. Instead of estimating the entire mixture globally, it was our major aim to provide an estimate for the wild-type first component only. The characteristics of this first component are not expected to change over time, once the wild-type population has been confidently identified for a given antimicrobial. With this purpose, we developed a new method based on the multinomial distribution, and we carry out a simulation study to study the properties of the new estimator. Because the new approach fits within the likelihood framework, we can compare distinct distributional assumptions in order to determine the most suitable distribution for the wild-type population. We determine the optimal parameters based on the AIC criterion, and attention is also paid to the model-averaged approach using the Akaike weights. The latter is thought to be very suitable to derive specific characteristics of the wild-type distribution and to determine limits for the wild-type MIC range. In this way, the new method provides an elegant means to compare distinct distributional assumptions and to quantify the wild-type MIC distribution of specific antibiotic-bacterium combinations.

  8. Age-dependent arginine phosphokinase activity changes in male vestigial and wild-type Drosophila melanogaster.

    Science.gov (United States)

    Baker, G T

    1975-01-01

    The activity of arginine phosphokinase, an important muscle enzyme in insects, was investigated with age in vestigial-winged and wild-type Drosophila melanogaster. Identical patterns of age-dependent activity changes were observed in the vestigial-winged flies as in the wild-type, even though vestigial-winged flies exhibit a 50% mortality approximately two thirds that of the wild-type as well as being incapable of flight. Results indicate that the age-dependent changes in arginine phosphokinase activity are intrinsically regulated within the cells of the flight muscle.

  9. After a cold conditioning swim, UCP2-deficient mice are more able to defend against the cold than wild type mice.

    Science.gov (United States)

    Abdelhamid, Ramy E; Kovács, Katalin J; Nunez, Myra G; Larson, Alice A

    2014-08-01

    Uncoupling protein 2 (UCP2) is widely distributed throughout the body including the brain, adipose tissue and skeletal muscles. In contrast to UCP1, UCP2 does not influence resting body temperature and UCP2-deficient (-/-) mice have normal thermoregulatory responses to a single exposure to cold ambient temperatures. Instead, UCP2-deficient mice are more anxious, exhibit anhedonia and have higher circulating corticosterone than wild type mice. To test the possible role of UCP2 in depressive behavior we exposed UCP2-deficient and wild type mice to a cold (26°C) forced swim and simultaneously measured rectal temperatures during and after the swim. The time that UCP2-deficient mice spent immobile did not differ from wild type mice and all mice floated more on day 2. However, UCP2-deficient mice were more able to defend against the decrease in body temperature during a second daily swim at 26°C than wild type mice (area under the curve for wild type mice: 247.0±6.4; for UCP2-deficient mice: 284.4±3.8, Pswim at 26°C correlated with their greater immobility whereas defense against the warmth during a swim at 41°C correlated better with greater immobility of UCP2-deficient mice. Together these data indicate that while the lack of UCP2 has no acute effect on body temperature, UCP2 may inhibit rapid improvements in defense against cold, in contrast to UCP1, whose main function is to promote thermogenesis.

  10. Voluntary sodium ingestion in wild-type and oxytocin knockout mice.

    Science.gov (United States)

    Vollmer, Regis R; Cai, Hou-Ming; Miedlar, Julie A; Amico, Janet A

    2013-01-01

    Oxytocin knockout (OT KO) mice acutely consume inappropriate amounts of sodium following overnight water deprivation suggesting that oxytocinergic neurons inhibit excessive sodium ingestion (Amico JA, Morris M, Vollmer RR. Mice deficient in oxytocin manifest increased saline consumption following overnight fluid deprivation. Am J Physiol - Regul Integr Comp Physiol 2001; 281:R1368-R1373). This study sought to determine whether oxytocin (OT) provides long-term regulation of voluntary sodium ingestion. Wild-type (WT) and oxytocin knockout male mice were provided choices between diets or drinking solutions that differed in their sodium content. Mice were given access for 1 week to two diets, one containing low sodium (0.01% sodium chloride [NaCl]) content and a second containing a normal sodium (1.0% NaCl) content. During the second week, the animals were given a choice between a low sodium diet and a high sodium (8.0% NaCl) diet. In the second week, mice consumed 4 times more sodium; however, there were no differences between WT and OT KO mice. In a second experiment, mice had access to a two-bottle choice of tap water and a 0.5 M NaCl solution made palatable by the addition of a 4.1% Intralipid emulsion. Both genotypes consumed large, but equivalent, volumes of the Intralipid/sodium solution. The ingestion of this sodium-rich solution stimulated thirst and enhanced the intake of water. Thus, the availability of palatable sodium-rich food or solutions can lead to excessive voluntary sodium ingestion. Compared with oxytocin knockout mice, enhanced voluntary ingestion of sodium-rich solid and liquid diets proceeded unimpeded in WT mice. Therefore, OT pathways may not be essential for regulating solute intake in this setting.

  11. ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

    Science.gov (United States)

    Morton, Derrick J; Patel, Divya; Joshi, Jugal; Hunt, Aisha; Knowell, Ashley E; Chaudhary, Jaideep

    2017-01-10

    Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

  12. Modeling the competition between antenna size mutant and wild type microalgae in outdoor mass culture.

    Science.gov (United States)

    de Mooij, Tim; Schediwy, Kira; Wijffels, René H; Janssen, Marcel

    2016-12-20

    Under high light conditions, microalgae are oversaturated with light which significantly reduces the light use efficiency. Microalgae with a reduced pigment content, antenna size mutants, have been proposed as a potential solution to increase the light use efficiency. The goal of this study was to investigate the competition between antenna size mutants and wild type microalgae in mass cultures. Using a kinetic model and literature-derived experimental data from wild type Chlorella sorokiniana, the productivity and competition of wild type cells and antenna size mutants were simulated. Cultivation was simulated in an outdoor microalgal raceway pond production system which was assumed to be limited by light only. Light conditions were based on a Mediterranean location (Tunisia) and a more temperate location (the Netherlands). Several wild type contamination levels were simulated in each mutant culture separately to predict the effect on the productivity over the cultivation time of a hypothetical summer season of 100days. The simulations demonstrate a good potential of antenna size reduction to increase the biomass productivity of microalgal cultures. However, it was also found that after a contamination with wild type cells the mutant cultures will be rapidly overgrown resulting in productivity loss.

  13. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    Science.gov (United States)

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

  14. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore

    Science.gov (United States)

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

  15. The mystery of oncogenic KRAS: Lessons from studying its wild-type counter part.

    Science.gov (United States)

    Chang, Yuan-I; Damnernsawad, Alisa; Kong, Guangyao; You, Xiaona; Wang, Demin; Zhang, Jing

    2016-07-22

    Using conditional knock-in mouse models, we and others have shown that despite the very high sequence identity between Nras and Kras proteins, oncogenic Kras displays a much stronger leukemogenic activity than oncogenic Nras in vivo. In this manuscript, we will summarize our recent work of characterizing wild-type Kras function in adult hematopoiesis and in oncogenic Kras-induced leukemogenesis. We attribute the strong leukemogenic activity of oncogenic Kras to 2 unique aspects of Kras signaling. First, Kras is required in mediating cell type- and cytokine-specific ERK1/2 signaling. Second, oncogenic Kras, but not oncogenic Nras, induces hyperactivation of wild-type Ras, which significantly enhances Ras signaling in vivo. We will also discuss a possible mechanism that mediates oncogenic Kras-evoked hyperactivation of wild-type Ras and a potential approach to down-regulate oncogenic Kras signaling.

  16. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    Science.gov (United States)

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  17. Liver steatosis study_PFAA treated Wild type and PPAR KO mouse data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data set 1 consists of the experimental data for the Wild Type and PPAR KO animal study and includes data used to prepare Figures 1-4 and Table 1 of the Das et al,...

  18. Horizontal and vertical transmission of wild-type and recombinant Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

    NARCIS (Netherlands)

    Zhou, M.; Sun, X.; Sun, X.C.; Vlak, J.M.; Hu, Z.H.; Werf, van der W.

    2005-01-01

    Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant

  19. Tendon fascicle gliding in wild type, heterozygous, and lubricin knockout mice.

    Science.gov (United States)

    Kohrs, Ross T; Zhao, Chunfeng; Sun, Yu-Long; Jay, Gregory D; Zhang, Ling; Warman, Matthew L; An, Kai-Nan; Amadio, Peter C

    2011-03-01

    The objective of this study was to investigate the role of lubricin in the lubrication of tendon fascicles. Lubricin, a glycoprotein, lubricates cartilage and tendon surfaces, but the function of lubricin within the tendon fascicle is unclear. We developed a novel method to assess the gliding resistance of a single fascicle in a mouse tail model and used it to test the hypothesis that gliding resistance would be increased in lubricin knockout mice. Thirty-six mouse tails were used from 12 wild type, 12 heterozygous, and 12 lubricin knockout mice. A 15 mm long fascicle segment was pulled proximally after being divided distally. The peak resistance during fascicle pullout and the fascicle perimeter were measured. Lubricin expression was evaluated by immunohistochemistry. The peak gliding resistance in the lubricin knockout mice was significantly higher than in the wild type (p < 0.05). Fascicles from heterozygous mice were intermediate in value, but not significantly different from either wild type or lubricin knockout fascicles in peak gliding resistance. No significant difference was found in fascicle perimeter among the three groups. No correlation was observed between fascicle perimeter and gliding resistance. While lubricin was detected by immunostaining on the fascicle surface in wild type and heterozygous mice, lubricin was not detectable in the tendons of knockout mice. We conclude that the absence of lubricin is associated with increased interfascicular friction and that lubricin may play an important role in interfascicular lubrication.

  20. Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Yoshiaki Furukawa

    2012-01-01

    Full Text Available Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1 gene cause a familial form of amyotrophic lateral sclerosis (ALS. While it remains controversial how SOD1 mutations lead to onset and progression of the disease, many in vitro and in vivo studies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations in sod1 gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

  1. Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

    Science.gov (United States)

    Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

    2009-07-01

    Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast

  2. Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs.

    Science.gov (United States)

    Yi, Li; Cheng, Shipeng; Xu, Hongli; Wang, Jianke; Cheng, Yuening; Yang, Shen; Luo, Bin

    2012-01-01

    A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.

  3. Inhibition of HIV type 1 infectivity by coexpression of a wild-type and a defective glycoprotein 120

    DEFF Research Database (Denmark)

    Lund, O S; Losman, B; Schønning, Kristian;

    1998-01-01

    model: when virion formation is saturated with envelope protein, expression and incorporation of a defective envelope protein imply a corresponding dilution of wild-type protein on the surface of virions. The cooperative function of wild-type envelope proteins is subsequently compromised, and a trans...

  4. Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

    NARCIS (Netherlands)

    Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.

    2009-01-01

    Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven ou

  5. In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains.

    Science.gov (United States)

    Pignolet, Béatrice; Duteyrat, Jean-Luc; Allemandou, Aude; Gelfi, Jacqueline; Foucras, Gilles; Bertagnoli, Stéphane

    2007-09-27

    Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1) and a vaccinal strain (SG33) of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC) occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies.

  6. A mutant chaperone converts a wild-type protein into a tumor-specific antigen.

    Science.gov (United States)

    Schietinger, Andrea; Philip, Mary; Yoshida, Barbara A; Azadi, Parastoo; Liu, Hui; Meredith, Stephen C; Schreiber, Hans

    2006-10-13

    Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.

  7. Panitumumab as a radiosensitizing agent in KRAS wild-type locally advanced rectal cancer.

    Science.gov (United States)

    Mardjuadi, Feby Ingriani; Carrasco, Javier; Coche, Jean-Charles; Sempoux, Christine; Jouret-Mourin, Anne; Scalliet, Pierre; Goeminne, Jean-Charles; Daisne, Jean-François; Delaunoit, Thierry; Vuylsteke, Peter; Humblet, Yves; Meert, Nicolas; van den Eynde, Marc; Moxhon, Anne; Haustermans, Karin; Canon, Jean-Luc; Machiels, Jean-Pascal

    2015-09-01

    Our goal was to optimize the radiosensitizing potential of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, when given concomitantly with preoperative radiotherapy in KRAS wild-type locally advanced rectal cancer (LARC). Based on pre-clinical studies conducted by our group, we designed a phase II trial in which panitumumab (6 mg/kg/q2 weeks) was combined with preoperative radiotherapy (45 Gy in 25 fractions) to treat cT3-4/N + KRAS wild-type LARC. The primary endpoint was complete pathologic response (pCR) (H0 = 5%, H1 = 17%, α = 0.05, β = 0.2). From 19 enrolled patients, 17 (89%) were evaluable for pathology assessment. Although no pCR was observed, seven patients (41%) had grade 3 Dworak pathological tumor regression. The regimen was safe and was associated with 95% of sphincter-preservation rate. No NRAS, BRAF, or PI3KCA mutation was found in this study, but one patient (5%) showed loss of PTEN expression. The quantification of plasma EGFR ligands during treatment showed significant upregulation of plasma TGF-α and EGF following panitumumab administration (p < 0.05). At surgery, patients with important pathological regression (grade 3 Dworak) had higher plasma TGF-α (p = 0.03) but lower plasma EGF (p = 0.003) compared to those with grade 0-2 Dworak. Our study suggests that concomitant panitumumab and preoperative radiotherapy in KRAS wild-type LARC is feasible and results in some tumor regression. However, pCR rate remained modest. Given that the primary endpoint of our study was not reached, we remain unable to recommend the use of panitumumab as a radiosensitizer in KRAS wild-type LARC outside a research setting.

  8. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E., E-mail: uhansmann@ou.edu [Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 (United States)

    2014-11-07

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  9. Nucleoprotein gene analysis of the wild-type measles viruses circulated in Beijing in 2001

    Institute of Scientific and Technical Information of China (English)

    谢正德; 申昆玲; 许文波; 照日格图; 朱贞

    2004-01-01

    @@ The hemagglutinin (H) and nucleoprotein (N) genes are the most variable regions on the genome of the measles virus. Nucleotide sequence analysis of the H and/or N genes has been used to describe different genetic groups of wild-type measles virus.1-3 This genetic information has been used in molecular epidemiological studies to identify the transmission pathways of measles virus. The present study was conducted to reveal the genotype of measles viruses which circulated in Beijing in 2001.

  10. HPLC ANALYSIS OF BIOACTIVE COMPOUNDS IN TEN DIFFERENT WILD TYPE UNDER-UTILIZED LEGUME GRAINS

    OpenAIRE

    Vellingiri Vadivel and Hans Konrad Biesalski

    2010-01-01

    In recent years, many food industries have been initiated the formulation of nutraceutical/functional foods by incorporating the bioactive ingredients for the prevention/treatment of certain chronic diseases. In this connection, certain promising wild type under-utilized legume grains received more attention, since they are naturally a rich source of L-Dopa (precursor of dopamine) and certain bioactive compounds including phenolics, tannins and phytic acid. In the present study, seed material...

  11. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    Science.gov (United States)

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

    2014-11-01

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  12. An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

    Directory of Open Access Journals (Sweden)

    Melissa S Rotunno

    2013-12-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1 protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.

  13. Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice

    DEFF Research Database (Denmark)

    Bang, Christian A; Bro, Susanne; Bartels, Emil D

    2007-01-01

    Wild-type mice normally do not develop atherosclerosis, unless fed cholic acid. Uremia is proinflammatory and increases atherosclerosis 6- to 10-fold in apolipoprotein E-deficient mice. This study examined the effect of uremia on lipoproteins, vascular inflammation, and atherosclerosis in wild...... in cholic acid-fed sham mice. The results suggest that moderate uremia neither induces aortic inflammation nor atherosclerosis in C57BL/6J mice despite increased LDL/HDL cholesterol ratio and altered HDL composition....

  14. Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants.

    Science.gov (United States)

    Vitha, S; Zhao, L; Sack, F D

    2000-02-01

    Root gravitropism in wild-type Arabidopsis and in two starchless mutants, pgm1-1 and adg1-1, was evaluated as a function of light position to determine the relative strengths of negative phototropism and of gravitropism and how much phototropism affects gravitropic measurements. Gravitropism was stronger than phototropism in some but not all light positions in wild-type roots grown for an extended period, indicating that the relationship between the two tropisms is more complex than previously reported. Root phototropism significantly influenced the time course of gravitropic curvature and the two measures of sensitivity. Light from above during horizontal exposure overestimated all three parameters for all three genotypes except the wild-type perception time. At the irradiance used (80 micromol m(-2) s(-1)), the shortest periods of illumination found to exaggerate gravitropism were 45 min of continuous illumination and 2-min doses of intermittent illumination. By growing roots in circumlateral light or by gravistimulating in the dark, corrected values were obtained for each gravitropic parameter. Roots of both starchless mutants were determined to be about three times less sensitive than prior estimates. This study demonstrates the importance of accounting for phototropism in the design of root gravitropism experiments in Arabidopsis.

  15. Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase.

    Science.gov (United States)

    Elkon, Hanock; Don, Jermy; Melamed, Eldad; Ziv, Ilan; Shirvan, Anat; Offen, Daniel

    2002-06-01

    Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

  16. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    Science.gov (United States)

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  17. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    Directory of Open Access Journals (Sweden)

    Eliton da Silva Vasconcelos

    2013-12-01

    Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  18. In Vitro intestinal mucosal epithelial responses to wild-typeSalmonella Typhi and attenuated typhoid vaccines

    Directory of Open Access Journals (Sweden)

    Maria eFiorentino

    2013-02-01

    Full Text Available Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4h post-exposure. S. Typhi triggered the secretion of interleukin (IL-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

  19. Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.

    Science.gov (United States)

    Iannuzzi, Clara; Carafa, Vincenzo; Altucci, Lucia; Irace, Gaetano; Borriello, Margherita; Vinciguerra, Roberto; Sirangelo, Ivana

    2015-11-01

    Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

  20. Mutant Forkhead L2 (FOXL2) proteins associated with premature ovarian failure (POF) dimerize with wild-type FOXL2, leading to altered regulation of genes associated with granulosa cell differentiation.

    Science.gov (United States)

    Kuo, Fang-Ting; Bentsi-Barnes, Ikuko K; Barlow, Gillian M; Pisarska, Margareta D

    2011-10-01

    Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding.

  1. Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains.

    Science.gov (United States)

    Wirth, H P; Beins, M H; Yang, M; Tham, K T; Blaser, M J

    1998-10-01

    Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after >/=20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.

  2. HPLC ANALYSIS OF BIOACTIVE COMPOUNDS IN TEN DIFFERENT WILD TYPE UNDER-UTILIZED LEGUME GRAINS

    Directory of Open Access Journals (Sweden)

    Vellingiri Vadivel and Hans Konrad Biesalski

    2010-05-01

    Full Text Available In recent years, many food industries have been initiated the formulation of nutraceutical/functional foods by incorporating the bioactive ingredients for the prevention/treatment of certain chronic diseases. In this connection, certain promising wild type under-utilized legume grains received more attention, since they are naturally a rich source of L-Dopa (precursor of dopamine and certain bioactive compounds including phenolics, tannins and phytic acid. In the present study, seed materials of certain promising wild type under-utilized food legume grains such as Abrus precatorius L., Acacia leucopholea Willd, Bauhinia varigata L., Canavalia gladiata (Jacq. DC., Cassia floribunda Cav., Entada scandens Benth., Indigofera linifolia (L.f. Retz., Mucuna monosperma DC. Ex Wight., Sesbania bispinosa (Jacq. Wight. and Tamarindus indica L., collected from Eastern and Western Ghats of South India, were investigated for certain bioactive compounds through HPLC technique. All the analysed samples were found to constitute a viable source of total free phenolics (4.23 – 8.75 g/100 g DM, tannins (1.04 – 5.41 g /100 g DM, L-Dopa (1.17 – 5.34 g/100 g DM and phytic acid (0.96 – 2.74 g/100 g DM and also the newly developed HPLC procedures were proven to be sensitive enough to detect these bioactive compounds even at tracer level. Further, such wild type legume grains could be recommended as a natural source of bioactive compounds in the dietary management of certain chronic diseases such as Parkinsonism, diabetes, obesity, cardiovascular diseases, cancer etc.

  3. Growth, seed development and genetic analysis in wild type and Def mutant of Pisum sativum L

    Directory of Open Access Journals (Sweden)

    Ayeh Kwadwo

    2011-11-01

    Full Text Available Abstract Background The def mutant pea (Pisum sativum L showed non-abscission of seeds from the funicule. Here we present data on seed development and growth pattern and their relationship in predicting this particular trait in wild type and mutant lines as well as the inheritance pattern of the def allele in F2 and F3 populations. Findings Pod length and seed fresh weight increase with fruit maturity and this may affect the abscission event in pea seeds. However, the seed position in either the distal and proximal ends of the pod did not show any difference. The growth factors of seed fresh weight (FW, width of funicles (WFN, seed width (SW and seed height (SH were highly correlated and their relationships were determined in both wild type and def mutant peas. The coefficient of determination R2 values for the relationship between WFN and FW, SW and SH and their various interactions were higher for the def dwarf type. Stepwise multiple regression analysis showed that variation of WFN was associated with SH and SW. Pearson's chi square analysis revealed that the inheritance and segregation of the Def locus in 3:1 ratio was significant in two F2 populations. Structural analysis of the F3 population was used to confirm the inheritance status of the Def locus in F2 heterozygote plants. Conclusions This study investigated the inheritance of the presence or absence of the Def allele, controlling the presence of an abscission zone (AZ or an abscission-less zone (ALZ forming in wild type and mutant lines respectively. The single major gene (Def controlling this phenotype was monogenic and def mutants were characterized and controlled by the homozygous recessive def allele that showed no palisade layers in the hilum region of the seed coat.

  4. Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

    Science.gov (United States)

    Collins, R A; Kelly, S M; Price, N C; Fothergill-Gilmore, L A; Muirhead, H

    1996-12-01

    A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

  5. Genetic variation of the transthyretin gene in wild-type transthyretin amyloidosis (ATTRwt)

    Science.gov (United States)

    Sikora, Jacquelyn L.; Logue, Mark W.; Chan, Gloria G.; Spencer, Brian H.; Prokaeva, Tatiana B.; Baldwin, Clinton T.; Seldin, David C.; Connors, Lawreen H.

    2014-01-01

    Wild-type transthyretin amyloidosis (ATTRwt), typically diagnosed as congestive heart failure in elderly Caucasian men, features myocardial amyloid deposits of wild-type plasma protein transthyretin (TTR). ATTRwt is sporadic, its pathogenesis is poorly understood, and currently there are no biomarkers for diagnosis or prognosis. Genetic studies of variant-associated transthyretin amyloidosis (ATTRm) have suggested that non-coding TTR gene variants modulate disease. We hypothesized that cis-acting regulatory elements in the TTR gene non-coding regions may modify expression, affecting ATTRwt onset and progression. We studied an ATTRwt cohort consisting of 108 Caucasian males ranging in age from 59–87 years with cardiomyopathy due to wild-type TTR deposition; results were compared to 118 anonymous controls matched by age, sex, and race. Four predicted non-coding regulatory regions and all exons in the TTR gene were sequenced using the Sanger method. Eleven common variants were identified; 3 variants were significantly associated with ATTRwt (p < 0.05), though only one, rs72922940, remained near significance (pcorrected = 0.083) after multiple testing correction. Exon analyses demonstrated the occurrence of the p.G26S (G6S) polymorphism in 7% of ATTRwt subjects and 12% of controls; this variant was predicted to be a protective factor (p = 0.051). Four variants were significantly associated with age at onset and survival. In this first genetic study of a large, well-characterized cohort of ATTRwt, non-coding and coding variants associated with disease, age at onset, and survival were identified. Further investigation is warranted to determine the prevalence of these variants in ATTRwt, their regulatory function, and potential role in assessing disease risk. PMID:25367359

  6. De novo establishment of wild-type song culture in the zebra finch.

    Science.gov (United States)

    Fehér, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P; Tchernichovski, Ofer

    2009-05-28

    Culture is typically viewed as consisting of traits inherited epigenetically, through social learning. However, cultural diversity has species-typical constraints, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song and geographically separated groups have local song dialects. Different species exhibit distinct song cultures, suggestive of genetic constraints. Without such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations. Here we report an experiment designed to determine whether wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and, if so, how this might happen and what type of social environment is required. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved towards the wild-type in three to four generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution. In analogy to models in quantitative genetics, we model song culture as a multigenerational phenotype partly encoded genetically in an isolate founding population, influenced by environmental variables and taking

  7. Structural and Morphometric Comparison of Lower Incisors in PACAP-Deficient and Wild-Type Mice.

    Science.gov (United States)

    Sandor, B; Fintor, K; Reglodi, D; Fulop, D B; Helyes, Z; Szanto, I; Nagy, P; Hashimoto, H; Tamas, A

    2016-06-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread distribution. PACAP plays an important role in the development of the nervous system, it has a trophic and protective effect, and it is also implicated in the regulation of various physiological functions. Teeth are originated from the mesenchyme of the neural crest and the ectoderm of the first branchial arch, suggesting similarities with the development of the nervous system. Earlier PACAP-immunoreactive fibers have been found in the odontoblastic and subodontoblastic layers of the dental pulp. Our previous examinations have shown that PACAP deficiency causes alterations in the morphology and structure of the developing molars of 7-day-old mice. In our present study, morphometric and structural comparison was performed on the incisors of 1-year-old wild-type and PACAP-deficient mice. Hard tissue density measurements and morphometric comparison were carried out on the mandibles and the lower incisors with micro-CT. For structural examination, Raman microscopy was applied on frontal thin sections of the mandible. With micro-CT morphometrical measurements, the size of the incisors and the relative volume of the pulp to dentin were significantly smaller in the PACAP-deficient group compared to the wild-type animals. The density of calcium hydroxyapatite in the dentin was reduced in the PACAP-deficient mice. No structural differences could be observed in the enamel with Raman microscopy. Significant differences were found in the dentin of PACAP-deficient mice with Raman microscopy, where increased carbonate/phosphate ratio indicates higher intracrystalline disordering. The evaluation of amide III bands in the dentin revealed higher structural diversity in wild-type mice. Based upon our present and previous results, it is obvious that PACAP plays an important role in tooth development with the regulation of morphogenesis, dentin, and enamel mineralization. Further studies are

  8. Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

    DEFF Research Database (Denmark)

    Ingversen, J.

    1975-01-01

    Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS...... with a granular component. Particles with the same structure were present in the protein body preparation from the mutant, where, however, the granular component was the most prominent. Amino-acid composition and SDS-polyacrylamide gel electrophoresis of the proteins from the protein body preparation revealed...

  9. Evidence for allosteric variants of wild-type p53, a tumour suppressor protein.

    OpenAIRE

    1990-01-01

    A tumour suppressor function for p53 is indicated in human lung cancer and in carcinoma of the colorectum. Loss of suppressor function, by mutation of the p53 gene, is associated with activation of p53 as an oncogene. The suppressor (wild type) and oncogenic (mutant) forms of the murine p53 protein are distinguishable at the molecular level by reactivity with anti-p53 monoclonal antibodies. For example, activated mutant p53 fails to react with PAb246 (p53-246 degrees). We now demonstrate that...

  10. Intronic T-DNA insertion in Arabidopsis NBR1 conditionally affects wild-type transcript level

    OpenAIRE

    Rodríguez, Milagros Collados; Wawrzyńska, Anna; Sirko, Agnieszka

    2014-01-01

    Abstract The SALK_135513 line of Arabidopsis thaliana is annotated by GenBank to have the T-DNA insertion in the fourth exon of NBR1 (At4g24690). Careful molecular analyses of the homozygous plants of SALK_135513 line indicated the place of T-DNA insertion in the fourth intron. Unexpectedly, 2 kinds of NBR1 transcripts, the wild-type and the mutated, resulting from alternative splicing events, were detected in those plants. Our findings explain the problems encountered by us with phenotypic e...

  11. Abnormal colonic motility in mice overexpressing human wild-type α-synuclein

    OpenAIRE

    2008-01-01

    The presynaptic protein α-synuclein (αSyn) has been implicated in both familial and sporadic forms of Parkinson’s disease. We examined whether human αSyn-overexpressing mice under Thy1 promoter (Thy1-αSyn) display alterations of colonic function. Basal fecal output was decreased in Thy1-αSyn mice fed ad libitum. Fasted/refed Thy1-αSyn mice had a slower distal colonic transit than the wild-type mice, as monitored by 2.2-fold increase in time to expel an intracolonic bead and 2.9-fold higher co...

  12. Rearing in seawater mesocosms improves the spawning performance of growth hormone transgenic and wild-type coho salmon.

    Directory of Open Access Journals (Sweden)

    Rosalind A Leggatt

    Full Text Available Growth hormone (GH transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination has not been fully ascertained. Hence, salmon were reared in large (350,000 L, semi-natural, seawater tanks (termed mesocosms designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic

  13. In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains

    Directory of Open Access Journals (Sweden)

    Foucras Gilles

    2007-09-01

    Full Text Available Abstract Myxoma virus (MYXV, a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1 and a vaccinal strain (SG33 of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies.

  14. Mycoplasma gallisepticum infection in wild-type turkeys living in close contact with domestic fowl.

    Science.gov (United States)

    Jessup, D A; DaMassa, A J; Lewis, R; Jones, K R

    1983-12-01

    Mycoplasma gallisepticum was isolated from 2 wild-type turkeys (Meleagris gallopavo) and 1 domestic turkey living in close contact on a farm in Tehama County, California. Sinusitis was detected in 2 of 14 wild-type turkeys and in 1 of 12 feral broad-breasted bronze turkeys, but in none of several chickens on the premises. The entire mixed flock was captured, sinus aspirates were collected from affected birds, and blood samples were obtained from all birds for serologic testing. Blood samples also were obtained from 10 domestic turkeys on adjacent premises from which breeding stock had been borrowed. The M gallisepticum isolated from sinus aspirates was typed and inoculated into susceptible chickens, resulting in airsacculitis. California wild turkeys with and without histories of exposure to domestic fowl and wild turkeys shipped into California from Texas for release were tested for antibodies to M gallisepticum, using the plate agglutination test. Evidence of M gallisepticum infection was not found in wild turkeys at any location other than the original premises.

  15. Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study.

    Directory of Open Access Journals (Sweden)

    Zhuo Song

    2011-11-01

    Full Text Available The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(- in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(- polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.

  16. Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study.

    Science.gov (United States)

    Song, Zhuo; Cao, Yang; Samuels, David C

    2011-11-01

    The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.

  17. Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains

    Directory of Open Access Journals (Sweden)

    Schouenborg Jens

    2008-05-01

    Full Text Available Abstract Background Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1 if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2 if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR. The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO2 laser heat pulses. Results While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different. Conclusion Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting.

  18. Durations and frequencies of free locomotion in wild type and GABAergic mutants of Caenorhabditis elegans.

    Science.gov (United States)

    Shingai, R

    2000-09-01

    We investigated how much time wild-type Caenorhabditis elegans (Bristol N2) nematodes and the GABA-deficient unc25 mutant and the vesicular GABA transporter-deficient unc47 mutant spent moving. The worms were allowed to move freely on the surface of agarose plates either with or without the food bacterium OP50. We identified forward movement, backward movement, resting and turns by watching images on video and computer displays. Forward movement lasted longer and rests were briefer without, than with, bacteria. Frequency distributions except for backward movement fitted a sum of two exponential functions. The duration of backward movement was not strongly influenced by exposure to bacteria, whereas the frequency of backward movements increased in their presence. The duration of forward movement of unc25 nematodes had no long component, thus differing from that of N2 and unc47 strain nematodes in treatments with and without bacteria. The durations of resting in these mutants were much longer than in the N2 strain, especially in the absence of bacteria. The turn frequency of unc47 nematodes had a higher short component than that of the wild type N2 and unc25 nematodes, in the absence of bacteria. A neural network model is discussed in conjunction with the features of mutants and current knowledge of GABAergic neural transmission.

  19. Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

    Institute of Scientific and Technical Information of China (English)

    YI Long; ZHOU Chang-yong

    2014-01-01

    The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

  20. Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants.

    Science.gov (United States)

    Oberpichler, Inga; Molina, Isabel; Neubauer, Olivia; Lamparter, Tilman

    2006-01-23

    Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.

  1. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    Science.gov (United States)

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  2. Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

    Science.gov (United States)

    Chen, Kuan-Hui Ethan; Walker, Ameae M

    2016-06-01

    Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

  3. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

    Science.gov (United States)

    Druzhinina, Irina S; Schmoll, Monika; Seiboth, Bernhard; Kubicek, Christian P

    2006-03-01

    The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.

  4. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    Science.gov (United States)

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection.

  5. The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice.

    Science.gov (United States)

    Reuvers, John; Thoreson, Andrew R; Zhao, Chunfeng; Zhang, Ling; Jay, Gregory D; An, Kai-Nan; Warman, Matthew L; Amadio, Peter C

    2011-10-01

    The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity. A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out ⁻/⁻, heterozygous ⁺/⁻ and wild-type ⁺/⁺. A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties. There was no significant difference in the Young's modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice. Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated.

  6. Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Wei Hou; Feng Wang; Cen Qiu; Yan Zhu; Ling Huang; Jing Zhao

    2012-01-01

    AIM:To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth.METHODS:The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry,and the results were correlated with clinicopathological parameters,including the mitotic count,proliferative index (Ki-67 immunohistochemical staining),mitotic index (phospho-histone H3 immunohistochemical staining)and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling).Using primary cultured GIST cells,the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting,methyl thiazolyl tetrazolium (MTT),and apoptosis assays.RESULTS:We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples,respectively,and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis,including larger tumor size (P =0.0118),higher mitotic count (P =0.0058),higher proliferative index (P =0.0012),higher mitotic index (P =0.0282),lower apoptosis index (P =0.0484),and increased National Institutes of Health risk level (P =0.0012).We also found that the introduction of exogenous SCF potently increased KIT kinase activity,stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation,while a KIT immunoblocking antibody suppressed proliferation (P =0.01) and promoted apoptosis (P < 0.01)in cultured GIST cells.CONCLUSION:SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth.The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

  7. Adolescent social defeat disturbs adult aggression-related impulsivity in wild-type rats.

    Science.gov (United States)

    Coppens, Caroline M; Coolen, Alex; de Boer, Sietse F; Koolhaas, Jaap M

    2014-10-01

    Adolescence is generally considered as a developmental period during which adverse social experiences may have lasting consequences in terms of an increased vulnerability to affective disorders. This study aimed at determining the individual susceptibility to adolescent social stress using a rat model. We used rats of the Wild-type Groningen strain, which are characterized by a broad variation in adult levels of aggression and impulsivity. We hypothesized that experience of social defeat in adolescence results in heightened aggression and impulsivity levels in adulthood. In contrast to our expectation, adolescent social defeat did not lead to a difference in the average adult level of aggression and impulsivity, but the significant correlation between offensive aggression and impulsivity found in control animals was not present in animals defeated during adolescence.

  8. Characterization of melanin produced by a wild-type strain of Bacillus cereus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jianping; CAI Jun; DENG Yinyue; CHEN Yuehua; REN Gaixin

    2007-01-01

    Bacillus cereus 58 (Bc58)is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine.The Fourier-transform infrared (FT-IR)spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma).A bioassay shows that the LC50 of a Bacillus thuringiensis (Bt)formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml,which is similar to that of the Bt formulation without UV treatment,however,it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58.The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation.This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation.

  9. The effects of wild-type and mutant SOD1 on smooth muscle contraction

    Directory of Open Access Journals (Sweden)

    Nikolić-Kokić Aleksandra

    2015-01-01

    Full Text Available In this work we compared the mutated liver copper zinc-containing superoxide dismutase (SOD1 protein G93A of the transgenic rat model of familial amyotrophic lateral sclerosis (FALS, to wild-type (WT rat SOD1. We examined their enzymatic activities and effects on isometric contractions of uteri of healthy virgin rats. G93A SOD1 showed a slightly higher activity than WT SOD1 and, in contrast to WT SOD1, G93A SOD1 did not induce smooth muscle relaxation. This result indicates that effects on smooth muscles are not related to SOD1 enzyme activity and suggest that heterodimers of G93A SOD1 form an ion-conducting pore that diminishes the relaxatory effects of SOD1. We propose that this type of pathogenic feedback affects neurons in FALS. [Projekat Ministarstva nauke Republike Srbije, br. 173014 i br. 175083

  10. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a.

    Science.gov (United States)

    Vanderslice, P; Copeland, W C; Robertus, J D

    1986-11-15

    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  11. Isolation and Translation of Hordein Messenger RNA from Wild Type and Mutant Endosperms in Barley

    DEFF Research Database (Denmark)

    Brandt, Anders Bøving; Ingwersen, J.

    1978-01-01

    Nucleotide sequences of four cDNA clones coding for the carboxy-terminal portion of at least two different B1 hordein polypeptides are presented. The open reading frame in the nucleotide sequence of the the largest clone (pc hor2-4, 720 nucleotides) translates into the 181 carboxy-terminal amino...... was found to be a major constituent of the total messenger RNA population of the endosperm cell. Polyadenylated hordein messenger RNA sedimented at 11S in sucrose gradients and electrophoretic analysis reveals the presence of at least three RNA species with apparent molecular weights of 0.45, 0.36 and 0.......30 megadaltons. The 11S messenger RNA was translated in vitro into hordein precursor polypeptides which are 2–4 kilodaltons larger than the native hordein polypeptides. The endosperm cell of mutant No. 1508 contained twice as much RNA as the wild type endosperm cell but the same amount of polyadenylated 11S RNA...

  12. Abnormal colonic motility in mice overexpressing human wild-type alpha-synuclein.

    Science.gov (United States)

    Wang, Lixin; Fleming, Sheila M; Chesselet, Marie-Françoise; Taché, Yvette

    2008-05-28

    The presynaptic protein alpha-synuclein (alphaSyn) has been implicated in both familial and sporadic forms of Parkinson's disease. We examined whether human alphaSyn-overexpressing mice under Thy1 promoter (Thy1-alphaSyn) display alterations of colonic function. Basal fecal output was decreased in Thy1-alphaSyn mice fed ad libitum. Fasted/refed Thy1-alphaSyn mice had a slower distal colonic transit than the wild-type mice, as monitored by 2.2-fold increase in time to expel an intracolonic bead and 2.9-fold higher colonic fecal content. By contrast, Thy1-alphaSyn mice had an increased fecal response to novelty stress and corticotropin releasing factor injected intraperipherally. These results indicate that Thy1-alphaSyn mice display altered basal and stress-stimulated propulsive colonic motility and will be a useful model to study gut dysfunction associated with Parkinson's disease.

  13. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form

    Science.gov (United States)

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme. PMID:26417201

  14. An Ultra-Violet Tolerant Wild-Type Strain of Melanin-Producing Bacillus thuringiensis

    Science.gov (United States)

    Sansinenea, Estibaliz; Salazar, Francisco; Ramirez, Melanie; Ortiz, Aurelio

    2015-01-01

    Background: Bacillus thuringiensis is the most successful biological control agent used in agriculture, forestry and mosquito control. However, the insecticidal activity of the B. thuringiensis formulation is not very stable and rapidly loses its biological activity under field conditions, due to the ultraviolet radiation in sunlight. Melanin is known to absorb radiation therefore photo protection of B. thuringiensis based on melanin has been extensively studied. Objectives: The aim of this study was to find a wild type strain of naturally melanin-producing B. thuringiensis to avoid any mutation or manipulation that can affect the Cry protein content. Materials and Methods: Bacillus thuringiensis strains were isolated from soils of different States of Mexico and pigment extraction was followed by lowering the pH to 2 using 1N HCl. Pigment was characterized by some chemical tests based on its solubility, bleaching by H2O2 and flocculation with FeCl3, and using an Infrared (IR) spectrum. Ultraviolet (UV) irradiation experiment was performed to probe the melanin efficacy. Results: ELI52 strain of B. thuringiensis was confirmed to naturally produce melanin. The Cry protein analysis suggested that ELI52 is probably a B. thuringiensis subsp. israelensis strain with toxic activity against the Diptera order of insects. Ultra Violet protection efficacy of melanin was probed counting total viable colonies after UV radiation and comparing the results with the non-producing melanin strain L-DOPA (L-3, 4-dihydroxyphenylalanine) was also detected in the culture. ELI52 strain showed an antagonistic effect over some common bacteria from the environment. Conclusions: ELI52 wild-type strain of B. thuringiensis is a good bio-insecticide that produces melanin with UV-resistance that is probably toxic against the Diptera order of insects and can inhibit the growth of other environmental bacteria. PMID:26421136

  15. Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

    Science.gov (United States)

    Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

    2016-02-19

    Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

  16. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor.

    Science.gov (United States)

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A; Metzeler, Klaus H; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-06-27

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.

  17. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

    Science.gov (United States)

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P.; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A.; Metzeler, Klaus H.; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  18. Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice.

    Science.gov (United States)

    Okada, Hiroyuki; Masujin, Kentaro; Miyazawa, Kohtaro; Yokoyama, Takashi

    2015-07-13

    L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP(Sc)) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrP(Sc) of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrP(Sc) glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice.

  19. Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain

    DEFF Research Database (Denmark)

    Pedersen, Henrik; Carlsen, Morten; Nielsen, Jens Bredal

    1999-01-01

    Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized,vith respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition...... or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain....

  20. Effects of salt stress on wild type and vte4 mutant Arabidopsis thaliana: Model plant to engineer tolerance towards salinity

    Directory of Open Access Journals (Sweden)

    Khalatbari Amir Ali

    2013-01-01

    Full Text Available One of the major environmental constraints impairing plant distribution and yield is believed to be salt stress. Additionally, engineered abiotic stress resistance or/and tolerance is considered as an indispensable target in order to enhance plant productivity. In this study, the effects of salinity on physiological and morphological of wild type (Columbia-0 and vte4 mutant Arabidopsis thaliana were investigated under different NaCl concentrations. These salt treatments, including control condition, 50mM and 100mM NaCl were imposed on the plants. Each salt treatment was replicated three times in a complete randomized design with factorial arrangement. Wild type and mutant A.thaliana plants were subjected to the abiotic stress (salinity for up to 11 days to evaluate the parameters of growth, development and water relations. As a result, the performance of wild type plants was stronger than vte4 mutant under different salt treatments. Under control condition, rosette dry weight, maximum quantum efficiency (PSII and specific leaf area obtained the highest values of 13.85 mg, considered, wild type A.thaliana recorded higher value of 0.82 gW/gFW for relative water content (RWC under 50mM NaCl whereas mutant plants gained the value of 0.78 gW/gFW under the same condition. However, root mass fraction indicated an increase for both wild type and vte4 mutant plants after 11 days of salt stress onset. The reduction of water potential was observed for wild type and mutant A.thaliana where it scored -1.3 MPa and -1.4, respectively. As a conclusion, these findings implied that under different salt treatments morphological and physiological responses of wild type and vte4 mutant were affected in which wild type plants showed more tolerance. Lack of γ-tocopherol methyltransferase (γ -TMT gene in vte4 seemed to impair defence mechanism of this mutant against salinity.

  1. Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

    Science.gov (United States)

    Huang, I-Hsiu; Sarker, Mahfuzur R

    2006-09-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

  2. Study on the mating compatibility of part pear varieties and wild types of Pyrus ussuriensis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To understand the mating compatibility of Pyrus ussuriensis Maxim.,we studied the fertility of pollen and conducted a hand-pollination trial in the field on some pear varieties and wild types.The results showed that about 53% of varieties among 32 tested genotypes were male sterile.Not only did the pollen vitalities in normal varieties show distinct differences,but pollen vitalities from flower forcing in a glasshouse were found to be lower than those from natural flowering in the field,which had no apparent effect on fruit setting of tested varieties.Most of the tested genotypes such as Nanguoli,Pingxiangli,and Hanxiangli showed selfincompatibility (SI).Honghuagaili could bear fruit after hand pollination,but there were abnormal seeds in its fruits.So we suggested it was a recessive SI that happened during embryo development.Longxiangli has the capacity of self-compatibility (SC) to some extent,its fruit setting rate of inflorescence could reach 23.3%.Manual self-pollination during bud flowering could improve the fruit setting rate of part tested genotypes with SI,but had no effect on the fruit setting rate 3 days after flowering.Mating between female parents with the variety selected from F1 generation showed that the majority of their combinations were compatible.There was one-way SC when Nanguoli was crossed with Hanhongli,while no fruits could be found after Hanhongli was crossed with Nanguoli.It may be related to the S-genotype or haplotype of Nanguoli.In addition,mating between the varieties derived from bud mutation with the female parent appeared incompatible.We concluded that P.ussuriensis Maxim.is similar to other grown pear systems with the characteristics of SI,the fruit setting rate of self pollination in some varieties and wild types can be improved by artificial self-pollination during bud flowering,and fruit cannot be developed through pollination between the varieties from bud mutation and the female parent.

  3. Cellulosic fuel ethanol: alternative fermentation process designs with wild-type and recombinant Zymomonas mobilis.

    Science.gov (United States)

    Lawford, Hugh G; Rousseau, Joyce D

    2003-01-01

    Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The "Iogen Process" for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity

  4. Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae.

    Science.gov (United States)

    Lukacin, Richard; Schreiner, Stephan; Silber, Katrin; Matern, Ulrich

    2005-02-01

    Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.

  5. Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression.

    Science.gov (United States)

    Sawatsky, Bevan; Wong, Xiao-Xiang; Hinkelmann, Sarah; Cattaneo, Roberto; von Messling, Veronika

    2012-04-01

    To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.

  6. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  7. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

    Science.gov (United States)

    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  8. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

    Science.gov (United States)

    Wang, Xinhe; McGovern, Gillian; Zhang, Yi; Wang, Fei; Zha, Liang; Jeffrey, Martin; Ma, Jiyan

    2015-07-01

    The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

  9. Comparative whole genome sequence analysis of wild-type and cidofovir-resistant monkeypoxvirus

    Directory of Open Access Journals (Sweden)

    Huggins John

    2010-05-01

    Full Text Available Abstract We performed whole genome sequencing of a cidofovir {[(S-1-(3-hydroxy-2-phosphonylmethoxy-propyl cytosine] [HPMPC]}-resistant (CDV-R strain of Monkeypoxvirus (MPV. Whole-genome comparison with the wild-type (WT strain revealed 55 single-nucleotide polymorphisms (SNPs and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV. These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies.

  10. Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics

    Science.gov (United States)

    Wong, Min Hao; Giraldo, Juan P.; Kwak, Seon-Yeong; Koman, Volodymyr B.; Sinclair, Rosalie; Lew, Tedrick Thomas Salim; Bisker, Gili; Liu, Pingwei; Strano, Michael S.

    2016-10-01

    Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors--single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal--embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

  11. Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

    Science.gov (United States)

    2016-01-01

    Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

  12. Wild-type p53 binds to MYC promoter G-quadruplex

    Science.gov (United States)

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejčí, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matej; Vorlíčková, Michaela; Brázdová, Marie

    2016-01-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320–393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes. PMID:27634752

  13. Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

    Science.gov (United States)

    Nam, Kyong-Hee; Kim, Do-Young; Shin, Hee Jae; Nam, Ki Jung; An, Joo Hee; Pack, In-Soon; Park, Jung-Ho; Jeong, Soon-Chun; Kim, Ho Bang; Kim, Chang-Gi

    2014-06-15

    Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions.

  14. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

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    Xinhe Wang

    2015-07-01

    Full Text Available The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p. inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

  15. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus

    Directory of Open Access Journals (Sweden)

    Camila Michele Appolinario

    2015-10-01

    Full Text Available ABSTRACTWe have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2 or vampire bat (hv3 variants and both groups were treated or leaved as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p = 0.0006. Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of Ngene in the same period (p < 0.0001. In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

  16. Comparative metabolomic analysis of wild type and mads3 mutant rice anthers

    Institute of Scientific and Technical Information of China (English)

    Guorun Qu; Sheng Quan; Palash Mondol; Jie Xu; Dabing Zhang; Jianxin Shi

    2014-01-01

    Rice (Oryza sativa L.) MADS3 transcription factor regulates the homeostasis of reactive oxygen species (ROS) during late anther development, and one MADS3 mutant, mads3-4, has defective anther wal s, aborted microspores and complete male sterility. Here, we report the untargeted metabolomic analysis of both wild type and mads3-4 mature anthers. Mutation of MADS3 led to an unbalanced redox status and caused oxidative stress that damages lipid, protein, and DNA. To cope with oxidative stress in mads3-4 anthers, soluble sugars were mobilized and carbohydrate metabolism was shifted to amino acid and nucleic acid metabolism to provide substrates for the biosynthesis of antioxidant proteins and the repair of DNA. Mutation of MADS3 also affected other aspects of rice anther development such as secondary metabolites associated with cuticle, cellwal , and auxin metabolism. Many of the discovered metabolic changes in mads3-4 anthers were corroborated with changes of expression levels of correspond-ing metabolic pathway genes. Altogether, this comparative metabolomic analysis indicated that MADS3 gene affects rice anther development far beyond the ROS homeostasis regulation.

  17. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    Science.gov (United States)

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  18. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants.

    Science.gov (United States)

    Herrera, Rito; Álvarez, María C; Gelis, Samuel; Ramos, José

    2013-09-15

    Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

  19. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    Science.gov (United States)

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

  20. Wild-type p53 binds to MYC promoter G-quadruplex.

    Science.gov (United States)

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejčí, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matej; Vorlíčková, Michaela; Brázdová, Marie

    2016-10-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

  1. Expression signature based on TP53 target genes doesn't predict response to TP53-MDM2 inhibitor in wild type TP53 tumors.

    Science.gov (United States)

    Sonkin, Dmitriy

    2015-10-22

    A number of TP53-MDM2 inhibitors are currently under investigation as therapeutic agents in a variety of clinical trials in patients with TP53 wild type tumors. Not all wild type TP53 tumors are sensitive to such inhibitors. In an attempt to improve selection of patients with TP53 wild type tumors, an mRNA expression signature based on 13 TP53 transcriptional target genes was recently developed (Jeay et al. 2015). Careful reanalysis of TP53 status in the study validation data set of cancer cell lines considered to be TP53 wild type detected TP53 inactivating alterations in 23% of cell lines. The subsequent reanalysis of the remaining TP53 wild type cell lines clearly demonstrated that unfortunately the 13-gene signature cannot predict response to TP53-MDM2 inhibitor in TP53 wild type tumors.

  2. Low-Dose Actinomycin-D Induces Redistribution of Wild-Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells.

    Science.gov (United States)

    Brodská, Barbora; Holoubek, Aleš; Otevřelová, Petra; Kuželová, Kateřina

    2016-06-01

    Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

  3. Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

    NARCIS (Netherlands)

    Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

    2006-01-01

    A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and tim

  4. Comparative metabolic flux analysis of an Ashbya gossypii wild type strain and a high riboflavin-producing mutant strain.

    Science.gov (United States)

    Jeong, Bo-Young; Wittmann, Christoph; Kato, Tatsuya; Park, Enoch Y

    2015-01-01

    In the present study, we analyzed the central metabolic pathway of an Ashbya gossypii wild type strain and a riboflavin over-producing mutant strain developed in a previous study in order to characterize the riboflavin over-production pathway. (13)C-Metabolic flux analysis ((13)C-MFA) was carried out in both strains, and the resulting data were fit to a steady-state flux isotopomer model using OpenFLUX. Flux to pentose-5-phosphate (P5P) via the pentose phosphate pathway (PPP) was 9% higher in the mutant strain compared to the wild type strain. The flux from purine synthesis to riboflavin in the mutant strain was 1.6%, while that of the wild type strain was only 0.1%, a 16-fold difference. In addition, the flux from the cytoplasmic pyruvate pool to the extracellular metabolites, pyruvate, lactate, and alanine, was 2-fold higher in the mutant strain compared to the wild type strain. This result demonstrates that increased guanosine triphosphate (GTP) flux through the PPP and purine synthesis pathway (PSP) increased riboflavin production in the mutant strain. The present study provides the first insight into metabolic flux through the central carbon pathway in A. gossypii and sets the foundation for development of a quantitative and functional model of the A. gossypii metabolic network.

  5. Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels.

    Directory of Open Access Journals (Sweden)

    William W Motley

    2011-12-01

    Full Text Available Charcot-Marie-Tooth disease type 2D (CMT2D is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS. In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.

  6. Wild-type mouse models to screen antisense oligonucleotides for exon-skipping efficacy in Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Limin Cao

    Full Text Available A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD, a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.

  7. Responses of Wild-Type and Resistant Strains of the Hyperthermophilic Bacterium Thermotoga maritima to Chloramphenicol Challenge▿ †

    Science.gov (United States)

    Montero, Clemente I.; Johnson, Matthew R.; Chou, Chung-Jung; Conners, Shannon B.; Geouge, Sarah G.; Tachdjian, Sabrina; Nichols, Jason D.; Kelly, Robert M.

    2007-01-01

    Transcriptomes and growth physiologies of the hyperthermophile Thermotoga maritima and an antibiotic-resistant spontaneous mutant were compared prior to and following exposure to chloramphenicol. While the wild-type response was similar to that of mesophilic bacteria, reduced susceptibility of the mutant was attributed to five mutations in 23S rRNA and phenotypic preconditioning to chloramphenicol. PMID:17557852

  8. Long-Term Treatment with Erlotinib for EGFR Wild-Type Non-Small Cell Lung Cancer: A Case Report.

    Science.gov (United States)

    Polychronidou, Genovefa; Papakotoulas, Pavlos

    2013-01-01

    The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are known to have greater efficacy in EGFR mutation-positive non-small cell lung cancer (NSCLC), although erlotinib also has activity in wild-type disease. We report the successful long-term maintenance treatment of a patient with EGFR wild-type NSCLC with gefitinib and later erlotinib. The patient (male; 44 years old; smoker) was diagnosed with EGFR wild-type NSCLC after computer tomography had revealed a mediastinal mass, and histology and mutation testing had identified the tumor as an EGFR wild-type grade 3 adenocarcinoma. The patient received multiple rounds of chemotherapy, followed by gefitinib maintenance (3 years). Later on, he received erlotinib maintenance and developed a persistent rash (grade 1/2) that lasted throughout the treatment. The patient's condition has remained stable on erlotinib for more than 5 years, with no evidence of progression. We describe the patient's disease course and treatment in the context of EGFR TKI therapy and the prognostic factors for long-term clinical outcomes of NSCLC, including the development of erlotinib-induced rash.

  9. PREPHENATE DEHYDRATASE OF THE ACTINOMYCETE AMYCOLATOPSIS-METHANOLICA - PURIFICATION AND CHARACTERIZATION OF WILD-TYPE AND DEREGULATED MUTANT PROTEINS

    NARCIS (Netherlands)

    EUVERINK, GJW; WOLTERS, DJ; DIJKHUIZEN, L

    1995-01-01

    Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric

  10. Mating compatibility and competitiveness of transgenic and wild type Aedes aegypti (L.) under contained semi-field conditions.

    Science.gov (United States)

    Lee, H L; Vasan, Seshadri; Ahmad, Nazni Wasi; Idris, Iswarti; Hanum, Norhaida; Selvi, S; Alphey, Luke; Murad, Shahnaz

    2013-02-01

    We conducted the world's first experiments under semi-field conditions (ACL-2 field house) to assess the mating competitiveness of genetically sterile RIDL male mosquitoes (513A strain). The field house is a state-of-the-art, fully-contained trial facility, simulating the living space for a household of 2-4 people in Peninsular Malaysia. Ten genetically sterile RIDL male A. aegypti mosquitoes competed with ten wild type males inside this field house to mate with ten wild type females. Hatched larvae from mated females were screened under a fluorescent microscope for genetic markers to determine if they were fathered by RIDL male or wild type male, and all results were cross-checked by PCR. Two such experiments were conducted, each repeated sufficient number of times. All strains were on a Malaysian lab strain background for the first experiment, while the RIDL males alone were on a recently-colonised Mexican strain background for the second experiment. A total of 52 % of the matings were with RIDL males in the first experiment, while 45 % of the matings were with RIDL (Mexican) males in the second experiment. Statistically, this is not significantly different from 50 % of the matings expected to take place with RIDL males if the latter were as competitive as that of the wild type males. This shows that A. aegypti RIDL-513A has excellent mating competitiveness under semi-field conditions, verifying earlier trends obtained in small lab cages. We also observed high mating compatibility between recently-colonised Mexican RIDL males and lab-reared Malaysian wild type females.

  11. Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.

    Science.gov (United States)

    Holstead, Ryan G; Li, Man-Song; Linsdell, Paul

    2011-10-01

    Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.

  12. Sequence analysis of VP4 genes of wild type and culture adapted human rotavirus G1P[8] strains

    Institute of Scientific and Technical Information of China (English)

    Ritu Arora; Ganesh S Dhale; Pooja R Patil; Shobha D Chitambar

    2011-01-01

    Objective:To conduct a comparative analysis of the VP4gene sequences of Indian wild type (06361,0613158, 061060and0715880) and cell culture adapted (06361-CA, 0613158-CA, 061060-CAand0715880-CA) G1P[8] rotavirus strains.Methods: Full-length VP4 genes of each of the four wild type G1P[8] rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected toRT-PCRamplification and nucleotide sequencing. Results: All four cell culture adaptedG1P[8]rotavirus strains showed nucleotide and amino acid substitutions in theVP4 gene as compared to their wild type strains. The number of substitutions however, varied from1-64and 1-13 respectively. The substitutions were distributed in both VP5*andVP8* subunits ofVP4gene respectively of permeabilization and hemagglutinating activity. The presence of unique amino acid substitutions was identified in two of the four wild type (V377G, S387N in 061060and I644Lin0715880) and all four cell culture adapted (A46Vin0613158-CA, T60R in06361-CA, L237V, G389V andQ480H in061060-CA andS615G andT625Pin0715880-CA) strains for the first time in theVP4 gene ofP[8]specificity. Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.Conclusions: Amino acid substitutions detected in the VP4 genes ofG1P[8]rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation, immunogenicity and conformation is needed for the development of newer rotavirus vaccines.

  13. Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.

    Science.gov (United States)

    Winter, J C; Amorosi, D J; Rice, Kenner C; Cheng, Kejun; Yu, Ai-Ming

    2011-09-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT.

  14. Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.

    Directory of Open Access Journals (Sweden)

    Jennifer J Jordan

    2008-06-01

    Full Text Available Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt, originally defined by the consensus RRRCWWGYYY (n = 0-13 RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs by wild type (WT and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2-(a single decamer and (3/4-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2- and (3/4-site REs greatly expands the p53 master regulatory network.

  15. Spaceflight influences both mucosal and peripheral cytokine production in PTN-Tg and wild type mice.

    Directory of Open Access Journals (Sweden)

    Justin L McCarville

    Full Text Available Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg were examined using the novel Mouse Drawer System (MDS aboard the International Space Station (ISS over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2 and transforming growth factor-beta1 (TGF-β1 were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice.

  16. Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ.

    Science.gov (United States)

    Peng, Cheng; Arthur, Dionne M; Sichani, Homa Teimouri; Xia, Qing; Ng, Jack C

    2013-11-01

    Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48 h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005-5 μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater.

  17. A novel response of wild-type duckweed (Lemna paucicostata Hegelm.) to heavy metals.

    Science.gov (United States)

    Li, Tianyu; Xiong, Zhiting

    2004-04-01

    The responses of wild-type Lemna paucicostata, used as the experimental stock, to seven heavy metals were investigated with a hydroponic culture in a culture facility. The results showed that six of the metals could induce L. paucicostata to release daughter fronds from the mother frond before maturity, resulting in breakup of the colony. This phenomenon is novel and has not been reported before. Six of the heavy metals investigated-copper, cadmium, nickel, zinc, mercury, and chromium-could induce this response, but sensitivity and response of the plant varied. When Cu(2+) at a concentration of 0.2 micromol/L was added, the response was significantly different from that of the control after a 2-h exposure. The response to cadmium (0.8 micromol/L) was significantly different after a 6-h exposure; to zinc (10 micromol/L) and nickel (5 micromol/L), after 5 and 7 h, respectively. But for lead, even at a concentration of 80 micromol/L for 96 h, no colony breakup was induced. The response to heavy metals occurred within 24 h; increasing the exposure by another 24 h had nearly no additional effect on the colony breakup. When the response was over, the number of colonies with fronds varied with the inducement capacity of the metal. Nutrients could affect the sensitivity of L. paucicostata to some heavy metals, such as mercury, chromium, zinc, and nickel, but not to copper and cadmium. Sensitivity was also changed when CuCl(2) was used as the copper source instead of CuSO(4). These results indicate that some heavy metals could serve as a toxicant for inducing L. paucicostata to release daughter fronds in advance. This change might involve a stress response such as ethylene production. Also discussed is the possibility of this response as a biomarker in a bioassay.

  18. Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

    Science.gov (United States)

    Breda, Ardala; Martinelli, Leonardo K B; Bizarro, Cristiano V; Rosado, Leonardo A; Borges, Caroline B; Santos, Diógenes S; Basso, Luiz A

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of

  19. Wild type p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress.

    Directory of Open Access Journals (Sweden)

    Carlos Farkas

    Full Text Available SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin receptors and showed that it plays a role in neuronal function, which does not necessarily explain why or how SALL2 is deregulated in cancer. Previous evidences indicate that SALL2 gene is regulated by the WT1 and AP4 transcription factors. Here, we identified SALL2 as a novel downstream target of the p53 tumor suppressor protein. Bioinformatic analysis of the SALL2 gene revealed several putative p53 half sites along the promoter region. Either overexpression of wild-type p53 or induction of the endogenous p53 by the genotoxic agent doxorubicin repressed SALL2 promoter activity in various cell lines. However R175H, R249S, and R248W p53 mutants, frequently found in the tumors of cancer patients, were unable to repress SALL2 promoter activity, suggesting that p53 specific binding to DNA is important for the regulation of SALL2. Electrophoretic mobility shift assay demonstrated binding of p53 to one of the identified p53 half sites in the Sall2 promoter, and chromatin immunoprecipitation analysis confirmed in vivo interaction of p53 with the promoter region of Sall2 containing this half site. Importantly, by using a p53ER (TAM knockin model expressing a variant of p53 that is completely dependent on 4-hydroxy-tamoxifen for its activity, we show that p53 activation diminished SALL2 RNA and protein levels during genotoxic cellular stress in primary mouse embryo fibroblasts (MEFs and radiosensitive tissues in vivo. Thus, our finding indicates that p53 represses SALL2 expression in a context-specific manner, adding knowledge to the understanding of SALL2 gene regulation, and to a potential mechanism for its deregulation in cancer.

  20. Wild-type phosphoribosylpyrophosphate synthase (PRS from Mycobacterium tuberculosis: a bacterial class II PRS?

    Directory of Open Access Journals (Sweden)

    Ardala Breda

    Full Text Available The 5-phospho-α-D-ribose 1-diphosphate (PRPP metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS. Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure

  1. Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Pallisgård, Niels;

    2013-01-01

    OBJECTIVE: The increasing number of negative trials for ovarian cancer treatment has prompted an evaluation of new biologic agents, which in combination with chemotherapy may improve survival. The aim of this study was to investigate the response rate in platinum-resistant, KRAS wild-type ovarian...... cancer patients treated with pegylated liposomal doxorubicin (PLD) supplemented with panitumumab. PATIENTS AND METHODS: Major eligibility criteria were relapsed ovarian/fallopian/peritoneal cancer patients with platinum-resistant disease, measurable disease by GCIG CA125 criteria and KRAS wild-type......-free and overall survival in the intention-to-treat population was 2.7 months (2.5-3.2 months, 95% confidence interval) and 8.1 months (5.6-11.7 months, 95% confidence interval), respectively. The most common treatment-related grade 3 toxicities included skin toxicity (42%), fatigue (19%), and vomiting (12...

  2. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    DEFF Research Database (Denmark)

    Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus;

    2011-01-01

    this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs...... of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis......Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...

  3. Automatic Assessment of Global Craniofacial Differences between Crouzon mice and Wild-type mice in terms of the Cephalic Index

    DEFF Research Database (Denmark)

    Ólafsdóttir, Hildur; Oubel, Estanislao; Frangi, Alejandro F.;

    2006-01-01

    This paper presents the automatic assessment of differences between Wild-Type mice and Crouzon mice based on high-resolution 3D Micro CT data. One factor used for the diagnosis of Crouzon syndrome in humans is the cephalic index, which is the skull width/length ratio. This index has traditionally...... been computed by time-consuming manual measurements that prevent large-scale populational studies. In this study, an automatic method to estimate cephalic index for this mouse model of Crouzon syndrome is presented. The method is based on constructing a craniofacial atlas of Wild-type mice...... and then registering each mouse to the atlas using affine transformations. The skull length and width are then measured on the atlas and propagated to all subjects to obtain automatic measurements of the cephalic index. The registration accuracy was estimated by RMS landmark errors. Even though the accuracy...

  4. The hypertonic environment differentially regulates wild-type CFTR and TNR-CFTR chloride channels.

    Science.gov (United States)

    Lassance-Soares, Roberta M; Cheng, Jie; Krasnov, Kristina; Cebotaru, Liudmila; Cutting, Garry R; Souza-Menezes, Jackson; Morales, Marcelo M; Guggino, William B

    2010-01-01

    This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla.

  5. A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

    Science.gov (United States)

    Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

    2014-05-21

    Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.

  6. Biomarkers predicting resistance to epidermal growth factor receptor-targeted therapy in metastatic colorectal cancer with wild-type KRAS

    Directory of Open Access Journals (Sweden)

    Liu J

    2016-01-01

    Full Text Available Jiang Liu,* Jing Hu,* Lei Cheng, Wei Ren, Mi Yang, Baorui Liu, Li Xie, Xiaoping Qian The Comprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, Jiangsu, People’s Republic of China *These authors contributed equally to this work Abstract: EGFR pathway is an important therapeutic target in human tumors, including metastatic colorectal cancer (mCRC. The advent of EGFR-targeted monoclonal antibodies panitumumab and cetuximab has generated promise for the treatment of mCRC and has largely improved patients’ progression-free survival (PFS and overall survival (OS. However, treatment with anti-EGFR monoclonal antibodies is only effective in a subset of mCRC patients with wild-type KRAS. This indicates that there are other factors affecting the efficacy of anti-EGFR monoclonal antibodies. Existing studies have demonstrated that among colorectal cancer patients with wild-type KRAS, harboring mutations of BRAF, PIK3CA, NRAS, or PTEN-null may demonstrate resistance to anti-EGFR-targeted therapy, and biomarkers detection can provide better-personalized treatment for mCRC patients. How to identify and reverse the secondary resistance to anti-EGFR monoclonal antibody therapy is also another great challenge to improve the anti-EGFR efficacy in wild-type KRAS mCRC patients. Finally, both of the molecular mechanisms of response and acquired resistance would be important for the directions of future research. This review focuses on how to further improve the predictive value of anti-EGFR therapies and how to also try and avoid futile treatment for wild-type KRAS colorectal cancer patients. Keywords: colorectal cancer, EGFR, BRAF, RAS, cetuximab, panitumumab

  7. Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

    OpenAIRE

    1993-01-01

    The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of ...

  8. Sex-Specific Diurnal Immobility Induced by Forced Swim Test in Wild Type and Clock Gene Deficient Mice

    OpenAIRE

    Ningyue Li; Yanhua Xu; Xiaojuan Chen; Qing Duan; Mei Zhao

    2015-01-01

    Objective: The link between alterations in circadian rhythms and depression are well established, but the underlying mechanisms are far less elucidated. We investigated the circadian characteristics of immobility behavior in wild type (WT) mice and mice with mutations in core Clock genes. Methods: All mice were tested with forced swim test (FST) at 4 h intervals. Results: These experiments revealed significant diurnal rhythms associated with immobility behavior in both male and female WT mice...

  9. Rescue of white egg 1 mutant by introduction of the wild-type Bombyx kynurenine 3-monooxygenase gene

    Institute of Scientific and Technical Information of China (English)

    GUO-XING QUAN; ISAO KOBAYASHI; KATSURA KOJIMA; KEIRO UCHINO; TOSHIO KANDA; HIDEKI SEZUTSU; TORU SHIMADA; TOSHIKI TAMURA

    2007-01-01

    In silkworms, the white egg 1 (w-1) mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase (KMO) activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter (A3KMO) or the native KMO gene promoter (KKMO). We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction(RT-PCR) analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.

  10. A Caenorhabditis elegans wild type defies the temperature-size rule owing to a single nucleotide polymorphism in tra-3.

    Directory of Open Access Journals (Sweden)

    Jan E Kammenga

    2007-03-01

    Full Text Available Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature-size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature-size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature-size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 x CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature-size rule, which has puzzled biologists for decades.

  11. Podocyte-specific overexpression of wild type or mutant trpc6 in mice is sufficient to cause glomerular disease.

    Directory of Open Access Journals (Sweden)

    Paola Krall

    Full Text Available Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6 gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.

  12. Assessment of 5-HT7 Receptor Agonists Selectivity Using Nociceptive and Thermoregulation Tests in Knockout versus Wild-Type Mice

    Directory of Open Access Journals (Sweden)

    Alex Brenchat

    2012-01-01

    Full Text Available No study has ever examined the effect of 5-HT7 receptor agonists on nociception by using 5-HT7 receptor knockout mice. Basal sensitivity to noxious heat stimuli and formalin-induced nociception in both phase I and II of the formalin test did not differ in 5-HT7 receptor knockout mice and paired wild-type controls. Similarly, there was no significant difference in basal body temperature between both genotypes. Subcutaneous administration of 5-HT7 receptor agonists AS-19 (10 mg/kg, E-57431 (10 mg/kg, and E-55888 (20 mg/kg significantly reduced formalin-induced licking/biting behavior during the phase II of the test in wild-type but not in 5-HT7 receptor knockout mice. At these active analgesic doses, none of the three 5-HT7 receptor agonists modified the basal body temperature neither in wild-type nor in 5-HT7 receptor knockout mice. However, a significant decrease in body temperature was observed at a higher dose (20 mg/kg of AS-19 and E-57431 in both genotypes. Our data strongly suggest that the 5-HT7 receptor agonists AS-19, E-57431, and E-55888 produce antinociception in the formalin test by activating 5-HT7 receptors. These results also strengthen the idea that the 5-HT7 receptor plays a role in thermoregulation, but by acting in concert with other receptors.

  13. Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

    Directory of Open Access Journals (Sweden)

    Pilar Eraso

    Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

  14. Sex-Specific Diurnal Immobility Induced by Forced Swim Test in Wild Type and Clock Gene Deficient Mice

    Directory of Open Access Journals (Sweden)

    Ningyue Li

    2015-03-01

    Full Text Available Objective: The link between alterations in circadian rhythms and depression are well established, but the underlying mechanisms are far less elucidated. We investigated the circadian characteristics of immobility behavior in wild type (WT mice and mice with mutations in core Clock genes. Methods: All mice were tested with forced swim test (FST at 4 h intervals. Results: These experiments revealed significant diurnal rhythms associated with immobility behavior in both male and female WT mice with sex-different circadian properties. In addition, male mice showed significantly less immobility during the night phase in comparison to female mice. Female Per1Brdm1 mice also showed significant rhythmicity. However, the timing of rhythmicity was very different from that observed in female wild type mice. Male Per1Brdm1 mice showed a pattern of rhythmicity similar to that of wild type mice. Furthermore, female Per1Brdm1 mice showed higher duration of immobility in comparison to male Per1Brdm1 mice in both daytime and early night phases. Neither Per2Brdm1 nor ClockΔ19 mice showed significant rhythmicity, but both female Per2Brdm1 and ClockΔ19 mice had lower levels of immobility, compared to males. Conclusions: This study highlights the differences in the circadian characteristics of immobility induced by FST in WT, ClockΔ19, Per1, and Per2 deficient mice.

  15. Uptake,Subcellular Distribution,and Chemical Forms of Cadmium in Wild-Type and Mutant Rice

    Institute of Scientific and Technical Information of China (English)

    HE Jun-Yu; ZHU Cheng; REN Yan-Fang; YAN Yu-Ping; CHENG Chang; JIANG De-An; SUN Zong-Xiu

    2008-01-01

    Wild-type (Zhonghua 11) and mutant rice (Oryza sativa L.) plants were used to investigate the effect of cadmium (Cd) application on biomass production,to characterize the influx of Cd from roots to shoots,and to determine the form,content,and subcellular distribution of Cd in the roots,leaf sheaths,and leaves of the rice plants.Seedlings were cultivated in a nutrient solution and were treated with 0.5 mmol L-1 of Cd2+ for 14 d.The sensitivity of rice plants to Cd toxicity was tested by studying the changes in biomass production and by observing the onset of toxicity symptoms in the plants.Both the wild-type and mutant rice plants developed symptoms of Cd stress.In addition,Cd application significantly (P ≤ 0.01) decreased dry matter production of roots,leaf sheaths,and leaves of both types,especially the mutant.The Cd content in roots of the mutant was significantly (P≤0.05) higher than that of the wild-type rice.However,there was no significant difference in the Cd content of roots,leaf sheaths,and leaves between the wild-type and mutant rice.Most of the Cd was bound to the cell wall of the roots,leaf sheaths,and leaves,and the mutant had greater Cd content in cell organelles than the wild type.The uneven subcellular distribution could be responsible for the Cd sensitivity of the mutant rice.Furthermore,different chemical forms of Cd were found to occur in the roots,leaf sheaths,and leaves of both types of rice plants.Ethanol-,water-,and NaCl-extractable Cd had greater toxicity than the other forms of Cd and induced stunted growth and chlorosls in the plants.The high Cd content of the toxic forms of Cd in the cell organelles could seriously damage the cells and the metabolic processes in mutant rice plants.

  16. The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants.

    Science.gov (United States)

    De Bolle, Miguel F C; Butaye, Katleen M J; Goderis, Inge J W M; Wouters, Piet F J; Jacobs, Anni; Delauré, Stijn L; Depicker, Ann; Cammue, Bruno P A

    2007-03-01

    Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a beta-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chi-MARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chi-MARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression

  17. Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent;

    2009-01-01

    Young mink kits (n = 8)were vaccinated withDNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodieswere induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres...... demonstrate that early life DNA vaccination with the H gene of a CDV vaccine strain induced robust protective immunity against a recent wild type CDV....

  18. Ratio of mutated versus wild-type coat protein sequences in Pepino mosaic virus determines the nature and severity of yellowing symptoms on tomato plants.

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Paeleman, Anneleen; Ortega-Parra, Nelia; Borodynko, Natasza; Minicka, Julia; Czerwoniec, Anna; Thomma, Bart P H J; Hanssen, Inge M

    2013-12-01

    Recently, Pepino mosaic virus (PepMV) infections causing severe yellowing symptoms in tomato plants have been reported in glasshouse tomato crops. When studying this phenomenon in commercial glasshouses, two different types of yellowing symptoms, occurring in adjacent plants, were distinguished: interveinal leaf yellowing and yellow mosaics. After several weeks, the interveinal leaf yellowing symptoms gradually disappeared and the plant heads became green again, with yellow mosaic patterns on the leaves as an intermediate stage. The sequencing of multiple isolates causing interveinal leaf yellowing identified two point mutations, occurring in positions 155 and 166 of the coat protein (CP), as unique to the yellowing pathotype. Site-directed mutagenesis of infectious clones confirmed that both CP mutations are determinants of the interveinal leaf yellowing symptoms. Sequencing of CP clones from plants or plant parts with the yellow mosaic symptoms resulted in a mixture of wild-type and mutated sequences, whereas sequencing of CP clones from the green heads of recovered plants resulted in only wild-type sequences. Yellow mosaic symptoms could be reproduced by inoculation of an artificial 1:1 mixture of RNA transcripts from the wild-type and mutated infectious clones. These results show that the ratio of mutated versus wild-type sequences can determine the nature and severity of symptom development. The gradual recovery of the plants, which coincides with the disappearance of the yellowing mutations, suggests that selection pressure acts to the advantage of the wild-type virus. Experiments with wild-type and mutated infectious clones showed that reverse mutation events from mutant to wild-type occur and that the wild-type virus does not have a replicative advantage over the mutant. These results suggest that reverse mutation events occur, with subsequent selection pressure acting in favour of the wild-type virus in the growing plant parts, possibly related to a lower

  19. Use of cassette dosing approach to examine the effects of P-glycoprotein on the brain and cerebrospinal fluid concentrations in wild-type and P-glycoprotein knockout rats.

    Science.gov (United States)

    Liu, Xingrong; Cheong, Jonathan; Ding, Xiao; Deshmukh, Gauri

    2014-04-01

    The study objectives were 1) to test the hypothesis that the lack of P-glycoprotein (P-gp) and the inhibition of breast cancer resistance protein (Bcrp) at the blood-brain barrier after cassette dosing of potent P-gp and Bcrp inhibitors were due to low plasma concentrations of those inhibitors and 2) to examine the effects of P-gp on the unbound brain (C(u,brain)) and cerebrospinal fluid (CSF) concentrations (C(u,CSF)) of P-gp substrates in rats. In vitro inhibition of 11 compounds (amprenavir, citalopram, digoxin, elacridar, imatinib, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], loperamide, prazosin, quinidine, sulfasalazine, and verapamil) on P-gp and Bcrp was examined in P-gp- and Bcrp-expressing Madin-Darby canine kidney cells, respectively. An in vivo study was conducted in wild-type and Mdr1a(-/-) rats after subcutaneous cassette dosing of the 11 compounds at 1-3 mg/kg, and the brain, CSF, and plasma concentrations of these compounds were determined. At the maximal unbound concentrations observed in rats at 1-3 mg/kg, P-gp and Bcrp were not inhibited by a cassette of the 11 compounds. For non-P-gp/Bcrp substrates, similar C(u,brain), C(u,CSF), and unbound plasma concentrations (C(u,plasma)) were observed in wild-type and P-gp knockout rats. For P-gp/Bcrp substrates, C(u,brain) ≤ C(u,CSF) ≤ C(u,plasma) in wild-type rats, but C(u,brain) and C(u,CSF) increased in the P-gp knockout rats and were within 3-fold of C(u,plasma) for six of the seven P-gp substrates. These results indicate that P-gp and Bcrp inhibition at the blood-brain barrier is unlikely in cassette dosing and also suggest that P-gp and Bcrp activity at the blood-CSF barrier is functionally not important in determination of the CSF concentration for their substrates.

  20. Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: a BOLD fMRI study

    Energy Technology Data Exchange (ETDEWEB)

    Suarez, S.V.; Changeux, J.P.; Granon, S. [Unite de Neurobiologie Integrative du Systeme Cholinergique, URA CNRS 2182, Institut Pasteur, Departement de Neuroscience, 25 rue du Dr Roux, 75015 Paris (France); Amadon, A.; Giacomini, E.; Le Bihan, D. [Service Hospitalier Frederic Joliot, 4 place du general Leclerc, 91400 Orsay (France); Wiklund, A. [Section of Anaesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm (Sweden)

    2009-07-01

    Rationale: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity {beta}2-containing nicotinic receptors ({beta}2*nAChRs) are located. Objectives We intend to see which brain circuits are activated when nicotine is given in animals naive for nicotine and whether the {beta}2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. Materials and methods: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and {beta}2 knockout (KO) mice. Results: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, {beta}2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via {alpha}7 nicotinic receptors. Conclusions: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on {beta}2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice. (authors)

  1. In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells

    Directory of Open Access Journals (Sweden)

    Vágvölgyi Csaba

    2011-05-01

    Full Text Available Abstract Background Candida parapsilosis typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, C. parapsilosis transforms itself into an opportunistic pathogen. Candida-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading C. parapsilosis. Thus, the aim of our study was to assess the function of dendritic cells in fighting C. parapsilosis and to determine the role that C. parapsilosis-derived lipase plays in the interaction with dendritic cells. Results Monocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively co-cultured with live wild type or lipase deficient C. parapsilosis strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed C. parapsilosis, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient C. parapsilosis cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells. Conclusions Our results show that DCs are activated by exposure to C. parapsilosis, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that C. parapsilosis derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.

  2. Photosystem II Activity of Wild Type Synechocystis PCC 6803 and Its Mutants with Different Plastoquinone Pool Redox States.

    Science.gov (United States)

    Voloshina, O V; Bolychevtseva, Y V; Kuzminov, F I; Gorbunov, M Y; Elanskaya, I V; Fadeev, V V

    2016-08-01

    To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox(-) mutant with naturally reduced PQ is characterized by slower QA(-) reoxidation and O2 evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (Fv/Fm) as compared to the wild type and SDH(-) mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox(-) mutant after dark adaptation. While light adaptation increases Fv/Fm in all tested strains, indicating PSII activation, by far the greatest increase in Fv/Fm and O2 evolution rates is observed in the Ox(-) mutant. Continuous illumination of Ox(-) mutant cells with low-intensity blue light, that accelerates QA(-) reoxidation, also increases Fv/Fm and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH(-) mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm.

  3. Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

    Institute of Scientific and Technical Information of China (English)

    任兆玉; 许晓明; 王水才; 辛越勇; 贺俊芳; 侯洵

    2003-01-01

    A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wildtype rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120ps, repetition rate of 4MHz and wavelength of 514nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wildtype. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

  4. Effect of Wild-type p53 Gene Transfection on the Growth and Radiotherapeutic Sensitivity of Human Glioma Cells

    Institute of Scientific and Technical Information of China (English)

    XIANG Wei; ZHU Xianli; ZHAO Hongyang

    2005-01-01

    To evaluate the effect of wild-type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251cells. p53 gene expression in transfected cells was detected by RT-PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells (inhibition rate (IR):(79.60±5.69) %). The killing effect of irradiation alone on U251 cells was not strong (IR: (17.06±4.35) %, (17.39±1.67) %, (18.73±4.68) %) and increased with the irradiation doses (3,6, 9 Gy). When combined treatment of wild-type p53 gene transfection and irradiation was used,the effect was significantly increased (IR:(80.60±5.35) %, (90.30±1.67) %, (91.30±2.01)%). The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38 %. The rate induced by irradiation increased (4. 61%, 4. 84 %, 5.40 %) with the irradiation doses (3, 6, 9Gy). The apoptosis rate was also significantly increased (17.80 %, 20.03 %, 22.34%) after combined treatment of p53 and irradiation with different doses (3, 6, 9 Gy). It is concluded that wildtype p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

  5. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    Directory of Open Access Journals (Sweden)

    Mary M Melia

    Full Text Available Signalling lymphocyte activation molecule (SLAM has been identified as an immune cell receptor for the morbilliviruses, measles (MV, canine distemper (CDV, rinderpest and peste des petits ruminants (PPRV viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4, also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF,for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  6. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    Science.gov (United States)

    Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

    2014-01-01

    Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  7. Pharmacological activation of wild-type p53 in the therapy of leukemia.

    Science.gov (United States)

    Kojima, Kensuke; Ishizawa, Jo; Andreeff, Michael

    2016-09-01

    The tumor suppressor p53 is inactivated by mutations in the majority of human solid tumors. Conversely, p53 mutations are rare in leukemias and are only observed in a small fraction of the patient population, predominately in patients with complex karyotype acute myeloid leukemia or hypodiploid acute lymphoblastic leukemia. However, the loss of p53 function in leukemic cells is often caused by abnormalities in p53-regulatory proteins, including overexpression of MDM2/MDMX, deletion of CDKN2A/ARF, and alterations in ATM. For example, MDM2 inhibits p53-mediated transcription, promotes its nuclear export, and induces proteasome-dependent degradation. The MDM2 homolog MDMX is another direct regulator of p53 that inhibits p53-mediated transcription. Several small-molecule inhibitors and stapled peptides targeting MDM2 and MDMX have been developed and have recently entered clinical trials. The clinical trial results of the first clinically used MDM2 inhibitor, RG7112, illustrated promising p53 activation and apoptosis induction in leukemia cells as proof of concept. Side effects of RG7112 were most prominent in suppression of thrombopoiesis and gastrointestinal symptoms in leukemia patients. Predictive biomarkers for response to MDM2 inhibitors have been proposed, but they require further validation both in vitro and in vivo so that the accumulated knowledge concerning pathological p53 dysregulation in leukemia and novel molecular-targeted strategies to overcome this dysregulation can be translated safely and efficiently into novel clinical therapeutics.

  8. Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

    Science.gov (United States)

    Lancki, D W; Fields, P; Qian, D; Fitch, F W

    1995-08-01

    The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was

  9. Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

    OpenAIRE

    Johnson, P; Gray, D.; Mowat, M; Benchimol, S

    1991-01-01

    Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine ...

  10. Drug-dependent functionalization of wild-type and mutant p53 in cisplatin-resistant human ovarian tumor cells.

    Science.gov (United States)

    Bhatt, Michelle; Ivan, Cristina; Xie, Xiaolei; Siddik, Zahid H

    2016-12-26

    Cisplatin (cis-Pt) resistance in tumor cells from p53 dysfunction is a significant clinical problem. Although mutation can inhibit p53 function, >60% of p53 mutants retain normal function according to literature reports. Therefore, we examined the status of p53 in cisplatin-resistant ovarian tumor models and its functional response to cis-Pt and the mechanistically-distinct non-cross-resistant oxaliplatin (oxali-Pt). Relative to sensitive A2780 cells harboring wild-type p53, the 2780CP/Cl-16, OVCAR-10, Hey and OVCA-433 cell lines were 10- to 30-fold resistant to cis-Pt, but was substantially circumvented by oxali-Pt. Mutant p53 in 2780CP/Cl-16 (p53V172F) and OVCAR-10 (p53V172F and p53G266R) cells, predicted as non-functional in p53 database, displayed attenuated response to cis-Pt, as did the polymorphic p53P72R (functionally equivalent to wild-type p53) in HEY and OVCA-433 cell lines. However, p53 was robustly activated by oxali-Pt in all cell lines, with resultant drug potency confirmed as p53-dependent by p53 knockout using CRISPR/Cas9 system. This p53 activation by oxali-Pt was associated with phosphorylation at Ser20 by MEK1/2 based on inhibitor and kinase studies. Cis-Pt, however, failed to phosphorylate Ser20 due to downregulated Chk2, and its clinical impact validated by reduced overall survival of ovarian cancer patients according to TCGA database. In conclusion, cis-Pt resistance occurs in both wild-type and mutant p53 ovarian cancer cells, but is associated with loss of Ser20 phosphorylation. However, these mutant p53, like polymorphic p53, are functional and activated by oxali-Pt-induced Ser20 phosphorylation. Thus, the potential exists for repurposing oxali-Pt or similar drugs against refractory cancers harboring wild-type or specific mutant p53.

  11. Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Lund, Bente

    , and head and neck cancer. No previous studies have evaluated the effect of panitumumab in OC based on KRAS mutation status. Methods: Eligibility criteria are confirmed stage I-IV primary epithelial ovarian/fallopian/peritoneal cancer patients with progression either during or within 6 months after end...... to a total of 33 patients. At present, 15 patients have been enrolled. The primary endpoint is to investigate the response rate in platinum-resistant, KRAS wild- type OC patients treated with PLD supplemented with panitumumab. Translational research is included as a secondary endpoint and tumor tissue...

  12. Establishment of a sensitized canine model for kidney transplantation

    Institute of Scientific and Technical Information of China (English)

    XIE Sen; XIA Sui-sheng; TANG Li-gong; CHENG Jun; CHEN Zhi-shui; ZHENG Shan-gen

    2005-01-01

    Objective:To establish a sensitized canine model for kidney transplantation. Methods:12 male dogs were averagely grouped as donors and recipients. A small number of donor canine lymphocytes was infused into different anatomic locations of a paired canine recipient for each time and which was repeated weekly. Specific immune sensitization was monitored by means of Complement Dependent Cytotoxicity (CDC) and Mixed Lymphocyte Culture (MLC) test. When CDC test conversed to be positive and MLC test showed a significant proliferation of reactive lymphocytes of canine recipients, the right kidneys of the paired dogs were excised and transplanted to each other concurrently. Injury of renal allograft function was scheduled determined by ECT dynamic kidney photography and pathologic investigation. Results :CDC test usually conversed to be positive and reactive lymphocytes of canine recipients were also observed to be proliferated significantly in MLC test after 3 to 4 times of canine donor lymphocyte infusions. Renal allograft function deterioration occurred 4 d post-operatively in 4 of 6 canine recipients, in contrast to none in control dogs. Pathologic changes suggested antibody-mediated rejection (delayed) or acute rejection in 3 excised renal allograft of sensitized dogs. Seven days after operation, all sensitized dogs had lost graft function, pathologic changes of which showed that the renal allografts were seriously rejected. 2 of 3 dogs in control group were also acutely rejected. Conclusion:A convenient method by means of repeated stimulation of canine lymphocyte may induce specific immune sensitization in canine recipients. Renal allografts in sensitized dogs will be earlier rejected and result in a more deteriorated graft function.

  13. The pigmentary system of developing axolotls. I. A biochemical and structural analysis of chromatophores in wild-type axolotls.

    Science.gov (United States)

    Frost, S K; Epp, L G; Robinson, S J

    1984-06-01

    A biochemical and transmission electron microscopic description of the wild-type pigment phenotype in developing Mexican axolotls (Ambystoma mexicanum) is presented. There are three pigment cell types found in adult axolotl skin - melanophores, xanthophores and iridophores. Both pigments and pigment cells undergo specific developmental changes in axolotls. Melanophores are the predominant pigment cell type throughout development; xanthophores occur secondarily and in fewer numbers than melanophores; iridophores do not appear until well into the larval stage and remain thereafter as the least frequently encountered pigment cell type. Ultrastructural differences in xanthophore organelle (pterinosome) structure at different developmental stages correlate with changes in the pattern of pteridine biosynthesis. Sepiapterin, a yellow pteridine, is present in larval axolotl skin but not in adults. Riboflavin (also yellow) is present in minimal quantities in larval skin and large quantities in adult axolotl skin. Pterinosomes undergo a morphological "reversion" at some point prior to or shortly after axolotls attain sexual maturity. Correlated with the neotenic state of the axolotl, certain larval pigmentary features are retained throughout development. Notably, the pigment cells remain scattered in the dermis such that no two pigment cell bodies overlap, although cell processes may overlap. This study forms the basis for comparison of the wild type pigment phenotype to the three mutant phenotypes-melanoid, axanthic and albino-found in the axolotl.

  14. Ablation of the Locus Coeruleus Increases Oxidative Stress in Tg-2576 Transgenic but Not Wild-Type Mice

    Directory of Open Access Journals (Sweden)

    Orest Hurko

    2010-01-01

    Full Text Available Mice transgenic for production of excessive or mutant forms of beta-amyloid differ from patients with Alzheimer's disease in the degree of inflammation, oxidative damage, and alteration of intermediary metabolism, as well as the paucity or absence of neuronal atrophy and cognitive impairment. Previous observers have suggested that differences in inflammatory response reflect a discrepancy in the state of the locus coeruleus (LC, loss of which is an early change in Alzheimer's disease but which is preserved in the transgenic mice. In this paper, we extend these observations by examining the effects of the LC on markers of oxidative stress and intermediary metabolism. We compare four groups: wild-type or Tg2576 A transgenic mice injected with DSP4 or vehicle. Of greatest interest were metabolites different between ablated and intact transgenics, but not between ablated and intact wild-type animals. The Tg2576_DSP4 mice were distinguished from the other three groups by oxidative stress and altered energy metabolism. These observations provide further support for the hypothesis that Tg2576 A transgenic mice with this ablation may be a more congruent model of Alzheimer's disease than are transgenics with an intact LC.

  15. Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534).

    Science.gov (United States)

    Li, Samuel Q; Cheuk, Adam T; Shern, Jack F; Song, Young K; Hurd, Laura; Liao, Hongling; Wei, Jun S; Khan, Javed

    2013-01-01

    Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

  16. Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534.

    Directory of Open Access Journals (Sweden)

    Samuel Q Li

    Full Text Available Rhabdomyosarcoma (RMS is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4 is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534 to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

  17. Molecular dynamics studies on the NMR structures of rabbit prion protein wild-type and mutants: surface electrostatic charge distributions

    CERN Document Server

    Zhang, Jiapu

    2014-01-01

    Prion is a misfolded protein found in mammals that causes infectious diseases of the nervous system in humans and animals. Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep and goats, cattle, deer, elk and humans etc. Recent studies have shown that rabbits have a low susceptibility to be infected by prion diseases with respect to other animals including humans. The present study employs molecular dynamics (MD) means to unravel the mechanism of rabbit prion proteins (RaPrPC) based on the recently available rabbit NMR structures (of the wild-type and its two mutants of two surface residues). The electrostatic charge distributions on the protein surface are the focus when analysing the MD trajectories. It is found that we can conclude that surface electrostatic charge distributions indeed contribute to the structural stability of wild-type RaPrPC; this may be useful for the medicinal treatment of prion diseases.

  18. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    Science.gov (United States)

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

    2015-07-01

    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  19. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    Science.gov (United States)

    Morkunas, Bernardas; Gal, Balint; Galloway, Warren R J D; Hodgkinson, James T; Ibbeson, Brett M; Sing Tan, Yaw; Welch, Martin

    2016-01-01

    Summary Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable. PMID:27559393

  20. Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells.

    Science.gov (United States)

    LaRocque, Jeannine R; Stark, Jeremy M; Oh, Jin; Bojilova, Ekaterina; Yusa, Kosuke; Horie, Kyoji; Takeda, Junji; Jasin, Maria

    2011-07-19

    Genomic integrity often is compromised in tumor cells, as illustrated by genetic alterations leading to loss of heterozygosity (LOH). One mechanism of LOH is mitotic crossover recombination between homologous chromosomes, potentially initiated by a double-strand break (DSB). To examine LOH associated with DSB-induced interhomolog recombination, we analyzed recombination events using a reporter in mouse embryonic stem cells derived from F1 hybrid embryos. In this study, we were able to identify LOH events although they occur only rarely in wild-type cells (≤2.5%). The low frequency of LOH during interhomolog recombination suggests that crossing over is rare in wild-type cells. Candidate factors that may suppress crossovers include the RecQ helicase deficient in Bloom syndrome cells (BLM), which is part of a complex that dissolves recombination intermediates. We analyzed interhomolog recombination in BLM-deficient cells and found that, although interhomolog recombination is slightly decreased in the absence of BLM, LOH is increased by fivefold or more, implying significantly increased interhomolog crossing over. These events frequently are associated with a second homologous recombination event, which may be related to the mitotic bivalent structure and/or the cell-cycle stage at which the initiating DSB occurs.

  1. Screening and Expression of a Silicon Transporter Gene (Lsi1 in Wild-Type Indica Rice Cultivars

    Directory of Open Access Journals (Sweden)

    Mahbod Sahebi

    2017-01-01

    Full Text Available Silicon (Si is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

  2. Response of wild-type and high pigment-1 tomato fruit to UV-B depletion: flavonoid profiling and gene expression.

    Science.gov (United States)

    Calvenzani, Valentina; Martinelli, Moira; Lazzeri, Valerio; Giuntini, Deborah; Dall'Asta, Chiara; Galaverna, Gianni; Tonelli, Chiara; Ranieri, Annamaria; Petroni, Katia

    2010-02-01

    The tomato high pigment-1 (hp-1) mutant is characterised by exaggerated photoresponsiveness and increased fruit pigmentation, and carries a mutation in the HP1/LeDDB1 gene, encoding the tomato homologue of the negative regulator of the light signal transduction DDB1a from Arabidopsis. Here, we investigated the molecular events underlying flavonoid accumulation in flesh and peel of wild-type and hp-1 fruits in presence or absence of UV-B light. In hp-1 peel, a twofold higher level of rutin and an earlier accumulation of flavonoids than in wild-type were observed, which correlated to the earlier activation of most flavonoid biosynthetic genes compared to wild-type. In hp-1 flesh, flavonoid content was up to 8.5-fold higher than in wild-type and correlated to the higher transcript level of flavonoid genes compared to wild-type. In both tissues, the expression of flavonoid genes was correlated with the anticipated and/or enhanced activation of the light signal transduction genes: LeCOP1LIKE, LeCOP1 and LeHY5. In wild-type, flavonoid content was severely reduced by UV-B depletion mostly in peel, whereas in hp-1 it was significantly increased in flesh. The activation of flavonoid and light signal transduction genes was UV-B dependent mostly at the mature green stage, whereas LeDDB1 expression was not regulated by UV-B.

  3. Hydroxycinnamic acids and UV-B depletion: Profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1.

    Science.gov (United States)

    Calvenzani, Valentina; Castagna, Antonella; Ranieri, Annamaria; Tonelli, Chiara; Petroni, Katia

    2015-06-01

    Hydroxycinnamic acids (HCAs) are phenolic compounds widely found in most plant families. Aim of the present work was to investigate their accumulation and biosynthetic gene expression in presence or absence of UV-B radiation in tomato fruits of wild-type and hp-1, a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression and HCAs content were higher in hp-1 than in wild type peel and UV-B depletion determined a decrease in HCAs accumulation in wild-type and an increase in hp-1 fruits, generally in accordance with biosynthetic gene expression. In flesh, despite a similar transcript level of most genes between the two genotypes, HCAs content was generally higher in wild type than in hp-1, although remaining at a lower level with respect to wild type peel. Under UV-B depletion, a general reduction of HCAs content was observed in wild-type flesh, whereas an increase in the content of p-coumaric acid and caffeic acid was observed in hp-1 flesh.

  4. Maintenance erlotinib in advanced nonsmall cell lung cancer: cost-effectiveness in EGFR wild-type across Europe

    Directory of Open Access Journals (Sweden)

    Walleser S

    2012-09-01

    Full Text Available Silke Walleser,1 Joshua Ray,2 Helge Bischoff,3 Alain Vergnenègre,4 Hubertus Rosery,5 Christos Chouaid,6 David Heigener,7 Javier de Castro Carpeño,8 Marcello Tiseo,9 Stefan Walzer21Health Economic Consultancy, Renens, Switzerland; 2F Hoffmann-La Roche Pharmaceuticals AG, Basel, Switzerland; 3Thoracic Hospital of Heidelberg, Heidelberg, Germany; 4Limoges University Hospital, Limoges, France; 5Assessment-in-Medicine GmbH, Loerrach, Germany; 6Hospital Saint Antoine, Paris, France; 7Hospital Grosshansdorf, Grosshansdorf, Germany; 8University Hospital La Paz, Madrid, Spain; 9University Hospital of Parma, Parma, ItalyBackground: First-line maintenance erlotinib in patients with locally advanced or metastatic nonsmall cell lung cancer (NSCLC has demonstrated significant overall survival and progression-free survival benefits compared with best supportive care plus placebo, irrespective of epidermal growth factor receptor (EGFR status (SATURN trial. The cost-effectiveness of first-line maintenance erlotinib in the overall SATURN population has been assessed and published recently, but analyses according to EGFR mutation status have not been performed yet, which was the rationale for assessing the cost-effectiveness of first-line maintenance erlotinib specifically in EGFR wild-type metastatic NSCLC.Methods: The incremental cost per life-year gained of first-line maintenance erlotinib compared with best supportive care in patients with EGFR wild-type stable metastatic NSCLC was assessed for five European countries (the United Kingdom, Germany, France, Spain, and Italy with an area-under-the-curve model consisting of three health states (progression-free survival, progressive disease, death. Log-logistic survival functions were fitted to Phase III patient-level data (SATURN to model progression-free survival and overall survival. The first-line maintenance erlotinib therapy cost (modeled for time to treatment cessation, medication cost in later lines, and

  5. Enamel Hypoplasia of Deciduous Canine

    OpenAIRE

    加納, 隆; 平出, 百合子; 舟津, 聡; 峯村, 隆一; 恩田, 千爾; 正木, 岳馬

    1993-01-01

    From observation of frequency and measurement of the lengths and widths of enamel hypoplasia on the maxillary and mandibular deciduous canines, extracted from 50 Indians' skulls, the following results were obtained. 1) Enamel hypoplasia occurred in 15% of the maxillary deciduous canines and 44% of the mandibular deciduous canines. 2) Symmetrical cases of enamel hypoplasia occurred in 8.0% of the maxillary deciduous canins and in 34% of the mandibular deciduous canines. The enamel hypoplasia o...

  6. Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice

    Directory of Open Access Journals (Sweden)

    Jackson George R

    2011-06-01

    Full Text Available Abstract Background The correlation between neurofibrillary tangles of tau and disease progression in the brains of Alzheimer's disease (AD patients remains an area of contention. Innovative data are emerging from biochemical, cell-based and transgenic mouse studies that suggest that tau oligomers, a pre-filament form of tau, may be the most toxic and pathologically significant tau aggregate. Results Here we report that oligomers of recombinant full-length human tau protein are neurotoxic in vivo after subcortical stereotaxic injection into mice. Tau oligomers impaired memory consolidation, whereas tau fibrils and monomers did not. Additionally, tau oligomers induced synaptic dysfunction by reducing the levels of synaptic vesicle-associated proteins synaptophysin and septin-11. Tau oligomers produced mitochondrial dysfunction by decreasing the levels of NADH-ubiquinone oxidoreductase (electron transport chain complex I, and activated caspase-9, which is related to the apoptotic mitochondrial pathway. Conclusions This study identifies tau oligomers as an acutely toxic tau species in vivo, and suggests that tau oligomers induce neurodegeneration by affecting mitochondrial and synaptic function, both of which are early hallmarks in AD and other tauopathies. These results open new avenues for neuroprotective intervention strategies of tauopathies by targeting tau oligomers.

  7. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Hansen Jacob B

    2011-05-01

    Full Text Available Abstract Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs, and retinoblastoma gene-deficient MEFs (Rb-/- MEFs. Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte

  8. In vivo rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement

    Directory of Open Access Journals (Sweden)

    Floros Joanna

    2011-10-01

    Full Text Available Abstract Background Mice lacking surfactant protein-A (SP-A-/-; knockout; KO exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL protein differences between KO and wild-type (WT are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining. Results We observed: a significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases. These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis, proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c that SP-A treatment altered cell size and F-actin distribution. Conclusions These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.

  9. Wild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in the YAC128 mouse model of Huntington disease

    Directory of Open Access Journals (Sweden)

    Leavitt Blair R

    2006-12-01

    Full Text Available Abstract Background Huntington disease (HD is an adult onset neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt protein. Htt function is essential for embryonic survival as well as normal function during the postnatal period. In addition to having roles in transcription and transport, recent evidence demonstrates that wild-type htt is neuroprotective in vivo. To determine whether treatment with wild-type htt would be beneficial in HD, we crossed the YAC128 mouse model of HD with mice that over-express wild-type htt (YAC18 mice to generate YAC128 mice that over-express wild-type htt (YAC18/128 mice. Results YAC18/128 mice were found to express mutant htt at the same level as YAC128 mice and wild-type htt at the same level as YAC18 mice. YAC18/128 mice show no significant behavioural improvement compared to YAC128 mice in the rotarod test of motor coordination or in an automated open field test. In the brain, YAC18/128 mice show no significant improvement in striatal volume, striatal neuronal numbers or striatal DARPP-32 expression compared to YAC128 mice. In contrast, striatal neuronal cross-sectional area showed significant improvement in YAC18/128 mice compared to YAC128 mice. Conclusion While the over-expression of wild-type htt results in a mild improvement in striatal neuropathology in YAC128 mice, our findings suggest that treatment with wild-type htt may not be sufficient to ameliorate the symptoms of HD in this model.

  10. Neuromuscular control of a single twitch muscle in wild type and mutant Drosophila, measured with an ergometer.

    Science.gov (United States)

    Harvey, Jennifer; Brunger, Holly; Middleton, C Adam; Hill, Julia A; Sevdali, Maria; Sweeney, Sean T; Sparrow, John C; Elliott, Christopher J H

    2008-06-01

    How do deficits in neuronal growth, aging or synaptic function affect the final, mechanical output of a single muscle twitch? We address this in vivo (indeed in situ) with a novel ergometer that records the output of a large specialised muscle, the Drosophila jump muscle. Here, we describe in detail the ergometer, its construction and use. We evaluated the ergometer by showing that adult fly jump muscle output varies little between 3 h and 7 days; but newly eclosed flies produce only 65%. In a mutant with little octopamine (Tbetah), jump muscle performance is reduced by 28%. The initial responses of synaptic growth mutants (highwire and spinster) do not differ from wild type, as expected on the homeostatic hypothesis. However, responses in highwire mutations gradually decline following repeated stimuli, suggesting physiological as well as anatomical abnormalities. We conclude that the assay is robust, sensitive and reliable with a good throughput.

  11. Silent reintroduction of wild-type poliovirus to Israel, 2013 - risk communication challenges in an argumentative atmosphere.

    Science.gov (United States)

    Kaliner, E; Moran-Gilad, J; Grotto, I; Somekh, E; Kopel, E; Gdalevich, M; Shimron, E; Amikam, Y; Leventhal, A; Lev, B; Gamzu, R

    2014-02-20

    Israel has been certified as polio-free by the World Health Organization and its routine immunisation schedule consists of inactivated poliovirus vaccine (IPV) only. At the end of May 2013, the Israeli Ministry of Health (MOH) has confirmed the reintroduction of wild-type poliovirus 1 into the country. Documented ongoing human-to-human transmission necessitated a thorough risk assessment followed by a supplemental immunisation campaign using oral polio vaccine (OPV). The unusual situation in which ongoing poliovirus transmission was picked up through an early warning system of sewage monitoring without active polio cases, brought about significant challenges in risk communication. This paper reviews the challenges faced by the MOH and the communication strategy devised, in order to facilitate and optimise the various components of the public health response, particularly vaccination. Lessons learned from our recent experience may inform risk communication approaches in other countries that may face a similar situation as global polio eradication moves towards the 'End game'.

  12. Phase II marker-driven trial of panitumumab and chemotherapy in KRAS wild-type biliary tract cancer

    DEFF Research Database (Denmark)

    Jensen, L H; Lindebjerg, J; Ploen, J;

    2012-01-01

    BACKGROUND: Combination chemotherapy has proven beneficial in biliary tract cancer and further improvements may be achieved by individualizing treatment based on biomarkers and by adding biological agents. We report the effect of chemotherapy with panitumumab as first-line therapy for KRAS wild-type......) and median overall survival was 10.0 months (95% CI 7.4-12.7 months). Toxicity was manageable including eight cases of epidermal growth factor receptor-related skin adverse events of grade 2 or more. CONCLUSIONS: Marker-driven patient selection is feasible in the systemic treatment of biliary tract cancer...... irresectable biliary tract cancer. PATIENTS AND METHODS: Patients were treated with gemcitabine 1000 mg/m(2), oxaliplatin 60 mg/m(2), and panitumumab 6 mg/kg i.v. every 2 weeks followed by two daily administrations of capecitabine 1000 mg/m(2) in 7 days. RESULTS: During 22 months, 46 patients were included...

  13. Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier.

    Science.gov (United States)

    Luo, J; Zhou, X; Diao, L; Wang, Z

    2010-01-01

    The transfection efficiency of wild-type p53 (wtp53) was investigated in retinoblastoma (RB) Y79 cells using an ultrasound microbubble technique. A human RB nude mouse xenograft tumour model was also used to investigate whether this technique could deliver wtp53 into solid tumours. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that wtp53 was successfully transfected into Y79 cells in the plasmid with microbubbles and ultrasound group and in the plasmid with liposomes group, but not in the plasmid with ultrasound group or in the untreated control group. Flow cytometry showed that apoptosis was highest in the microbubbles and ultrasound group (25.58%) compared with the plasmid with liposomes group (19.50%), and the other two groups (microbubbles and ultrasound group. This study provides preliminary evidence in support of a potential new approach to RB gene therapy.

  14. IFN-{gamma} enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease

    DEFF Research Database (Denmark)

    Baron, Rona; Nemirovsky, Anna; Harpaz, Idan

    2008-01-01

    mechanisms can generate immunity to such deficits in neuronal repair. We demonstrate that in contrast to primarily innate immunity cytokines, such as interleukin-6 and tumor necrosis factor-alpha, the adaptive immunity cytokine IFN-gamma enhances neurogenesis in the dentate gyrus of adult mice and improves...... model of Alzheimer's disease. We demonstrate that limited amounts of IFN-gamma in the brain shape the neuropoietic milieu to enhance neurogenesis, possibly representing the normal function of the immune system in controlling brain inflammation and repair.-Baron, R., Nemirovsky, A., Harpaz, I., Cohen, H......., Owens, T., Monsonego, A. IFN-gamma enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease....

  15. Metabolic tumor burden as marker of outcome in advanced EGFR wild-type NSCLC patients treated with erlotinib

    DEFF Research Database (Denmark)

    Winther-Larsen, Anne; Fledelius, Joan; Sorensen, Boe Sandahl;

    2016-01-01

    OBJECTIVES: Accurate estimation of the prognosis of advanced non-small cell lung cancer (NSCLC) patients is essential before initiation of palliative treatment; especially in the second and third-line setting. This study was conducted in order to evaluate tumor burden measured on an 2'-deoxy-2...... a prospectively collected cohort. An F-18-FDG-PET/CT scan was conducted prior to erlotinib treatment and tumor burden was measured in terms of metabolic tumor volume (MTV) and total lesion glycolysis (TLG). Median values of MTV and TLG were used for dichotomization of patients. Survival outcome was compared...... with shorter PFS and OS in advanced EGFR wild-type NSCLC patients treated with second or third-line erlotinib. Metabolic tumor burden is a highly promising clinical tool that may allow better patient selection for palliative treatment in the future....

  16. Inheritance of low pasting temperature in sweetpotato starch and the dosage effect of wild-type alleles

    Science.gov (United States)

    Katayama, Kenji; Tamiya, Seiji; Sakai, Tetsufumi; Kai, Yumi; Ohara-Takada, Akiko; Kuranouchi, Toshikazu; Yoshinaga, Masaru

    2015-01-01

    Sweetpotato (Ipomoea batatas (L.) Lam.), which is an outcrossing hexaploid, is one of the most important starch-producing crops in the world. During the last decade, new sweetpotato cultivars, e.g. ‘Quick Sweet’, which have approximately 20°C lower pasting temperature, slower retrogradation and higher digestibility of raw starch than ordinary cultivars, have been developed in Japan. Genetic analysis of these variants with low pasting temperature starch was conducted in this study. Using 8 variants and 15 normal clones, 26 families were generated. The results from analyzing these progenies suggested that this trait is a qualitative character controlled by one recessive allele (designated spt), which is inherited in a hexasomic manner. A dosage effect of the wild-type Spt allele was found for starch pasting temperature, although the effect was not linear. These results will aid breeders to develop sweetpotato cultivars with a range of starch pasting temperatures. PMID:26366119

  17. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2014-01-01

    Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  18. Comparison of the activities of wild type and mutant enhancing factor/mouse secretory phospholipase A2 proteins

    Indian Academy of Sciences (India)

    Bhakti M Kirtane; Rita Mulherkar

    2002-09-01

    Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities.

  19. Evaluation of the correlation of caspofungin MICs and treatment outcome in murine infections by wild type strains of Candida parapsilosis.

    Science.gov (United States)

    Salas, Valentina; Pastor, F Javier; Capilla, Javier; Sutton, Deanna A; Mayayo, Emilio; Fothergill, Annette W; Rinaldi, Michael G; Guarro, Josep

    2013-09-01

    We have evaluated the in vitro activity of caspofungin against 36 wild-type strains of Candida parapsilosis sensu stricto using 3 techniques: broth microdilution, disk diffusion, and the determination of minimal fungicidal concentration (MFC). The first 2 methods showed a good in vitro activity of caspofungin, but the MFCs were ≥2 dilutions above their corresponding MICs. In a murine model of disseminated infection, we evaluated the efficacy of caspofungin at 5 mg/kg against 8 strains of C. parapsilosis representing different degrees of in vitro susceptibility (0.12-1 μg/mL). All the isolates responded to treatment and (1→3)-β-D-glucan levels were reduced in all the cases; however, the study revealed differences among isolates, since caspofungin reduced the tissue burden of mice infected with isolates with MICs ≤0.5 μg/mL but was less effective against those with MICs of 1 μg/mL.

  20. Wild-type and molten globular chorismate mutase achieve comparable catalytic rates using very different enthalpy/entropy compensations

    Institute of Scientific and Technical Information of China (English)

    HU Hao

    2014-01-01

    The origin of the catalytic power of enzymes with a meta-stable native state,e.g.molten globular state,is an unsolved challenging issue in biochemistry.To help understand the possible differences between this special class of enzymes and the typical ones,we report here computer simulations of the catalysis of both the well-folded wild-type and the molten globular mutant of chorismate mutase.Using the ab initio quantum mechanical/molecular mechanical minimum free-energy path method,we determined the height of reaction barriers that are in good agreement with experimental measurements.Enzyme-substrate interactions were analyzed in detail to identify factors contributing to catalysis.Computed angular order parameters of backbone N–H bonds and side-chain methyl groups suggested site-specific,non-uniform rigidity changes of the enzymes during catalysis.The change of conformational entropy from the ground state to the transition state revealed distinctly contrasting entropy/enthalpy compensations in the dimeric wild-type enzyme and its molten globular monomeric variant.A unique catalytic strategy was suggested for enzymes that are natively molten globules:some may possess large conformational flexibility to provide strong electrostatic interactions to stabilize the transition state of the substrate and compensate for the entropy loss in the transition state.The equilibrium conformational dynamics in the reactant state were analyzed to quantify their contributions to the structural transitions enzymes needed to reach the transition states.The results suggest that large-scale conformational dynamics make important catalytic contributions to sampling conformational regions in favor of binding the transition state of substrate.

  1. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    Science.gov (United States)

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

  2. Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora.

    Science.gov (United States)

    Russell, P J; Wagner, S; Rodland, K D; Feinbaum, R L; Russell, J P; Bret-Harte, M S; Free, S J; Metzenberg, R L

    1984-01-01

    The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.

  3. Emergence of classical BSE strain properties during serial passages of H-BSE in wild-type mice.

    Directory of Open Access Journals (Sweden)

    Thierry Baron

    Full Text Available BACKGROUND: Two distinct forms of atypical spongiform encephalopathies (H-BSE and L-BSE have recently been identified in cattle. Transmission studies in several wild-type or transgenic mouse models showed that these forms were associated with two distinct major strains of infectious agents, which also differed from the unique strain that had been isolated from cases of classical BSE during the food-borne epizootic disease. METHODOLOGY/PRINCIPAL FINDINGS: H-BSE was monitored during three serial passages in C57BL/6 mice. On second passage, most of the inoculated mice showed molecular features of the abnormal prion protein (PrP(d and brain lesions similar to those observed at first passage, but clearly distinct from those of classical BSE in this mouse model. These features were similarly maintained during a third passage. However, on second passage, some of the mice exhibited distinctly different molecular and lesion characteristics, reminiscent of classical BSE in C57Bl/6 mice. These similarities were confirmed on third passage from such mice, for which the same survival time was also observed as with classical BSE adapted to C57Bl/6 mice. Lymphotropism was rarely detected in mice with H-BSE features. In contrast, PrP(d was detectable, on third passage, in the spleens of most mice exhibiting classical BSE features, the pattern being indistinguishable from that found in C57Bl/6 mice infected with classical BSE. CONCLUSION/SIGNIFICANCE: Our data demonstrate the emergence of a prion strain with features similar to classical BSE during serial passages of H-BSE in wild-type mice. Such findings might help to explain the origin of the classical BSE epizootic disease, which could have originated from a putatively sporadic form of BSE.

  4. On the mechanistic differences of benzene-induced leukemogenesis between wild type and p53 knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Hirabayashi, Yoko; Yoon, Byung-Il; Kawasaki, Yasushi; Li, Guang-Xun; Kanno, Jun; Inoue, Tohru [National Inst. of Health Sciences, Tokyo (Japan)

    2003-07-01

    Leukemia induction by benzene inhalation was first reported by Le Noire in 1887, described multiple cases of leukemia among Parisian cobblers. However, experimental induction of leukemia by benzene exposure was not succeeded for a hundred years, until Snyder et al. and our group reported it nearly 20 years ago. Nevertheless, the mechanistic background of benzene-induced leukemia was still an enigma until recently a benzene-induced peculiar cell kinetics of the stem/progenitor cells has been elucidated by our study, demonstrated a marked repeated oscillatory decrease in peripheral blood and bone marrow (BM) cellularity during and after benzene exposure, which epigenetically preceded and developed the leukemia more than a year later. We utilized the BUUV (bromodeoxyuridine + UV exposure) method to study stem/progenitor cell kinetics during and/or after benzene exposure. Using these methods, we were able to measure the labeling rate, cycling fraction of clonogenic progenitor cells, and other cell cycle parameters. The cycling fraction of stem/progenitor cells was found not to turn into an active hematopoiesis but to remain low during benzene inhalation and further we found evidence that the cycling fraction depression may be mediated in part by a slowing of stem/progenitor cell cycling perse by up-regulation of p21. The benzene induced leukemogenicity between mice carrying wild-type p53 and mice lacking p53 seem to differ from one another. In the case of p53 knockout mouse, DNA damage such as weak mutagenicity and or chromosomal damages are retained, and those damages participated in the induction of a consequent activation of proto-oncogenes and the like, which led cells to further neoplastic changes. In contrast, in the case of wild type mice, a dramatic oscillational change in the cell cycle of the stem cell compartment seems to be an important factor for mice carrying the p53 gene. (author)

  5. Morphometric analysis of the small intestine in wild type mice C57BL/6L -- a developmental study.

    Science.gov (United States)

    Gulbinowicz, Monika; Berdel, Bozena; Wójcik, Sławomir; Dziewiatkowski, Jerzy; Oikarinen, Seija; Mutanen, Marja; Kosma, Veli-Matti; Mykkänen, Hannu; Moryś, Janusz

    2004-11-01

    Recently the increasing prevalence of gastrointestinal diseases, including neoplasm, has resulted in the necessity of characterising not only the tumours, but also healthy mucosa. Research into the morphological changes of healthy mucosa under different experimental conditions, including drugs, special diets and the use of probiotic bacteria, is greatly facilitated by the availability of animal models. In spite of the widespread use of mice in gastrointestinal research, there is a lack of information on the qualitative and quantitative histological characteristics of the intestinal mucosa of the mouse. The aim of this study was to assess the morphological characteristics and the postnatal development of the small intestine of wild type mice -- C57BL/6J. The mice were aged either 5 weeks or 12 weeks. The 12-week-old mice had been weaned at the age of 5 weeks. After dissection the small intestine was divided into 5 equal portions and randomly chosen microscopical sections from each were stained with haematoxylin and eosin. The parameters describing the morphology of the small intestine (villus height, depth of the crypt, villus width near the crypt, width of the villus connective tissue near the crypt, thickness of the muscular layer and the height of the enterocytes and their nuclei) were evaluated under a light microscope. In both age groups the height and width of the villi decreased, while the thickness of the muscular layer increased in the distal direction. The height of the enterocytes decreased and the height of the enterocyte nucleus increased towards the colon in both age groups. The depth of the crypts was greater in the younger animals than in the older ones. Our data provides the baseline morphological description of the small intestinal mucosa in wild type mice, strain C57BL/6J, which can be used as a reference for testing the influence of drugs, toxins, nutrients and inborn mutations on the mouse intestine.

  6. Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium.

    Science.gov (United States)

    Krekeler, N; Marenda, M S; Browning, G F; Holden, K M; Charles, J A; Wright, P J

    2012-09-01

    Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.

  7. Characterization of a new dog isolate of canine distemper virus from China.

    Science.gov (United States)

    Qiao, J; Meng, Q; Chen, C; Xia, X; Cai, X; Ren, Y; Zhang, H

    2011-01-01

    Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

  8. Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

    DEFF Research Database (Denmark)

    Wandall-Frostholm, Christine; Skaarup, Lykke Moran; Sadda, Veeranjaneyulu

    2014-01-01

    as in pulmonary vessels with increased lumen size in partially-and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the K(Ca)2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked...... of the K(Ca)2.3 and K(Ca)3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of K(Ca)2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of K...

  9. Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, P.S.

    1984-01-01

    The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation. (orig.). 28 refs.; 4 figs.

  10. The Small Colony Variant Of Listeria Monocytogenes Is More Tolerant To Antibiotics And Grows Better Within Caco-2 Epithelial Cells Than The Wild Type

    DEFF Research Database (Denmark)

    Curtis, Thomas; Gram, Lone; Knudsen, Gitte Maegaard

    2015-01-01

    , the SCV E18 survived significantly better than the wild type N53-1 (one and three log10 higher CFU/ml) when exposed to super-MIC concentrations of most tested antibiotics, indicating a persister-like phenotype of the SCV. While SCV E18 displayed sensitivity towards oxygen, it was significantly more...... tolerant of 20mM H2O2 as compared to the wild type, with 6.3 log10 CFU/ml and 3.7 log10 CFU/ml, respectively. The SCV E18 had lower survival rate in unactivated macrophages, however, it was able to survive and multiply to almost 100-fold higher CFU/ml than the wild type in CaCo-2 epithelial cells...

  11. Combination of cetuximab and PP242 synergistically suppress the progression of wild-type KRAS colorectal carcinoma

    Directory of Open Access Journals (Sweden)

    Cheng L

    2015-11-01

    Full Text Available Lei Cheng,1,* Zuguang Xia,2,3,* Xinyu Bian,1 Guangchao Li,4 Jing Hu,1 Ya Cao,5 Qing Wang,1 Xiaoping Qian1 1Comprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, 2Department of Medical Oncology, Fudan University Shanghai Cancer Center, Shanghai, 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 4School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, 5Department of Biological Science and Technology and State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Mammalian target of rapamycin (mTOR has been shown to be overactive in human colorectal cancer, but the first-generation mTOR inhibitor, rapamycin, has failed to show clinical efficacy against colorectal cancer. On the other hand, although the second-generation mTOR inhibitor, PP242, has exerted substantial efficacy, it was revealed that independent inhibition by PP242 was transient, which could lead to positive-feedback loop to EGFR. Using wild-type KRAS colorectal cancer cells as models, we investigate the treatment efficacy of a widely used anti-EGFR monoclonal antibody, cetuximab, and PP242, alone or in combination in vitro and in vivo. Results of cell viability assays confirmed the synergistic inhibitory effect of PP242 and cetuximab on the survival of Caco-2 and HT-29 cells. Moreover, the ability of cancer-cell invasion and proliferation was also significantly inhibited by the combination therapy when compared with cetuximab or PP242 alone. Interestingly, the percentage of CD44-positive cancer cells was substantially decreased by the combination therapy in comparison with PP242 alone through fluorescence-activated cell sorting. The growth of cancer stem-like cell spheres in vitro was

  12. Impact of age on the cerebrovascular proteomes of wild-type and Tg-SwDI mice.

    Directory of Open Access Journals (Sweden)

    James L Searcy

    Full Text Available The structural integrity of cerebral vessels is compromised during ageing. Abnormal amyloid (Aβ deposition in the vasculature can accelerate age-related pathologies. The cerebrovascular response associated with ageing and microvascular Aβ deposition was defined using quantitative label-free shotgun proteomic analysis. Over 650 proteins were quantified in vessel-enriched fractions from the brains of 3 and 9 month-old wild-type (WT and Tg-SwDI mice. Sixty-five proteins were significantly increased in older WT animals and included several basement membrane proteins (nidogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, laminin subunit gamma-1 precursor and collagen alpha-2(IV chain preproprotein. Twenty-four proteins were increased and twenty-one decreased in older Tg-SwDI mice. Of these, increases in Apolipoprotein E (APOE and high temperature requirement serine protease-1 (HTRA1 and decreases in spliceosome and RNA-binding proteins were the most prominent. Only six shared proteins were altered in both 9-month old WT and Tg-SwDI animals. The age-related proteomic response in the cerebrovasculature was distinctly different in the presence of microvascular Aβ deposition. Proteins found differentially expressed within the WT and Tg-SwDI animals give greater insight to the mechanisms behind age-related cerebrovascular dysfunction and pathologies and may provide novel therapeutic targets.

  13. The clinical presentation of Marfan syndrome is modulated by expression of wild-type FBN1 allele.

    Science.gov (United States)

    Aubart, Mélodie; Gross, Marie-Sylvie; Hanna, Nadine; Zabot, Marie-Thérèse; Sznajder, Marc; Detaint, Delphine; Gouya, Laurent; Jondeau, Guillaume; Boileau, Catherine; Stheneur, Chantal

    2015-05-15

    Marfan syndrome is an autosomal dominant disorder mainly caused by mutations within FBN1 gene. The disease displays large variability in age of onset or severity and very poor phenotype/genotype correlations have been demonstrated. We investigated the hypothesis that phenotype severity could be related to the variable expression level of fibrillin-1 (FBN1) synthesized from the wild-type (WT) allele. Quantitative reverse-transcription and polymerase chain reaction was used to evaluate FBN1 levels in skin fibroblasts from 80 Marfan patients with premature termination codons and in skin fibroblasts from 80 controls. Results in controls showed a 3.9-fold variation in FBN1 mRNA synthesis level between subjects. A similar 4.4-fold variation was found in the Marfan population, but the mean level of FBN1 mRNA was a half of the control population. Differential allelic expression analysis in Marfan fibroblasts showed that over 90% of FBN1 mRNA was transcribed from the wild allele and the mutated allele was not detected. In the control population, independently of the expression level of FBN1, we observed steady-state equilibrium between the two allelic-mRNAs suggesting that FBN1 expression mainly depends on trans-acting regulators. Finally, we show that a low level of residual WT FBN1 mRNA accounts for a high risk of ectopia lentis and pectus abnormality and tends to increase the risk of aortic dilatation.

  14. Alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice.

    Science.gov (United States)

    Moran, Timothy P; Burgents, Joseph E; Long, Brian; Ferrer, Ivana; Jaffee, Elizabeth M; Tisch, Roland M; Johnston, Robert E; Serody, Jonathan S

    2007-09-04

    While dendritic cell (DC) vaccines can protect hosts from tumor challenge, their ability to effectively inhibit the growth of established tumors remains indeterminate. Previously, we have shown that human DCs transduced with Venezuelan equine encephalitis virus replicon particles (VRPs) were potent stimulators of antigen-specific T cells in vitro. Therefore, we investigated the ability of VRP-transduced DCs (VRP-DCs) to induce therapeutic immunity in vivo against tumors overexpressing the neu oncoprotein. Transduction of murine DCs with VRPs resulted in high-level transgene expression, DC maturation and secretion of proinflammatory cytokines. Vaccination with VRP-DCs expressing a truncated neu oncoprotein induced robust neu-specific CD8(+) T cell and anti-neu IgG responses. Furthermore, a single vaccination with VRP-DCs induced the regression of large established tumors in wild-type mice. Interestingly, depletion of CD4(+), but not CD8(+), T cells completely abrogated inhibition of tumor growth following vaccination. Taken together, our results demonstrate that VRP-DC vaccines induce potent immunity against established tumors, and emphasize the importance of the generation of both CD4(+) T cell and B cell responses for efficient tumor inhibition. These findings provide the rationale for future evaluation of VRP-DC vaccines in the clinical setting.

  15. Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants.

    Science.gov (United States)

    Caringella, Marissa A; Bongers, Franca J; Sack, Lawren

    2015-12-01

    Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (Kleaf ), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col-0) and four vein mutants (dot3-111 and dot3-134, and cvp1-3 and cvp2-1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean Kleaf . Across all genotypes, there was a strong correlation of Kleaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence Kleaf , indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing Kleaf .

  16. Extra Copper-mediated Enhancement of the DNA Cleavage Activity Supported with Wild-type Cu, Zn Superoxide Dismutase

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ruo-Yu; JIANG Wei; ZHANG Li-Na; WANG Li; LIU Chang-Lin

    2008-01-01

    It is well known that the primary function of wild type Cu, Zn superoxide dismutase (holo SOD) is to catalyze the conversion of the superoxide anion to H2O2 and O2 as an antioxidant enzyme. However, the aberrant copper-mediated oxidation chemistry in the enzyme (including its mutation forms) that damages nucleic acids, proteins including itself and cell membrane has attracted extensive attention in the past decade. The present study examined the hydrogen peroxide-dependent DNA cleavage activity supported with the combinations between holo SOD and extra copper (holo SOD+nCu(Ⅱ)). The results indicate that the presence of extra copper can enhance the DNA cleavage activity and a cooperative effect between holo SOD and the extra Cu(Ⅱ) occurs in DNA cleavage. The relative activity and kinetic assay showed that the DNA cleavage activity of holo SOD+nCu(Ⅱ) was enhanced upon addition of extra Cu(Ⅱ). The favorable pH regions for the DNA cleavage were observed to be 3.6-5.6 and 9.0-10, suggesting the species responsible for the DNA cleavage are different in different pH regions. In addition,to obtain an insight into DNA cleavage pathways, the effect of free radical scavengers and inhibitors on the DNA cleavage activity was probed.

  17. ARID1A alterations are associated with FGFR3-wild type, poor-prognosis, urothelial bladder tumors.

    Directory of Open Access Journals (Sweden)

    Cristina Balbás-Martínez

    Full Text Available Urothelial bladder cancer (UBC is heterogeneous at the clinical, pathological, genetic, and epigenetic levels. Exome sequencing has identified ARID1A as a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in UBC. Here, we assess ARID1A alterations in two series of patients with UBC. In the first tumor series, we analyze exons 2-20 in 52 primary UBC and find that all mutant tumors belong to the aggressive UBC phenotype (high grade non-muscle invasive and muscle invasive tumors (P = 0.05. In a second series (n = 84, we assess ARID1A expression using immunohistochemistry, a surrogate for mutation analysis, and find that loss of expression increases with higher stage/grade, it is inversely associated with FGFR3 overexpression (P = 0.03 but it is not correlated with p53 overexpression (P = 0.30. We also analyzed the expression of cytokeratins in the same set of tumor and find, using unsupervised clustering, that tumors with ARID1A loss of expression are generally KRT5/6-low. In this patient series, loss of ARID1A expression is also associated with worse prognosis, likely reflecting the higher prevalence of losses found in tumors of higher stage and grade. The independent findings in these two sets of patients strongly support the notion that ARID1A inactivation is a key player in bladder carcinogenesis occurring predominantly in FGFR3 wild type tumors.

  18. Current Approaches for Predicting a Lack of Response to Anti-EGFR Therapy in KRAS Wild-Type Patients

    Directory of Open Access Journals (Sweden)

    Tze-Kiong Er

    2014-01-01

    Full Text Available Targeting epidermal growth factor receptor (EGFR has been one of the most effective colorectal cancer strategies. Anti-EGFR antibodies function by binding to the extracellular domain of EGFR, preventing its activation, and ultimately providing clinical benefit. KRAS mutations in codons 12 and 13 are recognized prognostic and predictive biomarkers that should be analyzed at the clinic prior to the administration of anti-EGFR therapy. However, still an important fraction of KRAS wild-type patients do not respond to the treatment. The identification of additional genetic determinants of primary or secondary resistance to EGFR targeted therapy for further improving the selection of patients is urgent. Herein, we review the latest published literature highlighting the most important genes that may predict resistance to anti-EGFR monoclonal antibodies in colorectal cancer patients. According to the available findings, the evaluation of BRAF, NRAS, PIK3CA, and PTEN status could be the right strategy to select patients who are likely to respond to anti-EGFR therapies. In the future, the combination of those biomarkers will help establish consensus that can be introduced into clinical practice.

  19. Does the early social environment affect structure and consistency of personality in wild-type male's rat?

    Science.gov (United States)

    Gracceva, Giulia; Koolhaas, Jaap M; Groothuis, Ton G G

    2011-09-01

    Animal personality has been extensively studied from a functional and evolutionary point of view. Less attention has been paid to the development of personality, its phenotypic plasticity, and the influence of manipulation of early environmental factors. Here we describe the effects of manipulating the sex ratio of the litter, at postnatal day (pnd) 3, in wild-type rats, on personality traits in adulthood. We measured the treatment effects on aggression, defensive burying, and open field behavior at pnd 90 and 120, as well as on their contextual generality, and stability over time (differential and structural consistency). Main effects of litter composition were found on open field behavior at pnd 120 but not on the other behaviors. Since correlations between behaviors changed over time irrespective of the specific treatment, whereas in previous studies on unmanipulated litters this was not the case we suggest that early handling may disrupt adult personality traits. Overall the data indicate that personality is less stable over time that often assumed, having both proximate and ultimate implications.

  20. Wild type p53 increased chemosensitivity of drug-resistant human hepatocellular carcinoma Be17402 / 5-FU cells

    Institute of Scientific and Technical Information of China (English)

    Yu-xiuLI; Zhi-binLIN; Huan-ranTAN

    2004-01-01

    AIM: To study the effect of wild type (wt) p53 gene transfection on drug resistant human hepatocellular carcinoma(HCC) cells induced by 5-Fluorouracil (5-FU). METHODS: The cytotoxicity of anticancer drugs on Be17402 and Be17402/5-FU cells was assessed using SRB assay, p53 expression was detected at its mRNA level by RT-PCR assay and at its protein level Western blot or immunocytochemistry assay in Be17402/5-FU cells transfected with either control vector or wt p53. AnnexinV-FITC/PI double labeled assay was performed to detect apoptosis. The chemosensitivity of Be17402/5-FU cells transfected with wt p53 was assessed using SRB assay. RESULTS: Be17402/5-FU cells exhibited cross-resistance to vincristine, doxorubicin, paclitaxel, and so on. wt p53 gene transfection upregulated the expression of p53 in Be17402/5-FU cells, wt p53 was able to greatly inhibit cell proliferation and significantly induce apoptosis in Be17402/5-FU cells. Moreover, wt p53 gene transfection increased the chemosensitivity of Be17402/5-FU cells to some anticancer drugs. CONCLUSION: These results indicated that the wt p53 gene transfection not only induced suppression of cell growth, but also increased the sensitivity of Be17402/5-FU cells to 5-FU, vincristine, and doxorubicin.

  1. Beta Cell Formation in vivo Through Cellular Networking, Integration and Processing (CNIP) in Wild Type Adult Mice.

    Science.gov (United States)

    Doiron, Bruno; Hu, Wenchao; DeFronzo, Ralph A

    2016-01-01

    Insulin replacement therapy is essential in type 1 diabetic individuals and is required in ~40- 50% of type 2 diabetics during their lifetime. Prior attempts at beta cell regeneration have relied upon pancreatic injury to induce beta cell proliferation, dedifferentiation and activation of the embryonic pathway, or stem cell replacement. We report an alternative method to transform adult non-stem (somatic) cells into pancreatic beta cells. The Cellular Networking, Integration and Processing (CNIP) approach targets cellular mechanisms involved in pancreatic function in the organ's adult state and utilizes a synergistic mechanism that integrates three important levels of cellular regulation to induce beta cell formation: (i) glucose metabolism, (ii) membrane receptor function, and (iii) gene transcription. The aim of the present study was to induce pancreatic beta cell formation in vivo in adult animals without stem cells and without dedifferentiating cells to recapitulate the embryonic pathway as previously published (1-3). Our results employing CNIP demonstrate that: (i) insulin secreting cells can be generated in adult pancreatic tissue in vivo and circumvent the problem of generating endocrine (glucagon and somatostatin) cells that exert deleterious effects on glucose homeostasis, and (ii) longterm normalization of glucose tolerance and insulin secretion can be achieved in a wild type diabetic mouse model. The CNIP cocktail has the potential to be used as a preventative or therapeutic treatment or cure for both type 1 and type 2 diabetes.

  2. Identification of lipoxygenase (LOX) genes from legumes and their responses in wild type and cultivated peanut upon Aspergillus flavus infection

    Science.gov (United States)

    Song, Hui; Wang, Pengfei; Li, Changsheng; Han, Suoyi; Lopez-Baltazar, Javier; Zhang, Xinyou; Wang, Xingjun

    2016-01-01

    Lipoxygenase (LOX) genes are widely distributed in plants and play crucial roles in resistance to biotic and abiotic stress. Although they have been characterized in various plants, little is known about the evolution of legume LOX genes. In this study, we identified 122 full-length LOX genes in Arachis duranensis, Arachis ipaënsis, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus and Medicago truncatula. In total, 64 orthologous and 36 paralogous genes were identified. The full-length, polycystin-1, lipoxygenase, alpha-toxin (PLAT) and lipoxygenase domain sequences from orthologous and paralogous genes exhibited a signature of purifying selection. However, purifying selection influenced orthologues more than paralogues, indicating greater functional conservation of orthologues than paralogues. Neutrality and effective number of codons plot results showed that natural selection primarily shapes codon usage, except for C. arietinum, L. japonicas and M. truncatula LOX genes. GCG, ACG, UCG, CGG and CCG codons exhibited low relative synonymous codon usage (RSCU) values, while CCA, GGA, GCU, CUU and GUU had high RSCU values, indicating that the latter codons are strongly preferred. LOX expression patterns differed significantly between wild-type peanut and cultivated peanut infected with Aspergillus flavus, which could explain the divergent disease resistance of wild progenitor and cultivars. PMID:27731413

  3. Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild-Type and p53 Knockout HCT116 Cells

    Directory of Open Access Journals (Sweden)

    Kiheon Choi

    2017-01-01

    Full Text Available Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT or p53 knockout (KO HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

  4. Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice.

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    Benjamin Sanchez

    Full Text Available Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx. Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters.Eight wild-type (wt and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg-1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed.As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p 70 Hz, but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01. Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively.Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials.

  5. Zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice

    Science.gov (United States)

    Huang, Wen-Chin; Abraham, Rachy; Shim, Byoung-Shik; Choe, Hyeryun; Page, Damon T.

    2016-01-01

    Zika virus (ZIKV) infection in pregnant women has been established as a cause of microcephaly in newborns. Here we test the hypothesis that neurodevelopmental stages when the brain is undergoing rapid growth are particularly vulnerable to the effects of ZIKV infection. We injected ZIKV intracranially into wild type C57BL/6 mice at two different time points: early postnatal development, when the brain is growing at its maximal rate, and at weaning, when the brain has largely reached adult size. Both time points showed widespread immunoreactivity for ZIKV and cleaved caspase 3 (CC3, a marker of apoptosis) throughout the brain. However, in early postnatal ZIKV injected mice, some brain areas and cell types display particularly large increases in apoptosis that we did not observe in older animals. Corticospinal pyramidal neurons, a cell type implicated in human microcephaly associated with ZIKV infection, are an example of one such cell type. Proliferating cells in the ventricular zone stem cell compartment are also depleted. These findings are consistent with the hypothesis that periods of rapid brain growth are especially susceptible to neurodevelopmental effects of ZIKV infection, and establish a valuable model to investigate mechanisms underlying neurodevelopmental effects of ZIKV infection and explore candidate therapeutics. PMID:27713505

  6. Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells

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    Takeomi Mizutani

    2016-03-01

    Full Text Available This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1 locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]. To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article “Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85” [1].

  7. Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells.

    Science.gov (United States)

    Mizutani, Takeomi; Haga, Hisashi; Kawabata, Kazushige

    2016-03-01

    This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1) locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]). To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article "Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85" [1].

  8. Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis.

    Science.gov (United States)

    Poulsen, Christian Bjørn; Penkowa, Milena; Borup, Rehannah; Nielsen, Finn Cilius; Cáceres, Mario; Quintana, Albert; Molinero, Amalia; Carrasco, Javier; Giralt, Mercedes; Hidalgo, Juan

    2005-01-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays in wild-type and IL-6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post-lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with approximately 12,400 probe sets corresponding to approximately 10,000 different murine genes (MG_U74Av2). A robust, conventional statistical method (two-way anova) was employed to select the genes significantly affected. An orderly pattern of gene responses was clearly detected, with genes being up- or down-regulated at specific timings consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight the importance of IL-6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study.

  9. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function.

    Directory of Open Access Journals (Sweden)

    Fernando Aprile-Garcia

    Full Text Available The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln by arginine (Arg substitution at codon 460 of the purinergic P2X7 receptor (P2X7R has been associated with mood disorders. No change in function (loss or gain has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.

  10. ATLAS: A database linking binding affinities with structures for wild-type and mutant TCR-pMHC complexes.

    Science.gov (United States)

    Borrman, Tyler; Cimons, Jennifer; Cosiano, Michael; Purcaro, Michael; Pierce, Brian G; Baker, Brian M; Weng, Zhiping

    2017-02-03

    The ATLAS (Altered TCR Ligand Affinities and Structures) database (https://zlab.umassmed.edu/atlas/web/) is a manually curated repository containing the binding affinities for wild-type and mutant T cell receptors (TCRs) and their antigens, peptides presented by the major histocompatibility complex (pMHC). The database links experimentally measured binding affinities with the corresponding three dimensional (3D) structures for TCR-pMHC complexes. The user can browse and search affinities, structures, and experimental details for TCRs, peptides, and MHCs of interest. We expect this database to facilitate the development of next-generation protein design algorithms targeting TCR-pMHC interactions. ATLAS can be easily parsed using modeling software that builds protein structures for training and testing. As an example, we provide structural models for all mutant TCRs in ATLAS, built using the Rosetta program. Utilizing these structures, we report a correlation of 0.63 between experimentally measured changes in binding energies and our predicted changes. Proteins 2017. © 2017 Wiley Periodicals, Inc.

  11. Characterization of Hemodynamics in Great Arteries of Wild-Type Mouse Using Computational Fluid Dynamics Based on Ultrasound Images.

    Science.gov (United States)

    Chen, Zhuo; Zhou, Yue; Ma, Youcai; Wang, Jingying; He, Yihua; Li, Zhian

    2016-03-01

    Hemodynamic factors in cardiovascular system are hypothesized to play a significant role in causing structural heart development. It is thus important to improve our understanding of velocity characteristics and parameters. We present such a study on wild-type mouse to characterize the vessel geometry, flow pattern, and wall shear stress in great arteries. Microultrasound imaging for small animals was used to measure blood boundary and velocity of the great arteries. Subsequently, specimens' flow boundary conditions were used for 3-dimensional reconstructions of the great artery and aortic arch dimensions, and blood flow velocity data were input into subject-specific computational fluid dynamics for modeling hemodynamics. Measurement by microultrasound imaging showed that blood velocities in the great artery and aortic arch had strong correlations with vascular sizes, whereas blood pressure had a weak trend in relation to vascular size. Wall shear stress magnitude increased when closer to arterial branches and reduced proximally in the aortic root and distally in the descending aorta, and the parameters were related to the fluid mechanics in branches in some degree. We developed a method to investigate fluid mechanics in mouse arteries, using a combination of microultrasound and computational fluid dynamics, and demonstrated its ability to reveal detailed geometric, kinematic, and fluid mechanics parameters.

  12. Effects of different drought treatments on root and shoot development of the tomato wild type and flacca mutant

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    Prokić Ljiljana

    2011-01-01

    Full Text Available The effects of drought and partial root drying (PRD on shoot and root growth was assessed in the wild type Ailsa Craig (WT and the flacca tomato mutant deficient in the plant hormone ABA. Our results show that drought had an inhibitory effect on shoot growth in flacca and especially in WT; the most profound effect was observed in FI (full irrigation, then PRD and the smallest in D plants. Root development in both WT and flacca was stimulated after the 3rd day of the experiment following a decrease in the soil water content. On the 11th day of the experiment, when the soil water content was reduced by about 50% of full irrigation (FI, the root density was increased in the drying part of the PDR and on both sides of the drought treatment. On the basis of these results it can be assumed that increased root density and root length represent an adaptation or root adjustment to drought conditions.

  13. Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

    Science.gov (United States)

    Ludlow, M; Nguyen, D T; Silin, D; Lyubomska, O; de Vries, R D; von Messling, V; McQuaid, S; De Swart, R L; Duprex, W P

    2012-07-01

    The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

  14. Protective effect of recombinant human brain natriuretic peptide on acute renal injury induced by endotoxin in canines%rhB NP对内毒素介导的犬急性肾损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    李牧; 刘岩; 李辉

    2014-01-01

    干预组比较,血清HMGB-1水平显著性减少(P<0.05)。结论 rhBNP可以有效减轻内毒素介导的犬肾组织损伤,改善肾功能,并降低SVRI,且其治疗作用呈剂量-效应关系;rhBNP还能有效降低脓毒症犬血清HMGB-1水平,可能与晚期炎症因子HMGB1水平的降低有关。%Objective To observe the influence of recombinant human brain natriuretic peptide(rhBNP)on serum HMGB1 levels in canines'acute renal injury induced by endotoxin and explore its protective role of rhBNP in protecting canines'kidney against acute renal injury.Methods A total of 20 healthy dogs were randomly divided into four groups:blank group,sepsis shock group,low-dose intervention group and high dose intervention group, and there were 5 rats in each group.After establishing the model of canines'sepsis shock induced by endotoxin,15 canines (besides blank group)were randomly divided into 3 groups.As follows,5 μg/kg rhBNP was given to the low-dose intervention group,10μg/kg rhBNP was given to the high-dose intervention group.But nothing was given to control group.Systemic vascular resistance index(SVRI)at 0 h,2 h,4 h,8 h,12 h were observed by PICCO instrument.High mobility group box 1 protein (HMGB-1)and creatinine(CR)in blood samples at each time point were detected.After 12 hours,kidney samples were taken for histological examination.Results The results revealed that some renal tubulars epithelial cell were swelled,some epithelial cells were atrophy and interstitial cells swelled in control group under the light microscope.Kidney pathology score was 2-3.But these changes were improved in low-and high-dose intervention groups,and there were no significant difference in the latter two groups,kidney of both groups pathology score were 1-2.Compared with control group at the same point,CR of blood serum were significantly decreased in low-dose intervention group at 8 h,12 h(P<0.01), and high-dose intervention group significantly decreased at 4 h,8 h,12 h(P<0.01).Compared with

  15. Vaccines for Canine Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Faeze Foroughi-Parvar

    2014-01-01

    Full Text Available Leishmania infantum is the obligatory intracellular parasite of mammalian macrophages and causes zoonotic visceral leishmaniasis (ZVL. The presence of infected dogs as the main reservoir host of ZVL is regarded as the most important potential risk for human infection. Thus the prevention of canine visceral leishmaniasis (CVL is essential to stop the current increase of the Mediterranean visceral leishmaniasis. Recently considerable advances in achieving protective immunization of dogs and several important attempts for achieving an effective vaccine against CVL lead to attracting the scientists trust in its important role for eradication of ZVL. This paper highlights the recent advances in vaccination against canine visceral leishmaniasis from 2007 until now.

  16. Photosynthetic electron transport inhibition by 2-substituted 4-alkyl-6-benzylamino-1,3,5-triazines with thylakoids from wild- type and atrazine-resistant Chenopodium album

    NARCIS (Netherlands)

    Okano, R.; Ohki, A.; Ohki, S.; Kohno, H.; Rensen, van J.J.S.; Böger, P.; Wakabayashi, K.

    2002-01-01

    The effect of 2-benzylamino-1,3,5-triazines on photosynthetic electron transport (PET) was measured with thylakoids isolated from atrazine-resistant, wild-type Chenopodium album, and spinach to find novel 1,3,5-triazine herbicides bearing a strong PET inhibition. The PET inhibition assay with Chenop

  17. Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione.

    Science.gov (United States)

    Grabek-Lejko, Dorota; Kurylenko, Olena O; Sibirny, Vladimir A; Ubiyvovk, Vira M; Penninckx, Michel; Sibirny, Andriy A

    2011-11-01

    The ability of baker's yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.

  18. Contrasting colonization and plant growth promoting capacity between wild type and gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

    Energy Technology Data Exchange (ETDEWEB)

    Weyens N.; van der Lelie D.; Boulet, J.; Adriaensen, D.; Timmermans, J.-P.; Prinsen, E.; Van Oevelen, S.; D" Haen, J.; Smeets, K.; Taghavi, S.; Vangronsveld, J.

    2011-06-09

    This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

  19. Complete Genome Sequence of a Wild-Type Measles Virus Isolated during a 2016 Winter Outbreak in a Refugee Settlement in Calais, France

    Science.gov (United States)

    Hamel, Justine; Antona, Denise; Vabret, Astrid

    2017-01-01

    ABSTRACT Measles outbreaks are regularly reported in European countries despite efforts to improve vaccination coverage. In January 2016, an outbreak occurred in a refugee settlement in Calais, France. We report here the complete genome sequence of a wild-type measles virus isolated from a health care worker (MVi/Calais. FRA/01.16) infected during this outbreak. PMID:28280010

  20. Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

    DEFF Research Database (Denmark)

    Cox, David G; Simard, Jacques; Sinnett, Daniel

    2011-01-01

    carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence...

  1. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per

    1996-01-01

    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  2. Anticancer Effects of 15d-Prostaglandin-J(2) in Wild-Type and Doxorubicin-Resistant Ovarian Cancer Cells : Novel Actions on SIRT1 and HDAC

    NARCIS (Netherlands)

    de Jong, Edwin; Winkel, Peter; Poelstra, Klaas; Prakash, Jai

    2011-01-01

    15-deoxy-delta-12,14-prostaglandin-J(2) (15d-PGJ(2)), an arachidonic metabolite and a natural PPAR gamma agonist, is known to induce apoptosis in tumor cells. In this study, we investigated new therapeutic potentials of 15d-PGJ(2) by determining its anticancer effects in wild-type and doxorubicin-re

  3. Free energies of binding of R - and S -propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations

    NARCIS (Netherlands)

    Commandeur, Jan; Graaf, de Chris; Keizers, Peter; Oostenbrink, Chris; Vugt-Lussenburg, van Barbara; Vermeulen, Nico

    2007-01-01

    Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches t

  4. Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

    NARCIS (Netherlands)

    Dummer, R; Bergh, J; Karlsson, Y; Horovitz, JA; Mulder, NH; Huinin, DT; Burg, G; Hofbauer, G; Osanto, S

    2000-01-01

    p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector cont

  5. S-palmitoylation regulates biogenesis of core glycosylated wild-type and F508del CFTR in a post-ER compartment.

    Science.gov (United States)

    McClure, Michelle L; Wen, Hui; Fortenberry, James; Hong, Jeong S; Sorscher, Eric J

    2014-04-15

    Defects in CFTR (cystic fibrosis transmembrane conductance regulator) maturation are central to the pathogenesis of CF (cystic fibrosis). Palmitoylation serves as a key regulator of maturational processing in other integral membrane proteins, but has not been tested previously for functional effects on CFTR. In the present study, we used metabolic labelling to confirm that wild-type and F508del CFTR are palmitoylated, and show that blocking palmitoylation with the pharmacologic inhibitor 2-BP (2-bromopalmitate) decreases steady-state levels of both wild-type and low temperature-corrected F508del CFTR, disrupts post-ER (endoplasmic reticulum) maturation and reduces ion channel function at the cell surface. PATs (protein acyl transferases) comprise a family of 23 gene products that contain a DHHC motif and mediate palmitoylation. Recombinant expression of specific PATs led to increased levels of CFTR protein and enhanced palmitoylation as judged by Western blot and metabolic labelling. Specifically, we show that DHHC-7 (i) increases steady-state levels of wild-type and F508del CFTR band B, (ii) interacts preferentially with the band B glycoform, and (iii) augments radiolabelling by [3H]palmitic acid. Interestingly, immunofluorescence revealed that DHHC-7 also sequesters the F508del protein to a post-ER (Golgi) compartment. Our findings point to the importance of palmitoylation during wild-type and F508del CFTR trafficking.

  6. Biomarkers of aging, life span and spontaneous carcinogenesis in the wild type and HER-2 transgenic FVB/N female mice.

    Science.gov (United States)

    Panchenko, Andrey V; Popovich, Irina G; Trashkov, Alexandr P; Egormin, Peter A; Yurova, Maria N; Tyndyk, Margarita L; Gubareva, Ekaterina A; Artyukin, Ilia N; Vasiliev, Andrey G; Khaitsev, Nikolai V; Zabezhinski, Mark A; Anisimov, Vladimir N

    2016-04-01

    FVB/N wild type and transgenic HER-2/neu FVB/N female mice breed at N.N. Petrov Research Institute of Oncology were under observation until natural death without any special treatment. Age-related dynamics of body weight, food consumption and parameters of carbohydrate and lipid metabolism, level of nitric oxide, malonic dialdehyde, catalase, Cu, Zn-superoxide dismutase, vascular endothelial growth factor were studied in both mice strains. The parameters of life span and tumor pathology were studied as well. Cancer-prone transgenic HER-2/neu mice developed in 100 % multiple mammary adenocarcinomas and died before the age of 1 year. Forty tree percent of long-lived wild type mice survived the age of 2 years and 19 %-800 days. The total tumor incidence in wild type mice was 34 %. The age-associated changes in the level of serum IGF-1, glucose and insulin started much earlier in transgene HER-2/neu mice as compared with wild type FVB/N mice. It was suggested that transgenic HER-2/neu involves in initiation of malignization of mammary epithelial cells but also in acceleration of age-related hormonal and metabolic changes in turn promoting mammary carcinogenesis.

  7. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  8. Wild-Type Mitochondrial DNA Copy Number in Urinary Cells as a Useful Marker for Diagnosing Severity of the Mitochondrial Diseases.

    Directory of Open Access Journals (Sweden)

    Hui Liu

    Full Text Available The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.

  9. Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

    NARCIS (Netherlands)

    M. Ludlow (Martin); L.J. Rennick (Linda); S. Sarlang (Severine); G. Skibinski (Grzegorz); S. McQuaid (Stephen); T. Moore (Tara); R.L. de Swart (Rik); W.P. Duprex (Paul)

    2010-01-01

    textabstractThe lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entr

  10. A glycoprotein E deletion mutant of bovine herpesvirus 1 infects the same limited number of tissues in calves as wild-type virus, but for a shorter period

    NARCIS (Netherlands)

    Engelenburg, van F.A.C.; Kaashoek, M.J.; Oirschot, van J.T.; Rijsewijk, F.A.M.

    1995-01-01

    To gain insight into the role of glycoprotein E of bovine herpesvirus 1 (BHV-1), we compared the distribution of wild-type (wt) BHV-1 with that of a gE deletion mutant (gE-) in calves after intranasal inoculation. The wt-infected calves had severe clinical signs, but the gE--infected calves were vir

  11. Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole.

    NARCIS (Netherlands)

    Espinel-Ingroff, A.; Aller, A.I.; Canton, E.; Castanon-Olivares, L.R.; Chowdhary, A.; Cordoba, S.; Cuenca-Estrella, M.; Fothergill, A.; Fuller, J.; Govender, N.; Hagen, F.; Illnait-Zaragozi, M.T.; Johnson, E.; Kidd, S.; Lass-Florl, C.; Lockhart, S.R.; Martins, M.A.; Meis, J.F.G.M.; Melhem, M.S.; Ostrosky-Zeichner, L.; Pelaez, T.; Pfaller, M.A.; Schell, W.A.; St-Germain, G.; Trilles, L.; Turnidge, J.

    2012-01-01

    Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to

  12. Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine.

    NARCIS (Netherlands)

    Espinel-Ingroff, A.; Chowdhary, A.; Cuenca-Estrella, M.; Fothergill, A.; Fuller, J.; Hagen, F.; Govender, N.; Guarro, J.; Johnson, E.; Lass-Florl, C.; Lockhart, S.R.; Martins, M.A.; Meis, J.F.G.M.; Melhem, M.S.; Ostrosky-Zeichner, L.; Pelaez, T.; Pfaller, M.A.; Schell, W.A.; Trilles, L.; Kidd, S.; Turnidge, J.

    2012-01-01

    Clinical breakpoints (CBPs) are not available for the Cryptococcus neoformans-Cryptococcus gattii species complex. MIC distributions were constructed for the wild type (WT) to establish epidemiologic cutoff values (ECVs) for C. neoformans and C. gattii versus amphotericin B and flucytosine. A total

  13. Chemotherapy Plus Cetuximab versus Chemotherapy Alone for Patients with KRAS Wild Type Unresectable Liver-Confined Metastases Colorectal Cancer: An Updated Meta-Analysis of RCTs

    Science.gov (United States)

    Lv, W.; Zhang, G. Q.; Jiao, A.; Zhao, B. C.; Shi, Y.; Chen, B. M.

    2017-01-01

    Purpose. Our study analyses clinical trials and evaluates the efficacy of adding cetuximab in systematic chemotherapy for unresectable colorectal cancer liver-confined metastases patients. Materials and Methods. Search EMBASE, PubMed, and the Cochrane Central Register of Controlled Trials for RCTs comparing chemotherapy plus cetuximab with chemotherapy alone for KRAS wild type patients with colorectal cancer liver metastases (CRLMs). We calculated the relative risks (RRs) with 95% confidence interval and performed meta-analysis of hazard ratios (HRs) for the R0 resection rate, the overall response rate (ORR), the progression-free survival (PFS) and overall survival (OS). Results. 1173 articles were retrieved and 4 RCTs were available for our study. The four studies involved 504 KRAS wild type patients with CRLMs. The addition of cetuximab significantly improved all the 4 outcomes: the R0 resection rate (RR 2.03, p = 0.004), the ORR (RR 1.76, p < 0.00001), PFS (HR 0.63, p < 0.0001), and also OS (HR 0.74, p = 0.04); the last outcome is quite different from the conclusion published before. Conclusions. Although the number of patients analysed was limited, we found that the addition of cetuximab significantly improves the outcomes in KRAS wild type patients with unresectable colorectal cancer liver-confined metastases. Cetuximab combined with systematic chemotherapy perhaps suggests a promising choice for KRAS wild type patients with unresectable liver metastases. PMID:28167959

  14. Phylogenetic evidence of a new canine distemper virus lineage among domestic dogs in Colombia, South America.

    Science.gov (United States)

    Espinal, Maria A; Díaz, Francisco J; Ruiz-Saenz, Julian

    2014-08-06

    Canine distemper virus (CDV) is a highly contagious viral disease of carnivores affecting both wild and domestic populations. The hemagglutinin gene, encoding for the attachment protein that determines viral tropism, shows high heterogeneity among strains, allowing for the distinction of ten different lineages distributed worldwide according to a geographic pattern. We obtained the sequences of the full-length H gene of 15 wild-type CDV strains circulating in domestic dog populations from the Aburrá Valley, Colombia. A phylogenetic analysis of H gene nucleotide sequences from Colombian CDV viruses along with field isolates from different geographic regions and vaccine strains was performed. Colombian wild-type viruses formed a distinct monophyletic cluster clearly separated from the previously identified wild-type and vaccine lineages, suggesting that a novel genetic variant, quite different from vaccines and other lineages, is circulating among dog populations in the Aburrá Valley. We propose naming this new lineage as "South America 3". This information indicates that there are at least three different CDV lineages circulating in domestic and wild carnivore populations in South America. The first one, renamed Europe/South America 1, circulates in Brazil and Uruguay; the second, South America 2, appears to be restricted to Argentina; and the third, South America 3, which comprises all the strains characterized in this study, may also be circulating in other northern countries of South America.

  15. Salt stress-induced production of reactive oxygen- and nitrogen species and cell death in the ethylene receptor mutant Never ripe and wild type tomato roots.

    Science.gov (United States)

    Poór, Péter; Kovács, Judit; Borbély, Péter; Takács, Zoltán; Szepesi, Ágnes; Tari, Irma

    2015-12-01

    The salt stress triggered by sublethal, 100 mM and lethal, 250 mM NaCl induced ethylene production as well as rapid accumulation of superoxide radical and H2O2 in the root tips of tomato (Solanum lycopersicum cv. Ailsa Craig) wild type and ethylene receptor mutant, Never ripe (Nr/Nr) plants. In the wild type plants superoxide accumulation confined to lethal salt concentration while H2O2 accumulated more efficiently under sublethal salt stress. However, in Nr roots the superoxide production was higher and unexpectedly, H2O2 level was lower than in the wild type under sublethal salt stress. Nitric oxide production increased significantly under sublethal and lethal salt stress in both genotypes especially in mutant plants, while peroxynitrite accumulated significantly under lethal salt stress. Thus, the nitro-oxidative stress may be stronger in Nr roots, which leads to the programmed death of tissues, characterized by the DNA and protein degradation and loss of cell viability under moderate salt stress. In Nr mutants the cell death was induced in the absence of ethylene perception. Although wild type roots could maintain their potassium content under moderate salt stress, K(+) level significantly declined leading to small K(+)/Na(+) ratio in Nr roots. Thus Nr mutants were more sensitive to salt stress than the wild type and the viability of root cells decreased significantly under moderate salt stress. These changes can be attributed to a stronger ionic stress due to the K(+) loss from the root tissues.

  16. Canine pulmonary angiostrongylosis

    DEFF Research Database (Denmark)

    Koch, Jørgen; Willesen, Jakob Lundgren

    2009-01-01

    Canine pulmonary angiostrongylosis is an emerging snail-borne disease causing verminous pnemonia and coagulopathy in dogs. The parasite is fund in Europe, North and South America and Africa, covering tropical, subtropical and temperate regions. Its distribution has been characterised by isolated ...

  17. Placental glucose and amino acid transport in calorie-restricted wild-type and Glut3 null heterozygous mice.

    Science.gov (United States)

    Ganguly, Amit; Collis, Laura; Devaskar, Sherin U

    2012-08-01

    Calorie restriction (CR) decreased placenta and fetal weights in wild-type (wt) and glucose transporter (Glut) 3 heterozygous null (glut3(+/-)) mice. Because placental nutrient transport is a primary energy determinant of placentofetal growth, we examined key transport systems. Maternal CR reduced intra- and transplacental glucose and leucine transport but enhanced system A amino acid transport in wt mice. These transport perturbations were accompanied by reduced placental Glut3 and leucine amino acid transporter (LAT) family member 2, no change in Glut1 and LAT family member 1, but increased sodium coupled neutral amino acid transporter (SNAT) and SNAT2 expression. We also noted decreased total and active phosphorylated forms of mammalian target of rapamycin, which is the intracellular nutrient sensor, the downstream total P70S6 kinase, and pS6 ribosomal protein with no change in total and phosphorylated 4E-binding protein 1. To determine the role of placental Glut3 in mediating CR-induced placental transport changes, we next investigated the effect of gestational CR in glut3(+/-) mice. In glut3(+/-) mice, a key role of placental Glut3 in mediating transplacental and intraplacental glucose transport was established. In addition, reduced Glut3 results in a compensatory increase of leucine and system A transplacental transport. On the other hand, diminished Glut3-mediated intraplacental glucose transport reduced leucine transport and mammalian target of rapamycin and preserved LAT and enhancing SNAT. CR in glut3(+/-) mice further reduced transplacental glucose transport and enhanced system A amino acid transport, although the increased leucine transport was lost. In addition, increased Glut3 was seen and preserved Glut1, LAT, and SNAT. These placental changes collectively protect survival of wt and glut3(+/-) fetuses against maternal CR-imposed reduction of macromolecular nutrients.

  18. Investigating the structure and dynamics of the PIK3CA wild-type and H1047R oncogenic mutant.

    Directory of Open Access Journals (Sweden)

    Paraskevi Gkeka

    2014-10-01

    Full Text Available The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα, which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR experiments and Molecular Dynamics (MD simulations were carried out for both wild-type (WT and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.

  19. In situ dimerization of multiple wild type and mutant zinc transporters in live cells using bimolecular fluorescence complementation.

    Science.gov (United States)

    Lasry, Inbal; Golan, Yarden; Berman, Bluma; Amram, Noy; Glaser, Fabian; Assaraf, Yehuda G

    2014-03-14

    Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.

  20. Early cognitive/social deficits and late motor phenotype in conditional wild-type TDP-43 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Julio Armando Alfieri

    2016-12-01

    Full Text Available Frontotemporal Dementia (FTD and amyotrophic lateral sclerosis (ALS are two neurodegenerative diseases associated to mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43. To investigate in depth the behavioral phenotype associated with this proteinopathy, we used as a model transgenic mice conditionally overexpressing human wild-type TDP 43 protein (hTDP-43-WT in forebrain neurons. We previously characterized these mice at the neuropathological level and found progressive neurodegeneration and other features that evoke human TDP-43 proteinopathies of the FTD/ALS spectrum. In the present study we analyzed the behavior of mice at multiple domains, including motor, social and cognitive performance. Our results indicate that young hTDP-43-WT transgenic mice (1 month after post-weaning transgene induction present a normal motor phenotype compared to control littermates, as assessed by accelerated rotarod performance, spontaneous locomotor activity in the open field test and a mild degree of spasticity shown by a clasping phenotype. Analysis of social and cognitive behavior showed a rapid installment of deficits in social interaction, working memory (Y-maze test and recognition memory (novel object recognition test in the absence of overt motor abnormalities. To investigate if the motor phenotype worsen with age, we analyzed the behavior of mice after long-term (up to 12 months transgene induction. Our results reveal a decreased performance on the rotarod test and in the hanging wire test, indicating a motor phenotype that was absent in younger mice. In addition, long-term hTDP-43-WT expression led to hyperlocomotion in the open field test. In sum, these results demonstrate a time-dependent emergence of a motor phenotype in older hTDP-43-WT transgenic mice, recapitulating aspects of clinical FTD presentations with motor involvement in human patients, and providing a complementary animal model for studying TDP-43 proteinopathies.

  1. Dehydrocostuslactone, a sesquiterpene lactone activates wild-type and ΔF508 mutant CFTR chloride channel.

    Science.gov (United States)

    Wang, Xue; Zhang, Yao-Fang; Yu, Bo; Yang, Shuang; Luan, Jian; Liu, Xin; Yang, Hong

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) represents the main cAMP-activated Cl⁻ channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. The aim of this study was to identify natural compounds that are able to stimulate wild-type (wt) and ΔF508 mutant CFTR channel activities in CFTR-expressing Fischer rat thyroid (FRT) cells. We found that dehydrocostuslactone [DHC, (3aS, 6aR, 9aR, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b] furan-2(3H)-one)] dose dependently potentiates both wt and ΔF508 mutant CFTR-mediated iodide influx in cell-based fluorescent assays and CFTR-mediated Cl⁻ currents in short-circuit current studies, and the activations could be reversed by the CFTR inhibitor CFTRinh-172. Maximal CFTR-mediated apical Cl⁻ current secretion in CFTR-expressing FRT cells was stimulated by 100 μM DHC. Determination of intracellular cAMP content showed that DHC modestly but significantly increased cAMP level in FRT cells, but cAMP elevation effects contributed little to DHC-stimulated iodide influx. DHC also stimulated CFTR-mediated apical Cl⁻ current secretion in FRT cells expressing ΔF508-CFTR. Subsequent studies demonstrated that activation of CFTR by DHC is forskolin dependent. DHC represents a new class of CFTR potentiators that may have therapeutic potential in CFTR-related diseases.

  2. Comparative thoracic anatomy of the wild type and wingless (wg(1)cn(1)) mutant of Drosophila melanogaster (Diptera).

    Science.gov (United States)

    Fabian, Benjamin; Schneeberg, Katharina; Beutel, Rolf Georg

    2016-11-01

    Genetically modified organisms are crucial for our understanding of gene regulatory networks, physiological processes and ontogeny. With modern molecular genetic techniques allowing the rapid generation of different Drosophila melanogaster mutants, efficient in-depth morphological investigations become an important issue. Anatomical studies can elucidate the role of certain genes in developmental processes and point out which parts of gene regulatory networks are involved in evolutionary changes of morphological structures. The wingless mutation wg(1) of D. melanogaster was discovered more than 40 years ago. While early studies addressed the external phenotype of these mutants, the documentation of the internal organization was largely restricted to the prominent indirect flight muscles. We used SEM micrographs, histological serial sections, μ-computed tomography, CLSM and 3D reconstructions to study and document the thoracic skeletomuscular system of the wild type and mutant. A recently introduced nomenclature for the musculature of neopteran insects was applied to facilitate comparisons with closely or more distantly related taxa. The mutation is phenotypically mainly characterized by the absence of one or both wings and halteres. The wing is partly or entirely replaced by duplications of mesonotal structures, whereas the haltere and its associated muscles are completely absent on body sides showing the reduction. Both the direct and indirect mesothoracic flight muscles are affected by loss and reorientation of bundles or fibers. Our observations lead to the conclusion that the wingless mutation causes a homeotic transformation in the imaginal discs of wings and halteres with a direct effect on the development of skeletal structures and an indirect effect on the associated muscular system.

  3. Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS.

    Directory of Open Access Journals (Sweden)

    Sara Tavella

    Full Text Available Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt and pleiotrophin-transgenic (PTN-Tg mice exposed to a near-zero gravity on the International Space Station (ISS during the Mice Drawer System (MDS mission, to date, the longest mice permanence (91 days in space. The transgenic mouse strain over-expressing pleiotrophin (PTN in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

  4. Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1.

    Science.gov (United States)

    Samuilov, Sladjana; Lang, Friedericke; Djukic, Matilda; Djunisijevic-Bojovic, Danijela; Rennenberg, Heinz

    2016-09-01

    Growth and development of plants largely depends on their adaptation ability in a changing climate. This is particularly true on heavy metal contaminated soils, but the interaction of heavy metal stress and climate on plant performance has not been intensively investigated. The aim of the present study was to elucidate if transgenic poplars (Populus tremula x P. alba) with enhanced glutathione content possess an enhanced tolerance to drought and lead (Pb) exposure (single and in combination) and if they are good candidates for phytoremediation of Pb contaminated soil. Lead exposure reduced growth and biomass accumulation only in above-ground tissue of wild type poplar, although most of lead accumulated in the roots. Drought caused a decline of the water content rather than reduced biomass production, while Pb counteracted this decline in the combined exposure. Apparently, metals such as Pb possess a protective function against drought, because they interact with abscisic acid dependent stomatal closure. Lead exposure decreased while drought increased glutathione content in leaves of both plant types. Lead accumulation was higher in the roots of transgenic plants, presumably as a result of chelation by glutathione. Water deprivation enhanced Pb accumulation in the roots, but Pb was subject to leakage out of the roots after re-watering. Transgenic plants showed better adaptation under mild drought plus Pb exposure partially due to improved glutathione synthesis. However, the transgenic plants cannot be considered as a good candidate for phytoremediation of Pb, due to its small translocation to the shoots and its leakage out of the roots upon re-watering.

  5. Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice.

    Directory of Open Access Journals (Sweden)

    Jonas M Kristensen

    Full Text Available Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5'AMP activated protein kinase (AMPK has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α(2 (KD AMPK mice and wild type (WT littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

  6. Molecular alterations and expression of succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors.

    Science.gov (United States)

    Celestino, Ricardo; Lima, Jorge; Faustino, Alexandra; Vinagre, João; Máximo, Valdemar; Gouveia, António; Soares, Paula; Lopes, José Manuel

    2013-05-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing somatic KIT, PDGFRA and BRAF mutations. Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop Carney-Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, re-evaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex. As control, we used a series of well characterized 49 KIT/PDGFRA/BRAF-mutated GISTs. SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were significantly associated to younger age at diagnosis. A significant reduction in SDHB expression in WT GISTs was found when compared with KIT/PDGFRA/BRAF-mutated GISTs. No significant differences were found when comparing DOG-1 and c-KIT expression in WT, SDHB-mutated and KIT/PDGFRA/BRAF-mutated GISTs. Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to Carney-Stratakis dyad or Carney triad. Most importantly, the possibility of PGLs (Carney-Stratakis dyad) and/or pulmonary chondroma (Carney triad) should be addressed in these patients and their kindred.

  7. Early Cognitive/Social Deficits and Late Motor Phenotype in Conditional Wild-Type TDP-43 Transgenic Mice

    Science.gov (United States)

    Alfieri, Julio A.; Silva, Pablo R.; Igaz, Lionel M.

    2016-01-01

    Frontotemporal Dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two neurodegenerative diseases associated to mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43). To investigate in depth the behavioral phenotype associated with this proteinopathy, we used as a model transgenic (Tg) mice conditionally overexpressing human wild-type TDP 43 protein (hTDP-43-WT) in forebrain neurons. We previously characterized these mice at the neuropathological level and found progressive neurodegeneration and other features that evoke human TDP-43 proteinopathies of the FTD/ALS spectrum. In the present study we analyzed the behavior of mice at multiple domains, including motor, social and cognitive performance. Our results indicate that young hTDP-43-WT Tg mice (1 month after post-weaning transgene induction) present a normal motor phenotype compared to control littermates, as assessed by accelerated rotarod performance, spontaneous locomotor activity in the open field test and a mild degree of spasticity shown by a clasping phenotype. Analysis of social and cognitive behavior showed a rapid installment of deficits in social interaction, working memory (Y-maze test) and recognition memory (novel object recognition test) in the absence of overt motor abnormalities. To investigate if the motor phenotype worsen with age, we analyzed the behavior of mice after long-term (up to 12 months) transgene induction. Our results reveal a decreased performance on the rotarod test and in the hanging wire test, indicating a motor phenotype that was absent in younger mice. In addition, long-term hTDP-43-WT expression led to hyperlocomotion in the open field test. In sum, these results demonstrate a time-dependent emergence of a motor phenotype in older hTDP-43-WT Tg mice, recapitulating aspects of clinical FTD presentations with motor involvement in human patients, and providing a complementary animal model for studying TDP-43 proteinopathies. PMID:28066234

  8. Genotype-specific differences between mouse CNS stem cell lines expressing frontotemporal dementia mutant or wild type human tau.

    Directory of Open Access Journals (Sweden)

    Miranda E Orr

    Full Text Available Stem cell (SC lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS SC-containing neurosphere cultures for studying heritable neurodegenerative disease, we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD mutation, rTg(tau(P301L4510, with those expressing comparable levels of wild type human tau, rTg(tau(wt21221. rTg(tau(P301L4510 mice express the human tau(P301L variant in their forebrains and display cellular, histological, biochemical and behavioral abnormalities similar to those in human FTD, including age-dependent differences in tau phosphorylation that distinguish them from rTg(tau(wt21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tau(P301L4510 mice and from rTg(tau(wt21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice, validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tau(P301L4510 cultures was hypophosphorylated in comparison with rTg(tau(wt21221 as was seen in young adult mice. In addition, there were fewer human tau-expressing cells in rTg(tau(P301L4510 than in rTg(tau(wt21221 cultures. Following differentiation, neuronal filopodia-spine density was slightly greater in rTg(tau(P301L4510 than rTg(tau(wt21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns, the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms.

  9. Chronic allergic inflammation causes vascular remodeling and pulmonary hypertension in BMPR2 hypomorph and wild-type mice.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Mushaben

    Full Text Available Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2 gene have been identified in patients with heritable pulmonary arterial hypertension (PAH; however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT Balb/c/Byj mice were exposed to house dust mite (HDM allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.

  10. Randomized study of FOLFIRI plus either panitumumab or bevacizumab for wild-type KRAS colorectal cancer-WJOG 6210G.

    Science.gov (United States)

    Shitara, Kohei; Yonesaka, Kimio; Denda, Tadamichi; Yamazaki, Kentaro; Moriwaki, Toshikazu; Tsuda, Masahiro; Takano, Toshimi; Okuda, Hiroyuki; Nishina, Tomohiro; Sakai, Kazuko; Nishio, Kazuto; Tokunaga, Shoji; Yamanaka, Takeharu; Boku, Narikazu; Hyodo, Ichinosuke; Muro, Kei

    2016-12-01

    This randomized phase II trial compared panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) with bevacizumab plus FOLFIRI as second-line chemotherapy for wild-type (WT) KRAS exon 2 metastatic colorectal cancer (mCRC) and to explore the values of oncogenes in circulating tumor DNA (ctDNA) and serum proteins as predictive biomarkers. Patients with WT KRAS exon 2 mCRC refractory to first-line chemotherapy containing oxaliplatin and bevacizumab were randomly assigned to panitumumab plus FOLFIRI or bevacizumab plus FOLFIRI. Of 121 randomly assigned patients, 117 were eligible. Median overall survival (OS) for panitumumab plus FOLFIRI and bevacizumab plus FOLFIRI were 16.2 and 13.4 months [hazard ratio (HR), 1.16; 95% CI, 0.76-1.77], respectively. Progression-free survival (PFS) was also similar (HR, 1.14; 95% CI, 0.78-1.66). KRAS, NRAS, and BRAF status using ctDNA was successfully examined in 109 patients, and mutations were identified in 19 patients (17.4%). Panitumumab plus FOLFIRI showed favorable survival compared with bevacizumab plus FOLFIRI in WT patients and unfavorable survival in those with mutations (P for interaction = 0.026 in OS and 0.054 in PFS). OS with bevacizumab plus FOLFIRI was better than panitumumab plus FOLFIRI in patients with high serum vascular endothelial growth factor-A (VEGF-A) levels and worse in those with low levels (P for interaction = 0.016). Second-line FOLFIRI plus panitumumab and FOLFIRI plus bevacizumab showed a similar efficacy in patients with WT KRAS exon 2 mCRC. RAS and BRAF mutation in ctDNA could be a negative predictive marker for panitumumab.

  11. Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin.

    Science.gov (United States)

    El-Gedaily, A; Paesold, G; Krause, M

    1997-08-01

    The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quantitative immunoblotting. As expected, SpvA, SpvB, SpvC, and SpvD were not detectable before the late logarithmic growth phase and appeared in the sequence SpvA, SpvB, SpvC, and SpvD. In contrast to the transcriptional regulation, however, SpvA and SpvB reached their maximal expression shortly after induction and declined during further growth whereas SpvC and SpvD expression remained high throughout the stationary phase, indicating that the Spv proteins are individually regulated at a posttranscriptional level. To localize SpvABCD within the bacteria, the cells were fractionated into the periplasmic, cytoplasmic, inner membrane, and outer membrane components. The cell fractions and the culture supernatant were analyzed by immunoblotting. SpvA was present in the outer membrane, SpvB was present in the cytoplasm and the inner membrane, and SpvC was present in the cytoplasm. SpvD was secreted into the supernatant; however, a substantial portion of this protein was also detected in the cytoplasm and membranes. The molecular weights of SpvD in the supernatant and in the cytoplasm appeared to be equal, suggesting that SpvD is not cleaved upon secretion.

  12. Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

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    Maria Dolores Fernandez-Garcia

    2016-02-01

    Full Text Available The live attenuated yellow fever virus (YFV vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.

  13. Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

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    K.C. Médici

    2010-01-01

    Full Text Available Group B rotaviruses (RV-B were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8 were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

  14. Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms.

    Science.gov (United States)

    Emmons-Bell, Maya; Durant, Fallon; Hammelman, Jennifer; Bessonov, Nicholas; Volpert, Vitaly; Morokuma, Junji; Pinet, Kaylinnette; Adams, Dany S; Pietak, Alexis; Lobo, Daniel; Levin, Michael

    2015-11-24

    The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together

  15. Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms

    Directory of Open Access Journals (Sweden)

    Maya Emmons-Bell

    2015-11-01

    Full Text Available The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina. We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts, and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies

  16. Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS).

    Science.gov (United States)

    Tavella, Sara; Ruggiu, Alessandra; Giuliani, Alessandra; Brun, Francesco; Canciani, Barbara; Manescu, Adrian; Marozzi, Katia; Cilli, Michele; Costa, Delfina; Liu, Yi; Piccardi, Federica; Tasso, Roberta; Tromba, Giuliana; Rustichelli, Franco; Cancedda, Ranieri

    2012-01-01

    Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

  17. Prednisolone-induced differential gene expression in mouse liver carrying wild type or a dimerization-defective glucocorticoid receptor

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    Dokter Wim

    2010-06-01

    Full Text Available Abstract Background Glucocorticoids (GCs control expression of a large number of genes via binding to the GC receptor (GR. Transcription may be regulated either by binding of the GR dimer to DNA regulatory elements or by protein-protein interactions of GR monomers with other transcription factors. Although the type of regulation for a number of individual target genes is known, the relative contribution of both mechanisms to the regulation of the entire transcriptional program remains elusive. To study the importance of GR dimerization in the regulation of gene expression, we performed gene expression profiling of livers of prednisolone-treated wild type (WT and mice that have lost the ability to form GR dimers (GRdim. Results The GR target genes identified in WT mice were predominantly related to glucose metabolism, the cell cycle, apoptosis and inflammation. In GRdim mice, the level of prednisolone-induced gene expression was significantly reduced compared to WT, but not completely absent. Interestingly, for a set of genes, involved in cell cycle and apoptosis processes and strongly related to Foxo3a and p53, induction by prednisolone was completely abolished in GRdim mice. In contrast, glucose metabolism-related genes were still modestly upregulated in GRdim mice upon prednisolone treatment. Finally, we identified several novel GC-inducible genes from which Fam107a, a putative histone acetyltransferase complex interacting protein, was most strongly dependent on GR dimerization. Conclusions This study on prednisolone-induced effects in livers of WT and GRdim mice identified a number of interesting candidate genes and pathways regulated by GR dimers and sheds new light onto the complex transcriptional regulation of liver function by GCs.

  18. Charge recombination kinetics and protein dynamics in wild type and carotenoid-less bacterial reaction centers: studies in trehalose glasses.

    Science.gov (United States)

    Francia, Francesco; Malferrari, Marco; Sacquin-Mora, Sophie; Venturoli, Giovanni

    2009-07-30

    The coupling between electron transfer and protein dynamics has been investigated in reaction centers (RCs) from the wild type (wt) and the carotenoid-less strain R26 of the photosynthetic bacterium Rhodobacter sphaeroides. Recombination kinetics between the primary photoreduced quinone acceptor (QA-) and photoxidized donor (P+) have been analyzed at room temperature in RCs incorporated into glassy trehalose matrices of different water/sugar ratios. As previously found in R26 RCs, also in the wt RC, upon matrix dehydration, P+QA- recombination accelerates and becomes broadly distributed, reflecting the inhibition of protein relaxation from the dark-adapted to the light-adapted conformation and the hindrance of interconversion between conformational substates. While in wet trehalose matrices (down to approximately one water per trehalose molecule) P+QA- recombination kinetics are essentially coincident in wt and R26 RCs, more extensive dehydration leads to two-times faster and more distributed kinetics in the carotenoid-containing RC, indicating a stronger inhibition of the internal protein dynamics in the wt RC. Coarse-grained Brownian dynamics simulations performed on the two RC structures reveal a markedly larger flexibility of the R26 RC, showing that a rigid core of residues, close to the quinone acceptors, is specifically softened in the absence of the carotenoid. These experimental and computational results concur to indicate that removal of the carotenoid molecule has long-range effects on protein dynamics and that the structural/dynamical coupling between the protein and the glassy matrix depends strongly upon the local mechanical properties of the protein interior. The data also suggest that the conformational change stabilizing P+QA- is localized around the QA binding pocket.

  19. A transcriptomic approach to identify regulatory genes involved in fruit set of wild-type and parthenocarpic tomato genotypes.

    Science.gov (United States)

    Ruiu, Fabrizio; Picarella, Maurizio Enea; Imanishi, Shunsuke; Mazzucato, Andrea

    2015-10-01

    The tomato parthenocarpic fruit (pat) mutation associates a strong competence for parthenocarpy with homeotic transformation of anthers and aberrancy of ovules. To dissect this complex floral phenotype, genes involved in the pollination-independent fruit set of the pat mutant were investigated by microarray analysis using wild-type and mutant ovaries. Normalized expression data were subjected to one-way ANOVA and 2499 differentially expressed genes (DEGs) displaying a >1.5 log-fold change in at least one of the pairwise comparisons analyzed were detected. DEGs were categorized into 20 clusters and clusters classified into five groups representing transcripts with similar expression dynamics. The "regulatory function" group (685 DEGs) contained putative negative or positive fruit set regulators, "pollination-dependent" (411 DEGs) included genes activated by pollination, "fruit growth-related" (815 DEGs) genes activated at early fruit growth. The last groups listed genes with different or similar expression pattern at all stages in the two genotypes. qRT-PCR validation of 20 DEGs plus other four selected genes assessed the high reliability of microarray expression data; the average correlation coefficient for the 20 DEGs was 0.90. In all the groups were evidenced relevant transcription factors encoding proteins regulating meristem differentiation and floral organ development, genes involved in metabolism, transport and response of hormones, genes involved in cell division and in primary and secondary metabolism. Among pathways related to secondary metabolites emerged genes related to the synthesis of flavonoids, supporting the recent evidence that these compounds are important at the fruit set phase. Selected genes showing a de-regulated expression pattern in pat were studied in other four parthenocarpic genotypes either genetically anonymous or carrying lesions in known gene sequences. This comparative approach offered novel insights for improving the present

  20. Vaccines for canine leishmaniasis

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    Clarisa B. Palatnik-De-Sousa

    2012-04-01

    Full Text Available Leishmaniasis is the third most important vector-borne disease worldwide. Visceral leishmaniasis (VL is a severe and frequently lethal protozoan disease of increasing incidence and severity due to infected human and dog migration, new geographical distribution of the insect due to global-warming, co-infection with immunosuppressive diseases and poverty. The disease is an anthroponosis in India and Central Africa and a canid zoonosis (ZVL in the Americas, the Middle East, Central Asia, China and the Mediterranean. The ZVL epidemic has been controlled by one or more measures including the culling of infected dogs, treatment of human cases and insecticidal treatment of homes and dogs. However, the use of vaccines is considered the most cost-effective control tool for human and canine disease. Since the severity of the disease is related to the generation of T-cell immunosuppression, effective vaccines should be capable of sustaining or enhancing the T-cell immunity. In this review we summarize the clinical and parasitological characteristics of ZVL with special focus on the cellular and humoral canine immune response and review state-of-the-art vaccine development against human and canine visceral leishmaniasis. Experimental vaccination against leishmaniasis has evolved from the practice of leishmanization with living parasites to vaccination with crude lysates, native parasite extracts to recombinant and DNA vaccination. Although more than 30 defined vaccines have been studied in laboratory models no human formulation has been licensed so far; however three second-generation canine vaccines have already been registered. As expected for a zoonotic disease, the recent preventive vaccination of dogs in Brazil has led to a reduction in the incidence of canine and human disease. The recent identification of several Leishmania proteins with T-cell epitopes anticipates development of a multiprotein vaccine that will be capable of protecting both humans

  1. 超声引导下犬自体血栓栓塞性急性肺动脉高压模型的建立及意义%Establishment of a canine model with acute thrombo-embolic pulmonary hypertension guided by echocardiography

    Institute of Scientific and Technical Information of China (English)

    孙丹丹; 陈洪茂; 段云友; 尚福军; 梁宁南; 曹玮; 刘禧; 曹铁生

    2010-01-01

    目的 探讨超声引导下建立肺动脉血栓栓塞所致犬急性肺动脉高压模型的可行性.方法 模拟人体急性肺动脉血栓栓塞的病理生理过程,建立肺动脉血栓栓塞所致的犬急性肺动脉高压模型.超声引导下经股静脉穿刺,放置右心导管监测肺动脉压力,同时对侧股静脉推注自体血栓,以肺动脉收缩压超过30 mmHg为模型建立成功的标准.结果 超声可以准确引导右心导管的放置.实验用犬27只,成功建立急性肺动脉高压模型24只,成功率88.9%.结论 超声引导下建立肺动脉血栓栓塞所致犬急性肺动脉高压模型的方法具有良好的可行性和可重复性.%Objective To establish a canine model with acute thrombo-embolic pulmonary hypertension monitored by echocardiography. Methods Simulated the pathophysiologic process of acute thrombo-embolic pulmonary hypertension in human, the canine model was developed. The femoral vein puncture and the right heart catheterization to monitor pulmonary artery pressure were operated guided by echocardiography, while the autologous blood clot was injected to the contralateral femoral vein. The criteria of model was the pulmonary artery systolic pressure was higher than 30 mmHg. Results Echocardiography can accurately guide the placement of right heart catheterization and ancillarily observe the pressure testing of pulmonary artery. Of twenty seven healthy experiment dogs, twenty four models with acute thromboembolic pulmonary hypertension were successfully developed. The successful rate was 88.9 %. Conclusions This canine model with acute thrombo-embolic pulmonary hypertension guided by echocardiography is easy to operate and its repeatability is good.

  2. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

    Science.gov (United States)

    Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

  3. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

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    Sophie Le Poder

    2011-01-01

    Full Text Available A new human coronavirus responsible for severe acute respiratory syndrome (SARS was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses.

  4. An experimental study on application of ultrasound in canine model of acute cerebralcardiac syndrome%超声在急性脑损伤后脑心综合征实验研究中的应用

    Institute of Scientific and Technical Information of China (English)

    钱嵘; 孙兵; 赵宝珍; 吴炎

    2013-01-01

    Objective To investigate the application of real-time myocardial contrast echocardiography (RTMCE) on evaluating myocardial function and regional microcirculation in traumatic brain injury (TBI).Methods In 18 cases canine model of TBI,RTMCE and strain rate imaging were employed to measure the index including strain rate of different myocardial segments,the time-amplitude curves,the mean ascending slope of the curve and local myocardial blood flow with respectively 6 hours,1 day and 3 day pre and post-TBI.Results Compared with pre-TBI,the segmental myocardial strain rate,the maximal amplitude,the mean ascending slope and myocardial blood flow decreased in 3 cases immediately 6hour after TBI.In 3 days,more significant alteration of previous index was discovered in another 12 cases.Conclusions After acute TBI,myocardial dysfunction in heavy stress responses could result in the cerebral-cardiac syndrome,which develops in 3 days post-TBI.RTMCE and Strain rate imaging clinically provide an earlier way to detect myocardial damage and dysfunction in acute TBI patients.%目的 运用心肌超声造影技术、应变率显像技术评价急性脑损伤时心肌功能、心肌各节段微循环灌注情况.方法 建立18只犬急性颅脑损伤模型,于伤前及伤后6h、1d、3d利用实时心肌声学造影、应变率显像技术检测动物模型左室长轴方向各节段心肌应变率和由心肌再灌注后的时间-密度曲线得出的心肌显像峰值强度、平均斜率及局部心肌血流量等指标.结果 3只犬于伤后6h即出现节段性心肌应变率、心肌显像峰值强度、平均斜率及局部心肌血流量等指标的减低,另有12只犬均在3d内出现相应指标的改变.结论 急性脑损伤后,应激状态下中枢性心功能不全易导致心肌损伤继而并发脑心综合症,且多于伤后3d内发病.声学造影技术及应变率显像技术可为临床早期发现心肌损伤及心肌功能异常提供有效指标.

  5. Nanopore analysis of wild-type and mutant prion protein (PrP(C: single molecule discrimination and PrP(C kinetics.

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    Nahid N Jetha

    Full Text Available Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C in the central nervous system into the infectious isoform (PrP(Sc. The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc. A number of pathogenic PrP(C mutants exist that are characterized by an increased propensity for conversion into PrP(Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C, (a PrP(C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease, exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C capture event. Moreover, we present a four-state model to describe wild-type PrP(C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between

  6. Experimental Support for the Ecoimmunity Theory: Distinct Phenotypes of Nonlymphocytic Cells in SCID and Wild-Type Mice.

    Science.gov (United States)

    Ochayon, David E; Baranovski, Boris M; Malkin, Peter; Schuster, Ronen; Kalay, Noa; Ben-Hamo, Rotem; Sloma, Ido; Levinson, Justin; Brazg, Jared; Efroni, Sol; Lewis, Eli C; Nevo, Uri

    2016-01-01

    Immune tolerance toward "self" is critical in multiple immune disorders. While there are several mechanisms to describe the involvement of immune cells in the process, the role of peripheral tissue cells in that context is not yet clear. The theory of ecoimmunity postulates that interactions between immune and tissue cells represent a predator-prey relationship. A lifelong interaction, shaped mainly during early ontogeny, leads to selection of nonimmune cell phenotypes. Normally, therefore, nonimmune cells that evolve alongside an intact immune system would be phenotypically capable of evading immune responses, and cells whose phenotype falls short of satisfying this steady state would expire under hostile immune responses. This view was supported until recently by experimental evidence showing an inferior endurance of severe combined immunodeficiency (SCID)-derived pancreatic islets when engrafted into syngeneic immune-intact wild-type (WT) mice, relative to islets from WT. Here we extend the experimental exploration of ecoimmunity by searching for the presence of the phenotypic changes suggested by the theory. Immune-related phenotypes of islets, spleen, and bone marrow immune cells were determined, as well as SCID and WT nonlymphocytic cells. Islet submass grafting was performed to depict syngeneic graft functionality. Islet cultures were examined under both resting and inflamed conditions for expression of CD40 and major histocompatibility complex (MHC) class I/II and release of interleukin-1α (IL-1α), IL-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, and insulin. Results depict multiple pathways that appear to be related to the sculpting of nonimmune cells by immune cells; 59 SCID islet genes displayed relative expression changes compared with WT islets. SCID cells expressed lower tolerability to inflammation and higher levels of immune-related molecules, including MHC class I. Accordingly, islets exhibited a marked increase in insulin release upon

  7. Expression of wild-type Rp1 protein in Rp1 knock-in mice rescues the retinal degeneration phenotype.

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    Qin Liu

    Full Text Available Mutations in the retinitis pigmentosa 1 (RP1 gene are a common cause of autosomal dominant retinitis pigmentosa (adRP, and have also been found to cause autosomal recessive RP (arRP in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39 are located in the 4(th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd exon of RP1 (c.686delC; p.P229QfsX35 found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.

  8. Trajectory of chemotherapy for patients with EGFR wild-type advanced pulmonary adenocarcinoma: a single-institution retrospective study

    Science.gov (United States)

    Minami, Seigo; Ogata, Yoshitaka; Ihara, Shouichi; Yamamoto, Suguru; Komuta, Kiyoshi

    2017-01-01

    Background Pulmonary adenocarcinoma, recently benefited by new cytotoxic and molecularly targeted drugs, has been classified by driver mutations, such as EGFR mutations. The aim of this study was to research the proportions of patients treated with first- to third-line chemotherapy and to find influential factors for the introduction of chemotherapy and survival benefit from chemotherapy. Materials and methods Data were collected retrospectively on patients who met the following criteria: adenocarcinoma, diagnosed between June 2007 and March 2015 at our hospital, stage IIIB or IV, and EGFR wild type. A nonchemotherapy group of patients who did not receive chemotherapy was compared with a chemotherapy group of patients who received it. The patients who had received first- to third-line chemotherapy between June 2007 and November 2015 at our hospital were also analyzed. Results During the study period, 46 patients did not receive chemotherapy, while 148, 89, and 48 received first-, second- and third-line chemotherapy, respectively. As predictive factors for unlikely chemotherapy, multivariate logistic analysis detected Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≥2, hemoglobin <13.2 g/dL, creatinine clearance (Ccr) <50.4 mL/min, and CRP ≥0.53 mg/dL. As factors predicting shorter survival after chemotherapy, multivariate Cox proportional-hazard analyses detected age ≥75 years, ECOG PS ≥2, lower lymphocyte counts, and higher CRP for the first line; female, higher neutrophil counts, lower lymphocyte counts, reduced Ccr, hyponatremia, and shorter interval between first- and second-line chemotherapy for the second line; and age ≥75 years, body mass index <18.5 kg/m2, higher neutrophil counts, lower lymphocyte counts, hyponatremia, higher lactate dehydrogenase, and higher CRP for the third line. Conclusion Approximately 76% of patients were treated with first-line chemotherapy. Of those patients, 61% and 34% proceeded to second- and third

  9. Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

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    Sirec Teja

    2012-08-01

    Full Text Available Abstract Background The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process. Results We report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction. Conclusion Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure

  10. Parallel screening of wild-type and drug-resistant targets for anti-resistance neuraminidase inhibitors.

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    Kai-Cheng Hsu

    Full Text Available Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye, which inhibited WT NA and MDR NA with IC(50 values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high

  11. Lysogenic Streptococcus suis isolate SS2-4 containing prophage SMP showed increased mortality in zebra fish compared to the wild-type isolate.

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    Tang, Fang; Zhang, Wei; Lu, Chengping

    2013-01-01

    Streptococcus suis (S. suis) infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2) being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.

  12. Characterization of enteric neurons in wild-type and mutant zebrafish using semi-automated cell counting and co-expression analysis.

    Science.gov (United States)

    Simonson, Levi W; Ganz, Julia; Melancon, Ellie; Eisen, Judith S

    2013-06-01

    To characterize fluorescent enteric neurons labeled for expression of cytoplasmic markers in zebrafish mutants, we developed a new MATLAB-based program that can be trained by user input. We used the program to count enteric neurons and to analyze co-expression of the neuronal marker, Elavl, and the neuronal subtype marker, serotonin, in 3D confocal image stacks of dissected whole-mount zebrafish intestines. We quantified the entire population of enteric neurons and the serotonergic subpopulation in specific regions of the intestines of gutwrencher mutant and wild-type sibling larvae. We show a marked decrease in enteric neurons in gutwrencher mutants that is more severe at the caudal end of the intestine. We also show that gutwrencher mutants have the same number of serotonin-positive enteroendocrine cells in the intestine as wild types.

  13. Lysogenic Streptococcus suis isolate SS2-4 containing prophage SMP showed increased mortality in zebra fish compared to the wild-type isolate.

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    Fang Tang

    Full Text Available Streptococcus suis (S. suis infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2 being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.

  14. Synergism between wild-type Bacillus thuringiensis subsp. israelensis and B. sphaericus strains: a study based on isobolographic analysis and histopathology.

    Science.gov (United States)

    Sreshty, M Asha Latha; Kumar, K Pranay; Murty, U S N

    2011-04-01

    Prevention is the best resistance management strategy in integrated vector control programs. Combined use of insecticides of different classes that interact synergistically and show multi-site actions within the insect is recognized as a potential key strategy to be implemented even before the onset of resistance. The present study is aimed at harvesting the benefits of synergism between the wild-type Bacillus thuringiensis subsp. israelensis-H14 (Bti) and Bacillus sphaericus-2362 (Bs) strains by evaluating six different combinations of mixtures toxic to Aedes and Culex mosquito larvae. Isobolographic analysis was performed to distinguish the synergistic combinations of Bti and Bs, followed by determination of the degree of synergism through synergy and improvement factors. Furthermore, the speed of activity of Bs when combined with Bti is studied by histopathological investigations on the fate of midgut muscles of mosquito larvae upon exposure to individual wild-type strains as well as their mixtures.

  15. Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

    DEFF Research Database (Denmark)

    Cox, David G; Simard, Jacques; Sinnett, Daniel

    2011-01-01

    Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly...... instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation...... carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence...

  16. First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar

    DEFF Research Database (Denmark)

    Dubois, A. E. J.; Pagliarani, G.; Brouwer, R. M.;

    2015-01-01

    BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples...... which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual...... analogue scale (VAS). RESULTS: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges...

  17. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation

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    Ze-Yi Zheng

    2015-07-01

    Full Text Available Basal-like breast cancers (BLBCs are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

  18. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation.

    Science.gov (United States)

    Zheng, Ze-Yi; Tian, Lin; Bu, Wen; Fan, Cheng; Gao, Xia; Wang, Hai; Liao, Yi-Hua; Li, Yi; Lewis, Michael T; Edwards, Dean; Zwaka, Thomas P; Hilsenbeck, Susan G; Medina, Daniel; Perou, Charles M; Creighton, Chad J; Zhang, Xiang H-F; Chang, Eric C

    2015-07-21

    Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

  19. Dietary Calcium and Dairy Modulation of Oxidative Stress and Mortality in aP2-Agouti and Wild-type Mice

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    Antje Bruckbauer

    2009-08-01

    Full Text Available Oxidative and inflammatory stress have been implicated as major contributors to the aging process. Dietary Ca reduced both factors in short-term interventions, while milk exerted a greater effect than supplemental Ca. In this work, we examined the effects of life-long supplemental and dairy calcium on lifespan and life-span related biomarkers in aP2-agouti transgenic (model of diet-induced obesity and wild-type mice fed obesigenic diets until their death. These data demonstrate that dairy Ca exerts sustained effects resulting in attenuated adiposity, protection against age-related muscle loss and reduction of oxidative and inflammatory stress in both mouse strains. Although these effects did not alter maximum lifespan, they did suppress early mortality in wild-type mice, but not in aP2-agouti transgenic mice.

  20. Genotype-temperature interaction in the regulation of development, growth, and morphometrics in wild-type, and growth-hormone transgenic coho salmon.

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    Mare Lõhmus

    Full Text Available BACKGROUND: The neuroendocrine system is an important modulator of phenotype, directing cellular genetic responses to external cues such as temperature. Behavioural and physiological processes in poikilothermic organisms (e.g. most fishes, are particularly influenced by surrounding temperatures. METHODOLOGY/PRINCIPAL FINDINGS: By comparing the development and growth of two genotypes of coho salmon (wild-type and transgenic with greatly enhanced growth hormone production at six different temperatures, ranging between 8 degrees and 18 degrees C, we observed a genotype-temperature interaction and possible trend in directed neuroendocrine selection. Differences in growth patterns of the two genotypes were compared by using mathematical models, and morphometric analyses of juvenile salmon were performed to detect differences in body shape. The maximum hatching and alevin survival rates of both genotypes occurred at 12 degrees C. At lower temperatures, eggs containing embryos with enhanced GH production hatched after a shorter incubation period than wild-type eggs, but this difference was not apparent at and above 16 degrees C. GH transgenesis led to lower body weights at the time when the yolk sack was completely absorbed compared to the wild genotype. The growth of juvenile GH-enhanced salmon was to a greater extent stimulated by higher temperatures than the growth of the wild-type. Increased GH production significantly influenced the shape of the salmon growth curves. CONCLUSIONS: Growth hormone overexpression by transgenesis is able to stimulate the growth of coho salmon over a wide range of temperatures. Temperature was found to affect growth rate, survival, and body morphology between GH transgenic and wild genotype coho salmon, and differential responses to temperature observed between the genotypes suggests they would experience different selective forces should they ever enter natural ecosystems. Thus, GH transgenic fish would be expected to

  1. Near-Infrared Spectroscopy, a Rapid Method for Predicting the Age of Male and Female Wild-Type and Wolbachia Infected Aedes aegypti

    Science.gov (United States)

    Milali, Masabho P.; Henry, Michael; Wirtz, Robert A.; Hugo, Leon E.; Dowell, Floyd E.; Devine, Gregor J.

    2016-01-01

    Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS) technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS) regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (Pfemale Ae. aegypti could be identified as female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284) and 78% (N = 229), respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253), 83% (N = 277) and 78% (N = 234), respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs. PMID:27768689

  2. Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability

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    Estacion Mark

    2010-06-01

    Full Text Available Abstract Background A direct role of sodium channels in pain has recently been confirmed by establishing a monogenic link between SCN9A, the gene which encodes sodium channel Nav1.7, and pain disorders in humans, with gain-of-function mutations causing severe pain syndromes, and loss-of-function mutations causing congenital indifference to pain. Expression of sodium channel Nav1.8 in DRG neurons has also been shown to be essential for the manifestation of mutant Nav1.7-induced neuronal hyperexcitability. These findings have confirmed key roles of Nav1.7 and Nav1.8 in pain and identify these channels as novel targets for pain therapeutic development. Ranolazine preferentially blocks cardiac late sodium currents at concentrations that do not significantly reduce peak sodium current. Ranolazine also blocks wild-type Nav1.7 and Nav1.8 channels in a use-dependent manner. However, ranolazine's effects on gain-of-function mutations of Nav1.7 and on DRG neuron excitability have not been investigated. We used voltage- and current-clamp recordings to evaluate the hypothesis that ranolazine may be effective in regulating Nav1.7-induced DRG neuron hyperexcitability. Results We show that ranolazine produces comparable block of peak and ramp currents of wild-type Nav1.7 and mutant Nav1.7 channels linked to Inherited Erythromelalgia and Paroxysmal Extreme Pain Disorder. We also show that ranolazine, at a clinically-relevant concentration, blocks high-frequency firing of DRG neurons expressing wild-type but not mutant channels. Conclusions Our data suggest that ranalozine can attenuate hyperexcitability of DRG neurons over-expressing wild-type Nav1.7 channels, as occurs in acquired neuropathic and inflammatory pain, and thus merits further study as an alternative to existing non-selective sodium channel blockers.

  3. Identifying the Integrated Neural Networks Involved in Capsaicin-Induced Pain Using fMRI in Awake TRPV1 Knockout and Wild-Type Rats

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    Jason Richard Yee

    2015-02-01

    Full Text Available In the present study, we used functional MRI in awake rats to investigate the pain response that accompanies intradermal injection of capsaicin into the hindpaw. To this end, we used BOLD imaging together with a 3D segmented, annotated rat atlas and computational analysis to identify the integrated neural circuits involved in capsaicin-induced pain. The specificity of the pain response to capsaicin was tested in a transgenic model that contains a biallelic deletion of the gene encoding for the transient receptor potential cation channel subfamily V member 1 (TRPV1. Capsaicin is an exogenous ligand for the TRPV1 receptor, and in wild-type rats, activated the putative pain neural circuit. In addition, capsaicin-treated wild-type rats exhibited activation in brain regions comprising the Papez circuit and habenular system, systems that play important roles in the integration of emotional information, and learning and memory of aversive information, respectively. As expected, capsaicin administration to TRPV1-KO rats failed to elicit the robust BOLD activation pattern observed in wild-type controls. However, the intradermal injection of formalin elicited a significant activation of the putative pain pathway as represented by such areas as the anterior cingulate, somatosensory cortex, parabrachial nucleus, and periaqueductal gray. Notably, comparison of neural responses to capsaicin in wild-type versus knock-out rats uncovered evidence that capsaicin may function in an antinociceptive capacity independent of TRPV1 signaling. Our data suggest that neuroimaging of pain in awake, conscious animals has the potential to inform the neurobiological basis of full and integrated perceptions of pain.

  4. The Asp245-->Asn mutant of Coprinus cinereus peroxidase. Characterization by 1H-NMR spectroscopy and comparison with the wild-type enzyme.

    Science.gov (United States)

    Veitch, N C; Gao, Y; Welinder, K G

    1996-11-12

    The resting, fluoride-ligated and cyanide-ligated states of the Asp245-->Asn mutant of Coprinus cinereus peroxidase (D245N CIP) have been characterized using 1H-NMR spectroscopy in conjunction with parallel studies of the wild-type enzyme. Analysis of the spectra of resting state D245N CIP over the pH range 5-10 has uncovered the existence of three high-spin species in dynamic equilibrium with each other. The predominant species at neutral pH is six-coordinate high-spin (6-c HS), with a distal water molecule as the sixth ligand. This species is in slow exchange on the NMR time scale with a second six-coordinate high-spin species (6-c HS*) and a five-coordinate high-spin species (5-c HS**), toward acidic and alkaline pH values, respectively. The 6-c HS* species appears to be unique and is proposed to differ from the 6-c HS species by protonation of the proximal His residue, whereas the 5-c HS** species lacks the proximal His ligand and is coordinated by a hydroxyl group. In sharp contrast, wild-type CIP is a five-coordinate high-spin (5-c HS) species over the same pH range. The D245N CIP mutant also exhibits a greater affinity for fluoride than wild-type CIP. The 1H-NMR spectrum of cyanide-ligated D245N CIP, assigned using two-dimensional methods, differs significantly from that of the wild-type enzyme. Perturbations to heme and heme-linked proton resonances are rationalised in terms of the loss or significant weakening of the hydrogen bond between His183 N delta 1H and the side-chain of residue 245 when Asp is replaced by Asn. This subtle interaction directly affects the heme pocket structure of CIP both proximal and distal to the heme plane.

  5. Rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Pan, Chu-Hsiang; Jong, Ming-Hwa; Huang, Yu-Liang; Huang, Tien-Shine; Chao, Parn-Hwa; Lai, Shiow-Suey

    2008-07-01

    A simple one-step reverse transcription polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild type and vaccine strains of Classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.

  6. The expression analysis of cysteine proteinase-like protein in wild-type and nm2 mutant silkworm (Lepidoptera: Bombyx mori).

    Science.gov (United States)

    Wu, Fan; Kang, Lequn; Wang, Pingyang; Zhao, Qiaoling

    2016-07-15

    The mutant of non-molting in the 2nd instar (nm2) is a recently discovered mutant of Bombyx mori. The mutant cannot molt and exuviate and died successively in premolting of 2nd instar. In this study, two dimensional gel electrophoresis (2-DE) was performed to screen the differential expression of epidermis proteins in pre-molting larvae of 2nd instar between the wild-type and nm2 mutant. Interestingly, a cysteine proteinase-like (BmCP-like) protein in nm2 was significantly higher than that of the wild-type. The transcription profiles of BmCP-like gene were investigated by quantitative real-time PCR (qRT-PCR), and the result revealed that BmCP-like mRNA was remarkably higher in nm2 than that of the wild-type. The transcription level of BmCP-like was high in the epidermis while low in the midgut and hemocytes, and fluctuate with development, while the highest in the newly molted larvae of 3rd and lowest in the pre-molting of the 1st and 2nd instar. The body of injected BmCP-like RNAi of 2nd larvae formed a dark spots around the injection place. These results suggested the BmCP-like gene play a key role in the degradation of the cuticle and epidermis layer during molting of 1st and 2nd instar silkworm. Furthermore, the ORF of BmCP-like gene in nm2 was the same to the wild-type. These studies give us a hint that BmCP-like gene maybe not the major gene responsible for nm2, but BmCP-like gene might participate in the immune systems of silkworm, and the upregulation of BmCP-like transcription in the nm2 mutant might be induced by the disadvantages that limit the growth and development of silkworm in order to survive.

  7. A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

    Science.gov (United States)

    Middelhoven, W J; van Eijk, J; van Renesse, R; Blijham, J M

    1978-01-01

    NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

  8. Iron and zinc complexation in wild-type and ferritin-expressing wheat grain: implications for mineral transport into developing grain

    DEFF Research Database (Denmark)

    Neal, Andrew L; Geraki, Kalotina; Borg, Søren

    2013-01-01

    We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells...... of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain....

  9. Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

    Directory of Open Access Journals (Sweden)

    Ming Liu

    Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

  10. Differential inhibition of ex-vivo tumor kinase activity by vemurafenib in BRAF(V600E and BRAF wild-type metastatic malignant melanoma.

    Directory of Open Access Journals (Sweden)

    Andliena Tahiri

    Full Text Available BACKGROUND: Treatment of metastatic malignant melanoma patients harboring BRAF(V600E has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. METHODOLOGY: In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. RESULTS: Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. CONCLUSIONS: Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

  11. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis.

    Science.gov (United States)

    Culver, Brady P; Savas, Jeffrey N; Park, Sung K; Choi, Jeong H; Zheng, Shuqiu; Zeitlin, Scott O; Yates, John R; Tanese, Naoko

    2012-06-22

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.

  12. Density and composition of an insect population in a field trial of chitinase transgenic and wild-type silver birch (Betula pendula) clones.

    Science.gov (United States)

    Vihervuori, Liisa; Pasonen, Hanna-Leena; Lyytikäinen-Saarenmaa, Päivi

    2008-12-01

    Fifteen silver birch (Betula pendula Roth) lines carrying a sugar beet chitinase IV gene and eight wild-type birch clones were grown in a field trial. The composition and density of the insect population and the leaf damage caused by insects were monitored and compared between transgenic and wild-type trees. The most abundant insect group in all trees was aphids, and the variation in total insect densities was mainly explained by the variation in aphid densities. Insect densities were generally higher in the transgenic than in the control trees, indicating that the expression of the sugar beet chitinase IV gene had an influence on the suitability of birch leaves to aphids. The level of leaf damage was higher among transgenic than among control trees. Chewing damage was the most common type of leaf damage in all trees. The number of different damage types was higher among the wild-type clones than among the transgenic lines or their controls. The results indicate that the chitinase transgenic trees are more susceptible to aphids and suffer higher levels of leaf damage than the control trees. In the composition of the damage types, the control trees were more similar to the transgenic than to other wild-type trees, indicating that the composition was mostly linked to the genotype of the tree and not to the expression of the transgene. This study provides important information on the ecological interactions of chitinase transgenic trees in the field trial. No clear harmful effects of transgenic chitinase on the biodiversity of insect population were detected.

  13. Genetic analysis of two OsLpa1-like genes in Arabidopsis reveals that only one is required for wild-type seed phytic acid levels

    Science.gov (United States)

    Phytic acid (inositol-1,2,3,4,5,6-hexakisphosphate or InsP6) is the primary storage form of phosphorus in plant seeds. The rice OsLpa1 encodes a novel protein required for wild-type levels of seed InsP6 and was identified from a low phytic acid (lpa) mutant exhibiting a 45-50% reduction in seed InsP...

  14. An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein.

    Science.gov (United States)

    Dettloff, M; Weers, P M; Niere, M; Kay, C M; Ryan, R O; Wiesner, A

    2001-03-13

    Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

  15. Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

    Science.gov (United States)

    Uyar, Basar; Gürgan, Muazzez; Özgür, Ebru; Gündüz, Ufuk; Yücel, Meral; Eroglu, Inci

    2015-10-01

    Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.

  16. [Canine histoplasmosis in Japan].

    Science.gov (United States)

    Sano, Ayako; Miyaji, Makoto

    2003-01-01

    Histoplasmosis is a fungal infection caused by Histoplasma capsulatum and is distributed a worldwide. Although the disease has been treated as an imported mycosis, some autochthonous human, 1 equine and 4 canine cases suggested that the disease is endemic. Histoplasmosis is classified depending on the variety of causative agent. Histoplasmosis farciminosi known as pseudofarcy, is manifested only in Perissodactyla where it invades lymph nodes and lymph ducts, and is recognized by isolation from horses. Historically, Japan was one of the endemic areas of pseudofarcy before World War II, and more than 20,000 cases were recorded in horses used by the military. Interestingly, Japanese canine histoplasmosis uniformly showed skin ulcers and granulomatous lesions on the skin without pulmonary or gastrointestinal involvement, both of which were very similar to pseudofarcy. It was diagnosed as histoplasmosis by the detection of internal transcribed spacer legions of rRNA gene of H. capsulatum from paraffin embedded tissue samples. Furthermore, the fungal isolate from the human case with no history of going abroad or immigrating was identified as H. capsulatum var. farciminosum by a gene sequence. These facts indicated that pseudofarcy is not only an infectious disease in horses, but also a zoonotic fungal infection. Japanese autochthonous canine histoplasmosis might be a heteroecism of pseudofarcy because of its likeness to the human case, the similarity of clinical manifestations and the historical background at this stage.

  17. Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

    Science.gov (United States)

    Radtanakatikanon, Araya; Keawcharoen, Juthatip; Charoenvisal, Na Taya; Poovorawan, Yong; Prompetchara, Eakachai; Yamaguchi, Ryoji; Techangamsuwan, Somporn

    2013-09-27

    Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand.

  18. Comparative Study on Growth Performance of Transgenic (Over-ExpressedOsNHX1) and Wild-Type Nipponbare under Different Salinity Regimes

    Institute of Scientific and Technical Information of China (English)

    Nurul Kahrani ISHAK; Zohrah SULAIMAN; Kushan U TENNAKOON

    2015-01-01

    Transgenic Nipponbarewhich over-expressed a Na+/H+ antiporter geneOsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

  19. Effect of S-1 + cisplatin and gemcitabine + cisplatin on malignant indexes in serum and tumor tissue of advanced NSCLC patients with wild-type EGFR

    Institute of Scientific and Technical Information of China (English)

    Can Chen; Qing Fang; Qing-Yuan Zhou

    2016-01-01

    Objective:To study the effect of S-1 + cisplatin and gemcitabine + cisplatin on malignant indexes in serum and tumor tissue of advanced NSCLC patients with wild-type EGFR. Methods: The medical records of 58 advanced non-small cell lung cancer patients with wild-type EGFR were retrospectively analyzed, patients who received S-1 + cisplatin chemotherapy were included in the experimental group, patients who received gemcitabine + cisplatin chemotherapy were included in the control group, and then the levels of tumor markers in serum and the expression levels of proto-oncogenes and tumor suppressor genes in tumor tissue were determined.Results:After treatment, serum tumor markers CEA, NSE, Cyfra21-1, PCDGF, VEGF and TK1 levels of experimental group were significantly lower than those of control group; Pim-1, LUNX, PFKFB3, PFKFB4 and Ki67 levels in tumor tissue of experimental group were significantly lower than those of control group, and TCF21, PDCD5, ASPP1, ASPP2, p53 and EFEMP1 levels were significantly higher than those of control group. Conclusions: S-1 + cisplatin has better curative effect on advanced NSCLC with wild-type EGFR than gemcitabine + cisplatin, and can more effectively kill cancer cells and regulate the expression of proto-oncogenes and tumor suppressor genes.

  20. High-level expression of wild-type p53 in melanoma cells is frequently associated with inactivity in p53 reporter gene assays.

    Directory of Open Access Journals (Sweden)

    Roland Houben

    Full Text Available BACKGROUND: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5-8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. CONCLUSIONS/SIGNIFICANCE: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.

  1. Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

    DEFF Research Database (Denmark)

    Jensen, T G; Andresen, B S; Bross, P;

    1992-01-01

    An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

  2. Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk.

    Directory of Open Access Journals (Sweden)

    Vesa-Matti Pohjanen

    Full Text Available Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790 and +1196 (rs4986791 in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001 and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390. Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

  3. Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk

    Science.gov (United States)

    Pohjanen, Vesa-Matti; Koivurova, Olli-Pekka; Huhta, Heikki; Helminen, Olli; Mäkinen, Johanna M.; Karhukorpi, Jari M.; Joensuu, Tapio; Koistinen, Pentti O.; Valtonen, Jarno M.; Niemelä, Seppo E.; Karttunen, Riitta A.; Karttunen, Tuomo J.

    2015-01-01

    Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion. PMID:26161647

  4. Maximizing bone formation in posterior spine fusion using rhBMP-2 and zoledronic acid in wild type and NF1 deficient mice.

    Science.gov (United States)

    Bobyn, Justin; Rasch, Anton; Kathy, Mikulec; Little, David G; Schindeler, Aaron

    2014-08-01

    Spinal pseudarthrosis is a well described complication of spine fusion surgery in NF1 patients. Reduced bone formation and excessive resorption have been described in NF1 and anti-resorptive agents may be advantageous in these individuals. In this study, 16 wild type and 16 Nf1(+/-) mice were subjected to posterolateral fusion using collagen sponges containing 5 µg rhBMP-2 introduced bilaterally. Mice were dosed twice weekly with 0.02 mg/kg zoledronic acid (ZA) or sterile saline. The fusion mass was assessed for bone volume (BV) and bone mineral density (BMD) by microCT. Co-treatment using rhBMP-2 and ZA produced a significant increase (p Nf1(+/-) mice (+174%). Co-treatment also produced a significantly higher total BMD of the fusion mass compared to rhBMP-2 alone in both groups (p Nf1(+/-) deficient mice still generated less bone than wild type controls. TRAP staining on histological sections indicated an increased osteoclast surface/bone surface (Oc.S/BS) in Nf1(+/-) mice relative to wild type mice, and this was reduced with ZA treatment.

  5. Engineered zinc-finger proteins can compensate genetic haploinsufficiency by transcriptional activation of the wild-type allele: application to Willams-Beuren syndrome and supravalvular aortic stenosis.

    Science.gov (United States)

    Zhang, Pei; Huang, Angela; Morales-Ruiz, Manuel; Starcher, Barry C; Huang, Yan; Sessa, William C; Niklason, Laura E; Giordano, Frank J

    2012-11-01

    Williams-Beuren syndrome (WBS) and supravalvular aortic stenosis (SVAS) are genetic syndromes marked by the propensity to develop severe vascular stenoses. Vascular lesions in both syndromes are caused by haploinsufficiency of the elastin gene. We used these distinct genetic syndromes as models to evaluate the feasibility of using engineered zinc-finger protein transcription factors (ZFPs) to achieve compensatory expression of haploinsufficient genes by inducing augmented expression from the remaining wild-type allele. For complex genes with multiple splice variants, this approach could have distinct advantages over cDNA-based gene replacement strategies. Targeting the elastin gene, we show that transcriptional activation by engineered ZFPs can induce compensatory expression from the wild-type allele in the setting of classic WBS and SVAS genetic mutations, increase elastin expression in wild-type cells, induce expression of the major elastin splice variants, and recapitulate their natural stoichiometry. Further, we establish that transcriptional activation of the mutant allele in SVAS does not overcome nonsense-mediated decay, and thus ZFP-mediated transcriptional activation is not likely to induce production of a mutant protein, a crucial consideration. Finally, we show in bioengineered blood vessels that ZFP-mediated induction of elastin expression is capable of stimulating functional elastogenesis. Haploinsufficiency is a common mechanism of genetic disease. These findings have significant implications for WBS and SVAS, and establish that haploinsufficiency can be overcome by targeted transcriptional activation without inducing protein expression from the mutant allele.

  6. Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-β plaque formation in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Hellwig, Sabine; Masuch, Annette; Nestel, Sigrun; Katzmarski, Natalie; Meyer-Luehmann, Melanie; Biber, Knut

    2015-01-01

    The role of microglia in amyloid-β (Aβ) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to Aβ plaque formation. We found that microglia ingested Aβ, thereby preventing plaque formation in OHSCs. Conversely, Aβ deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent Aβ plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of Aβ clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high Aβ1-42 burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial Aβ clearance capacity. These data may therefore explain why Aβ plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study Aβ plaque formation in a cell culture setting.

  7. Differential regulation of p21 (waf1) protein half-life by DNA damage and Nutlin-3 in p53 wild-type tumors and its therapeutic implications.

    Science.gov (United States)

    Chang, Li-Ju; Eastman, Alan

    2012-09-01

    DNA damage induces the canonical p53 pathway including elevation of p21 (waf1) resulting in arrest of cell cycle progression. This can protect cells from subsequent Chk1 inhibition. Some p53 wild-type cancer cells such as HCT116 and U2OS exhibit attenuated p21 (waf1) induction upon DNA damage due to translational inhibition, and are incapable of maintaining arrest upon Chk1 inhibition. The purpose of this study was to determine whether this attenuated p21 (waf1) induction also occurred with the non-DNA damaging agent Nutlin-3 which induces p53 by disrupting binding to its negative regulator MDM2. We find that Nutlin-3 circumvented the attenuated induction of p21 (waf1) protein by increasing its half-life which led to G 1 and G 2 arrest in both cell lines. Interestingly, the p21 (waf1) protein half-life remained short on Nutlin-3 in p53 wild-type MCF10A cells; these cells achieve high p21 (waf1) levels through transcriptional upregulation. Consequently, all three p53 wild-type cells but not p53 mutant MDA-MB-231 cancer cells were protected from subsequent incubation with a combination of DNA damage plus a checkpoint inhibitor.

  8. Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems.

    Science.gov (United States)

    Dadashipour, Mohammad; Fukuta, Yasuhisa; Asano, Yasuhisa

    2011-05-01

    Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.

  9. Homomeric and heteromeric interactions between wild-type and mutant phenylalanine hydroxylase subunits: evaluation of two-hybrid approaches for functional analysis of mutations causing hyperphenylalaninemia.

    Science.gov (United States)

    Waters, P J; Scriver, C R; Parniak, M A

    2001-07-01

    Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH), while mutations in genes encoding the two enzymes (dihydropteridine reductase, DHPR, and pterin-4-alpha-carbinolamine dehydratase, PCD) required for recycling of its cofactor, tetrahydrobiopterin (BH(4)), cause other rarer disease forms of hyperphenylalaninemia. We have applied a yeast two-hybrid method, in which protein--protein interactions are measured by four reporter gene constructs, to the analysis of six PKU-associated PAH missense mutations (F39L, K42I, L48S, I65T, A104D, and R157N). By studying homomeric interactions between mutant PAH subunits, we show that this system is capable of detecting quite subtle aberrations in PAH oligomerization caused by missense mutations and that the observed results generally correlate with the severity of the mutation as determined by other expression systems. The mutant PAH subunits are also shown in this system to be able to interact with wild-type PAH subunits, pointing to an explanation for apparent dominant negative effects previously observed in obligate heterozygotes for PKU mutations. Based on our findings, the applications and limitations of two-hybrid approaches in understanding mechanisms by which PAH missense mutations exert their pathogenic effects are discussed. We have also used this technique to demonstrate homomeric interactions between wild-type DHPR subunits and between wild-type PCD subunits. These data provide a basis for functional studies on HPA-associated mutations affecting these enzymes.

  10. Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk.

    Science.gov (United States)

    Pohjanen, Vesa-Matti; Koivurova, Olli-Pekka; Huhta, Heikki; Helminen, Olli; Mäkinen, Johanna M; Karhukorpi, Jari M; Joensuu, Tapio; Koistinen, Pentti O; Valtonen, Jarno M; Niemelä, Seppo E; Karttunen, Riitta A; Karttunen, Tuomo J

    2015-01-01

    Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

  11. Species specific behavioural patterns (digging and swimming and reaction to novel objects in wild type, Wistar, Sprague-Dawley and Brown Norway rats.

    Directory of Open Access Journals (Sweden)

    Rafał Stryjek

    Full Text Available BACKGROUND: The purpose of the present study was to analyse species-specific forms of behaviour (digging and swimming and response to novelty in laboratory rats and their wild type counterparts at a very early stage of laboratorization. Three behavioural phenomena were taken into account: burrowing, spontaneous swimming, and neophobic behaviour. PRINCIPAL FINDINGS: Wild-type rats and three strains of laboratory rats were involved in experiments: Warsaw-Wild-Captive-Pisula-Stryjek (WWCPS, Wistar, Sprague-Dawley, and Brown Norway rats were compared in spontaneous swimming test, while WWCPS and Wistar rats were studied in burrowing and neophobia experiments. Wild rats were found to be faster at building tunnels than Wistar rats and at constructing more complex burrow systems. The experiment on neophobia showed that Wistar rats exhibited less neophobic responses and were more often trapped. WWCPS rats showed highly neophobic behaviour and were rarely trapped in this experiment. The experiment on swimming showed that WWCPS rats showed more complex water tank related activity than their laboratory counterparts. They swam and explored under surface environment. CONCLUSIONS: The three experiments showed profound behavioural differences in quasi-natural forms of behaviour between wild type rats (WWCPS and three laboratory strains frequently used in behavioural studies.

  12. Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion.

    Science.gov (United States)

    Lazzeri, Valerio; Calvenzani, Valentina; Petroni, Katia; Tonelli, Chiara; Castagna, Antonella; Ranieri, Annamaria

    2012-05-16

    Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.

  13. Mice Deficient in the Gene for Cytochrome P450 (CYP)1A1 Are More Susceptible Than Wild-Type to Hyperoxic Lung Injury: Evidence for Protective Role of CYP1A1 Against Oxidative Stress

    Science.gov (United States)

    Wang, Lihua; Wang, Gangduo; Couroucli, Xanthi I.; Shivanna, Binoy; Welty, Stephen E.; Barrios, Roberto; Khan,  M. Firoze; Nebert, Daniel W.; Roberts, L. Jackson; Moorthy, Bhagavatula

    2014-01-01

    Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome in adults and bronchopulmonary dysplasia in premature infants. Cytochrome P450 (CYP)1A1 has been shown to modulate hyperoxic lung injury. The mechanistic role(s) of CYP1A1 in hyperoxic lung injury in vivo is not known. In this investigation, we hypothesized that Cyp1a1(–/–) mice would be more susceptible to hyperoxic lung injury than wild-type (WT) mice, and that the protective role of CYP1A1 is in part due to CYP1A1-mediated decrease in the levels of reactive oxygen species-mediated lipid hydroperoxides, e.g., F2-isoprostanes/isofurans, leading to attenuation of oxidative damage. Eight- to ten-week-old male WT (C57BL/6J) or Cyp1a1(–/–) mice were exposed to hyperoxia (>95% O2) or room air for 24–72 h. The Cyp1a1(–/–) mice were more susceptible to oxygen-mediated lung damage and inflammation than WT mice, as evidenced by increased lung weight/body weight ratio, lung injury, neutrophil infiltration, and augmented expression of IL-6. Hyperoxia for 24–48 h induced CYP1A expression at the mRNA, protein, and enzyme levels in liver and lung of WT mice. Pulmonary F2-isoprostane and isofuran levels were elevated in WT mice after hyperoxia for 24 h. On the other hand, Cyp1a1(–/–) mice showed higher levels after 48–72 h of hyperoxia exposure compared to WT mice. Our results support the hypothesis that CYP1A1 protects against hyperoxic lung injury by decreasing oxidative stress. Future research could lead to the development of novel strategies for prevention and/or treatment of acute lung injury. PMID:24893714

  14. Restoration of missing or misplaced canines.

    Science.gov (United States)

    Bower, C F; Reinhardt, R A

    1985-06-01

    Restorative treatments for canines were discussed to correct three clinical abnormalities: (1) fully erupted permanent canine in the lateral incisor position, (2) missing permanent canines, and (3) partially exposed canines in normal arch position. The primary concerns are the development of esthetics, anterior guidance, and adequate support for fixed restorations.

  15. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

    OpenAIRE

    Sophie Le Poder

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious ...

  16. Feline and canine coronaviruses : common genetic and pathobiological features

    OpenAIRE

    Sophie Le Poder

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious ...

  17. Effect of G gene-deleted recombinant viral hemorrhagic septicemia virus (rVHSV-ΔG) on the replication of wild type VHSV in a fish cell line and in olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Kim, Min Sun; Choi, Seung Hyuk; Kim, Ki Hong

    2016-07-01

    In an earlier study, we generated a replicon viral hemorrhagic septicemia virus (VHSV) particle that was lacking the G gene in the genome (rVHSV-ΔG), and proved the potential of it as a protective vaccine through the immunization of olive flounder (Paralichthys olivaceus) fingerlings. Safety is the most important preconsideration for the development of recombinant live vaccines, and a major concern of propagation-incompetent viral particles would be the possible harmful effect to hosts through the interaction with wild-type viruses. Thus, in the present study, we analyzed the replication of rVHSV-ΔG in the presence of wild-type VHSV and the effect of rVHSV-ΔG on the replication of wild-type VHSV in Epithelioma papulosum cyprini (EPC) cells and in olive flounder fingerlings. The replication of wild-type VHSV in EPC cells was severely suppressed when the MOI of rVHSV-ΔG was 0.1 or 0.01, on the other hand, the titers of rVHSV-ΔG were not increased and stayed in a relatively constant according to time lapse. Furthermore, the replication of other novirhabdoviruses, IHNV and HIRRV, was also inhibited by co-infection with high titers of rVHSV-ΔG. There were no big differences in mortalities between groups infected with wild-type VHSV plus rVHSV-ΔG and groups infected with wild-type VHSV alone, when the challenged wild-type VHSV was more than 10(2) PFU/fish. However, a group of fish infected with 10 PFU/fish of wild-type VHSV plus rVHSV-ΔG showed significantly lower and slowly progressing cumulative mortality than a group of fish infected with 10 PFU/fish of wild-type VHSV alone. This result suggests that rVHSV-ΔG has an ability to attenuate the disease progression caused by wild-type VHSV when co-infected with relatively low titers of wild-type VHSV. These results indicate that the propagation-incompetent rVHSV-ΔG would not worsen but attenuate the progression of a disease caused by wild-type VHSV infection. Therefore, rVHSV-ΔG-based vaccines can provide a

  18. 野生型Parkin基因对肝癌细胞生长的影响%Effect of wild type parkin gene on growth of hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    陈钢; 施红旗; 单云峰; 沈文状; 朱智; 刘顺芳

    2010-01-01

    目的 探讨野生型及突变型Parkin基因表达对人肝癌细胞株Huh-7在体内外生长情况的影响.方法 利用脂质体介导的基因转染法将野生型及突变型Parkin基因真核表达载体转染肝癌细胞株Huh-7,筛选稳定表达细胞株,通过逆转录-聚合酶链反应(RT-PCR)进行鉴定并送测序分析.细胞增殖实验和裸鼠致瘤性实验检测各稳定表达株的体内外生长情况.结果 成功建立了稳定表达野生型和突变型Parkin基因的Huh-7细胞株.以转染空载体的Huh-7细胞作为对照,野生型Parkin基因的表达可明显抑制肝癌细胞在体外的生长(t=3.875,P=0.031),可显著减缓裸鼠皮下瘤的生长速度并减小其体积(t=8.228,P=0.003).突变型Parkin基因的表达对肝癌细胞的生长影响不大(P>0.05).结论 野生型Parkin的重表达有助于肝癌细胞恶性表型的逆转.野生型Parkin基因是一个肝癌相关的抑癌基因.%Objective To explore the effect of wild type or mutant parkin gene expression on the growth of human hepatocellular carcinoma cell line Huh-7. Methods The parkin (wild type or mutant) expression vector and empty vector were transferred into Huh-7 cell lines with LipofectAMINE 2000 reagents. The positive clones that expressed parkin gene stably were chosen by G418 and checked by reverse transcription-polymerase chain reaction (RT-PCR) to check the DNA sequences. The cytobiological behaviors of those positive clones were analyzed by cell proliferation assay and tumorigenesis in nude mice. Results Huh-7 cell lines that expressed wild type or mutant parkin gene stably were successfully established. The growth of wild type parkin-expressed cells was obviously inhibited compared with the control cells transfected with empty vectors(t= 3. 875, P= 0. 031).The volume of tumor formed by wild type parkin-expressing cells in nude mice was also significantly reduced (t=8. 228,P=-0. 003). Mutant parkin gene expression had a slight effect on the

  19. Canine mammary gland tumors.

    Science.gov (United States)

    Sorenmo, Karin

    2003-05-01

    The National Consensus Group recommends that all women with tumors larger than 1 cm be offered chemotherapy regardless of tumor histology of lymph node status. This recommendation is to ensure that everyone at risk for failing, even though the risk may be low in women with relatively small tumors and favorable histology, has a choice and receives the benefit of adjuvant chemotherapy. This type of treatment recommendation may also be made in dogs based on recognized, well-accepted prognostic factors such as tumor size, stage, type, and histologic differentiation. Based on the limited clinical information available in veterinary medicine, the drugs that are effective in human breast cancer, such as cyclophosphamide, 5-fluorouracil, and doxorubicin, may also have a role in the treatment of malignant mammary gland tumors in dogs. Randomized prospective studies are needed, however, to evaluate the efficacy of chemotherapy in dogs with high-risk mammary gland tumors and to determine which drugs and protocols are the most efficacious. Until such studies are performed, the treatment of canine mammary gland tumors will be based on the individual oncologist's understanding of tumor biology, experience, interpretation of the available studies, and a little bit of gut-feeling. Table 2 is a proposal for treatment guidelines for malignant canine mammary gland tumors according to established prognostic factors, results from published veterinary studies, and current recommendations for breast cancer treatment in women.

  20. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses.

    Directory of Open Access Journals (Sweden)

    Tassilo Förg

    Full Text Available The homodimeric transmembrane receptor endoglin (CD105 plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC, immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing

  1. Investigation of endoglin wild-type and missense mutant protein heterodimerisation using fluorescence microscopy based IF, BiFC and FRET analyses.

    Science.gov (United States)

    Förg, Tassilo; Hafner, Mathias; Lux, Andreas

    2014-01-01

    The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other

  2. The Effects of Wild-type p53 Gene Transfection on the Growth and Chemotherapeutic Sensitivity of Human Gl ioma Cells

    Institute of Scientific and Technical Information of China (English)

    项炜; 朱贤立; 赵洪洋

    2002-01-01

    To evaluate the effects of wild-type p53 gene on the growth and chemotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, the cell growth inhibition and apoptosis in either the absence or the presence of cisplatin was assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate,IR (79.60±5.69) %]. The killing effects of cisplatin by itself on U251 cells was not strong [IR (19.40±6. 69) %, (24.41±2. 68) %, (51.84±13. 38) %, (66. 22±5.02) %] and increased with the increase of cisplatin concentration (1, 2, 4, 8 μg/ml). When combined treatment of wildtype p53 gene transfection and cisplatin was used, that was significantly increased [IR (91.64+1.00) %, (94. 98±1.67) %, (95.32±2.01)%, (95. 65±1.00) %]. The apoptosis rate of U251cells induced by p53 gene transfection was 17.38%. That induced by cisplatin increased (5.71 %,5. 93 %, 6.27 %, and 6.81%) with the increase of cisplatin concentration (1, 2, 4, 8 μg/ml).The apoptosis rate was also significantly increased (23.50 %, 23. 54 %, 23.89 %, and 28.88 %)after combined treatment of p53 and cisplatin with different concentration (1, 2, 4, 8 μg/ml). It is concluded that wild-type p53 gene and cisplatin could result in synergistic inhibition effects on the growth of human glioma cells.

  3. The conditional nature of genetic interactions: the consequences of wild-type backgrounds on mutational interactions in a genome-wide modifier screen.

    Directory of Open Access Journals (Sweden)

    Sudarshan Chari

    Full Text Available The phenotypic outcome of a mutation cannot be simply mapped onto the underlying DNA variant. Instead, the phenotype is a function of the allele, the genetic background in which it occurs and the environment where the mutational effects are expressed. While the influence of genetic background on the expressivity of individual mutations is recognized, its consequences on the interactions between genes, or the genetic network they form, is largely unknown. The description of genetic networks is essential for much of biology; yet if, and how, the topologies of such networks are influenced by background is unknown. Furthermore, a comprehensive examination of the background dependent nature of genetic interactions may lead to identification of novel modifiers of biological processes. Previous work in Drosophila melanogaster demonstrated that wild-type genetic background influences the effects of an allele of scalloped (sd, with respect to both its principal consequence on wing development and its interactions with a mutation in optomotor blind. In this study we address whether the background dependence of mutational interactions is a general property of genetic systems by performing a genome wide dominant modifier screen of the sd(E3 allele in two wild-type genetic backgrounds using molecularly defined deletions. We demonstrate that ~74% of all modifiers of the sd(E3 phenotype are background-dependent due in part to differential sensitivity to genetic perturbation. These background dependent interactions include some with qualitative differences in the phenotypic outcome, as well as instances of sign epistasis. This suggests that genetic interactions are often contingent on genetic background, with flexibility in genetic networks due to segregating variation in populations. Such background dependent effects can substantially alter conclusions about how genes influence biological processes, the potential for genetic screens in alternative wild-type

  4. Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

    Science.gov (United States)

    Suchland, Robert J; Jeffrey, Brendan M; Xia, Minsheng; Bhatia, Ajay; Chu, Hencelyn G; Rockey, Daniel D; Stamm, Walter E

    2008-12-01

    Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

  5. Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

    Directory of Open Access Journals (Sweden)

    Lei-Ming Sun

    Full Text Available MicroRNAs (miRNAs are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf. and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

  6. Protection of Cattle against Rinderpest by Vaccination with Wild-Type but Not Attenuated Strains of Peste des Petits Ruminants Virus

    Science.gov (United States)

    Holzer, Barbara; Hodgson, Sophia; Logan, Nicola; Willett, Brian

    2016-01-01

    ABSTRACT Although rinderpest virus (RPV) has been eradicated in the wild, efforts are still continuing to restrict the extent to which live virus is distributed in facilities around the world and to prepare for any reappearance of the disease, whether through deliberate or accidental release. In an effort to find an alternative vaccine which could be used in place of the traditional live attenuated RPV strains, we have determined whether cattle can be protected from rinderpest by inoculation with vaccine strains of the related morbillivirus, peste des petits ruminants virus (PPRV). Cattle were vaccinated with wild-type PPRV or either of two established PPRV vaccine strains, Nigeria/75/1 or Sungri/96. All animals developed antibody and T cell immune responses to the inoculated PPRV. However, only the animals given wild-type PPRV were protected from RPV challenge. Animals given PPRV/Sungri/96 were only partially protected, and animals given PPRV/Nigeria/75/1 showed no protection against RPV challenge. While sera from animals vaccinated with the vaccine strain of RPV showed cross-neutralizing ability against PPRV, none of the sera from animals vaccinated with any strain of PPRV was able to neutralize RPV although sera from animals inoculated with wild-type PPRV were able to neutralize RPV-pseudotyped vesicular stomatitis virus. IMPORTANCE Rinderpest virus has been eradicated, and it is only the second virus for which this is so. Significant efforts are still required to ensure preparedness for a possible escape of RPV from a laboratory or its deliberate release. Since RPV vaccine protects sheep and goats from PPRV, it is important to determine if the reverse is true as this would provide a non-RPV vaccine for dealing with suspected RPV outbreaks. This is probably the last in vivo study with live RPV that will be approved. PMID:26984722

  7. The GAP Portion of Pseudomonas Aeruginosa Type III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

    Directory of Open Access Journals (Sweden)

    Deepali N. Tukaye

    2013-02-01

    Full Text Available Background: Pseudomonas aeruginosa (PA infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP and C-terminal ADP rybosyl transferase (ADPRT activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP. In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP mediates cellular toxicity. Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system. Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na+/K+ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K, known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR. Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.

  8. Full life-cycle assessment of gene flow consistent with fitness differences in transgenic and wild-type Japanese medaka fish (Oryzias latipes).

    Science.gov (United States)

    Pennington, Kelly M; Kapuscinski, Anne R; Morton, Michael S; Cooper, Anne M; Miller, Loren M

    2010-01-01

    Transgenic fish in development for aquaculture could escape from farms and interbreed with wild relatives in the nearby environment. Predicting whether escapes would result in transgene introgression is a major challenge in assessing environmental risks of transgenic fish. Previous studies have simulated gene flow from transgenic fish using mathematical modeling of fitness traits to predict the relative selective value of transgenic genotypes. Here, we present the first study of gene flow over the full life cycle in openly-breeding populations of transgenic animals, along with measurement of fitness traits. We conducted two invasion experiments in which we released two lines of growth-enhanced transgenic fish (T67 and T400), Japanese medaka (Oryzias latipes), into populations of wild-type (W) medaka in structured mesocosms. After several generations, the frequency of transgenic fish varied across replicates in the first invasion experiment (6 months), but the frequency of transgenic fish decreased in the second experiment (19 months). We also measured selected fitness traits in transgenic and wild-type medaka because these traits could be used to predict the relative selective value of a genotype. We found that: T400 males were more fertile than W males; offspring of W females lived longer than those with transgenic mothers; and W and T67 females reached sexual maturity sooner than T400 females. In contrast with other research that reported larger transgenic males had a mating advantage, we found that W males obtained more matings with females than T males; genetic background effects may account for our differing results as we compared W and T fish derived from different strains. The decreasing frequency of transgenic fish in the second invasion experiment suggests that transgenic fish had a selective disadvantage in the experimental environment. Our finding of transgenic advantage of some fitness traits and wild-type advantage in others is consistent with our

  9. Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells.

    Science.gov (United States)

    Shi, Ming-Zhu; Xie, De-Yu

    2011-04-01

    We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

  10. Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

    Science.gov (United States)

    Fierobe, H P; Mirgorodskaya, E; McGuire, K A; Roepstorff, P; Svensson, B; Clarke, A J

    1998-03-17

    Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

  11. Transgenic Eimeria mitis Expressing Chicken IL-4 Mediated Decrease in Pathogenicity Compared to Wild Type Eimeria mitis Strains in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    Jamal Muhammad Khan1,2, Saeed El-Ashram, Hong Bin Liu3, Sher Hayat Khan4, Adnan Ayan5, Xian Yong Liu1,2, Hui Wang2, Xin Ming Tang1,2, Xun Suo1,2* and Mohammad Farooque Hassan4,6

    2016-11-01

    Full Text Available The present study was designed to establish transgenic Eimeria mitis (tE. mitis expressing chicken interleukin-4 (ChIL-4 and further exploitation of tE. mitis as a novel vaccine vector to control coccidiosis. For this purpose we compared the clinico-hematological alterations induced by wild type and tE. mitis carrying ChIL-4 in broilers birds. Wild type E. mitis (wE. mitis oocysts were nucleofected with double cassette vector and transgenic oocysts were propagated for stable transfection. Stability of the transfected E. mitis was observed by fluorescent microscopy and PCR analysis revealed the appearance of 411bp band suggestive of presence of IL-4 into the genome of tE. mitis. Birds inoculated with transgenic EmiChIL-4 excreted fewer oocysts and fecundity potential of tE. mitis turned out to be significantly lower than that of the wild type parent strain. Moderate clinical and behavioral signs such as restlessness, diarrhea, emaciation, decreased feed intake and loss of body weight was observed in birds inoculated with wE. mitis oocysts. The erythrocyte counts, Hb conc, PCV and MCHC decreased in birds infected with wE. mitis as compared to those infected with tE. mitis. The total leukocyte count including heterophils, eosinophil and basophils increased while lymphocytes were decreased in birds inoculated with wE. mitis as compared to birds in control group and those infected with tE.mitis. It was concluded that tE. mitis incorporated with ChIL-4 functions well as an adjuvant, conferring reduced fertility and compromised pathogenicity of tE. mitis. This discrimination, may be applied as a potential vaccine vector to control coccidiosis as well as pathogens from which antigens are expressed by tE. mitis.

  12. Comparison of sugar, acids, and volatile composition in raspberry bushy dwarf virus-resistant transgenic raspberries and the wild type 'meeker' (rubus idaeus L.).

    Science.gov (United States)

    Malowicki, Sarah M M; Martin, Robert; Qian, Michael C

    2008-08-13

    Raspberry bushy dwarf virus (RBDV) causes a significant reduction in yield and quality in raspberry and raspberry-blackberry hybrid. Genetic modifications were made to 'Meeker' red raspberries to impart RBDV resistance. The RBDV-resistant transgenic and wild type 'Meeker' plants were grown in Oregon and Washington, and the fruits were harvested in the 2004 and 2005 growing seasons. Year-to-year and site-to-site variations were observed for the degrees Brix and titratable acidity, with Oregon raspberries having slightly higher degrees Brix and lower titratable acidity than Washington raspberries. Twenty-nine volatile compounds were quantified using stir bar sorptive extraction (SBSE) paired with gas chromatography-mass spectrometry (GC-MS). There were very few differences in volatile concentrations between the transgenic varieties and the wild type 'Meeker'. Much larger variations were observed between sites and harvest seasons. Raspberries grown in Oregon appeared to have higher concentrations of delta-octalactone, delta-decalactone, geraniol, and linalool. Chiral analysis of alpha-ionone, alpha-pinene, linalool, terpinen-4-ol, delta-octalactone, and delta-decalactone demonstrated a much higher percentage of one isomer over the other, particularly alpha-ionone, alpha-pinene, delta-octalactone, and delta-decalactone, with more than 90% of one isomer, while a racemic mixture was observed for linalool. The isomeric analysis revealed very little variation between varieties, locations, or years. The flavor compounds tested in this study did not show any difference between the transgenic lines and the wild type 'Meeker' raspberry.

  13. Helium-neon laser preirradiation induces protection against UVC radiation in wild-type E. coli strain K12AB1157.

    Science.gov (United States)

    Kohli, R; Gupta, P K; Dube, A

    2000-02-01

    We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E. coli strain K12AB1157. The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation. The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m(2), 100 W/m(2) and 1 h, respectively. The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.

  14. Channel-opening kinetic mechanism for human wild-type GluK2 and the M867I mutant kainate receptor.

    Science.gov (United States)

    Han, Yan; Wang, Congzhou; Park, Jae Seon; Niu, Li

    2010-11-02

    GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms

  15. Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states

    Energy Technology Data Exchange (ETDEWEB)

    Bodenheimer, Annette M. [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA; Meilleur, Flora [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA

    2016-11-07

    Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

  16. Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels.

    Directory of Open Access Journals (Sweden)

    Christine Wandall-Frostholm

    Full Text Available OBJECTIVE: In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. APPROACH AND RESULT: Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. CONCLUSION: Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a

  17. Homology modeling of wild-type, D516V, and H526L Mycobacterium tuberculosis RNA polymerase and their molecular docking study with inhibitors.

    Science.gov (United States)

    Josa, Daniela; da Cunha, Elaine F F; Ramalho, Teodorico C; Souza, Thais C S; Caetano, Melissa S

    2008-02-01

    Rifamicyns (Rifs) are antibiotic widely used for the treatment of tuberculosis (TB); nevertheless, their efficacy has been limited by a high percentage of mutations, principally in the rpoB gene. In this work, the first three-dimensional molecular model of the hypothetical structures for the wild-type and D516V and H526L mutants of Mycobacterium tuberculosis (mtRNAP) were elucidated by a homology modeling method. In addition, the orientations and binding affinities of some Rifs with those new structures were investigated. Our findings could be helpful for the design of new more potent rifamycin analogs.

  18. Panitumumab and pegylated liposomal doxorubicin to platinum-resistant epithelial ovarian cancer with KRAS wild-type: An ongoing, nonrandomized, multicenter, phase II trial

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Lund, B

    2010-01-01

    , and head and neck cancer. No previous studies have evaluated the effect of panitumumab in OC based on KRAS mutation status. Methods: Eligibility criteria are confirmed stage I-IV primary epithelial ovarian/fallopian/peritoneal cancer patients with progression either during or within 6 months after end...... to a total of 33 patients. At present, 15 patients have been enrolled. The primary endpoint is to investigate the response rate in platinum-resistant, KRAS wild- type OC patients treated with PLD supplemented with panitumumab. Translational research is included as a secondary endpoint and tumor tissue...

  19. Increasing levels of wild-type CREB up-regulates several activity-regulated inhibitor of death (AID genes and promotes neuronal survival

    Directory of Open Access Journals (Sweden)

    Tan Yan-Wei

    2012-05-01

    Full Text Available Abstract Background CREB (cAMP-response element binding protein is the prototypical signal-regulated transcription factor. In neurons, it is the target of the synaptic activity-induced nuclear calcium-calcium/calmodulin dependent protein kinase (CaMK IV signaling pathway that controls the expression of genes important for acquired neuroprotection as well as other long-lasting adaptive processes in the nervous system. The function of CREB as a transcriptional activator is controlled by its phosphorylation on serine 133, which can be catalyzed by CaMKIV and leads to the recruitment of the co-activator, CREB binding protein (CBP. Activation of CBP function by nuclear calcium-CaMKIV signaling is a second regulatory step required for CREB/CBP-mediated transcription. Results Here we used recombinant adeno-associated virus (rAAV to increase the levels of wild type CREB or to overexpress a mutant version of CREB (mCREB containing a serine to alanine mutation at position amino acid 133 in mouse hippocampal neurons. Increasing the levels of CREB was sufficient to boost neuroprotective activity even under basal conditions (i.e., in the absence of stimulation of synaptic activity. In contrast, overexpression of mCREB increased cell death. The ratio of phospho(serine 133CREB to CREB immunoreactivity in unstimulated hippocampal neurons was similar for endogenous CREB and overexpressed wild type CREB and, as expected, dramatically reduced for overexpressed mCREB. A gene expression analysis revealed that increased expression of CREB but not that of mCREB in hippocampal neurons led to elevated expression levels of bdnf as well as that of several members of a previously characterized set of Activity-regulated Inhibitor of Death (AID genes, which include atf3, btg2, gadd45β, and gadd45γ. Conclusions Our findings indicate that the expression levels of wild type CREB are a critical determinant of the ability of hippocampal neurons to survive harmful conditions

  20. Epidemiological Survey on Canine Parvovirus Disease in Taizhou Region,Jiangsu Province,China

    Institute of Scientific and Technical Information of China (English)

    Yuan; Weifeng; Liu; Jing; Zhao; Xuegang; Lu; Jiang; Zheng; Xiaoliang; Zhu; Hongfei

    2014-01-01

    The canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). It is clinically characterized by severe vomiting,hemorrhagic enteritis,significant reduction in white blood cells and myocarditis. The disease with high incidence,highly infectious and high mortality has become one of the serious infectious diseases threatening dog raising industry in China. In this research,260 cases of canine parvovirus case from an Aite Pet Clinic in Taizhou City during January 2010 and March 2011 were analyzed. This study discloses the epidemiology of CPV in Taizhou region of Jiangsu Province,i. e.,the incidence of CPV and canine motility are closely correlated with age,breed,immune inoculation and season. This study provides useful guide for the clinical treatment of CPV in the future.

  1. Seroepidemiology of Canine parvovirus infection in dogs

    Directory of Open Access Journals (Sweden)

    Indrawati Sendow

    2004-10-01

    Full Text Available Canine parvovirus is an acute and fatal viral disease in dogs. A total of 209 local, cross breed and breed dogs sera from Kodya Bogor, Kabupaten Bogor, Sukabumi, and Jakarta, had been tested using Haemagglutination Inhibition Test (HI with pig red blood cells. A total of 64 breed and cross breed dogs from Sukabumi and Kodya Bogor, were used as a sentinel dogs to study the epidemiology of Canine parvovirus (CPV infection and its immunological responses caused by vaccination. The results indicated that 78% (95 breed and cross bred dogs and 59% (51 local dogs had antibody to CPV. Sentinel dogs results indicated that dogs had been vaccinated showed antibody response with the varied titre dependant upon prevaccination titre. Low prevaccinated titre gave better response than protective level titre. From 19 puppies observed, Maternal antibodi were still detected until 5 weeks old puppies. First vaccination given at less than 3 months old, should be boosted after 3 months old puppied. Antibodi titre produced by natural infection will keep untill 2 years. These data concluded that the dog condition and time of vaccination will affect the optimum antibody response.

  2. Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c{sub 552} and its b-type variant

    Energy Technology Data Exchange (ETDEWEB)

    Tozawa, Kaeko; Ferguson, Stuart J.; Redfield, Christina, E-mail: christina.redfield@bioch.ox.ac.uk [University of Oxford, Department of Biochemistry (United Kingdom); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [University of Oxford, Department of Chemistry (United Kingdom)

    2015-06-15

    Cytochrome c{sub 552} from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, {sup 15}N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high {sup 15}N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

  3. Contribution of heavy metals to toxicity of coal combustion related fine particulate matter (PM2.5) in Caenorhabditis elegans with wild-type or susceptible genetic background.

    Science.gov (United States)

    Sun, Lingmei; Wu, Quli; Liao, Kai; Yu, Peihang; Cui, Qiuhong; Rui, Qi; Wang, Dayong

    2016-02-01

    Contribution of chemical components in coal combustion related fine particulate matter (PM2.5) to its toxicity is largely unclear. We focused on heavy metals in PM2.5 to investigate their contribution to toxicity formation in Caenorhabditis elegans. Among 8 heavy metals examined (Fe, Zn, Pb, As, Cd, Cr, Cu, and Ni), Pb, Cr, and Cu potentially contributed to PM2.5 toxicity in wild-type nematodes. Combinational exposure to any two of these three heavy metals caused higher toxicity than exposure to Pb, Cr, or Cu alone. Toxicity from the combinational exposure to Pb, Cr, and Cu at the examined concentrations was higher than exposure to PM2.5 (100 mg/L). Moreover, mutation of sod-2 or sod-3 gene encoding Mn-SOD increased susceptibility in nematodes exposed to Fe, Zn, or Ni, although Fe, Zn, or Ni at the examined concentration did not lead to toxicity in wild-type nematodes. Our results highlight the potential contribution of heavy metals to PM2.5 toxicity in environmental organisms.

  4. Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method.

    Science.gov (United States)

    Ngo, Son Tung; Mai, Binh Khanh; Hiep, Dinh Minh; Li, Mai Suan

    2015-10-01

    The binding mechanism of AC1NX476 to HIV-1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double-annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25-A, Asp29-A, Asp30-A, Ile47-A, Gly48-A, and Val50-A from chain A, and Asp25-B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor-ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV.

  5. Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

    Directory of Open Access Journals (Sweden)

    Felix K M Lorenz

    Full Text Available Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

  6. Photoinactivation of Photosystem II in wild-type and chlorophyll b-less barley leaves: which mechanism dominates depends on experimental circumstances.

    Science.gov (United States)

    He, Jie; Yang, Wenquan; Qin, Lin; Fan, Da-Yong; Chow, Wah Soon

    2015-12-01

    Action spectra of photoinactivation of Photosystem II (PS II) in wild-type and chlorophyll b-less barley leaf segments were obtained. Photoinactivation of PS II was monitored by the delivery of electrons from PS II to PS I following single-turnover flashes superimposed on continuous far-red light, the time course of photoinactivation yielding a rate coefficient k i. Susceptibility of PS II to photoinactivation was quantified as the ratio of k i to the moderate irradiance (I) of light at each selected wavelength. k i/I was very much higher in blue light than in red light. The experimental conditions permitted little excess light energy absorbed by chlorophyll (not utilized in photochemical conversion or dissipated in controlled photoprotection) that could lead to photoinactivation of PS II. Therefore, direct absorption of light by Mn in PS II, rather than by chlorophyll, was more likely to have initiated the much more severe photoinactivation in blue light than in red light. Mutant leaves were ca. 1.5-fold more susceptible to photoinactivation than the wild type. Neither the excess-energy mechanism nor the Mn mechanism can explain this difference. Instead, the much lower chlorophyll content of mutant leaves could have exerted an exacerbating effect, possibly partly due to less mutual shading of chloroplasts in the mutant leaves. In general, which mechanism dominates depends on the experimental conditions.

  7. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

    Directory of Open Access Journals (Sweden)

    Cavazzoni Andrea

    2012-12-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  8. Oncogenic K-Ras Signals through Epidermal Growth Factor Receptor and Wild-Type H-Ras to Promote Radiation Survival in Pancreatic and Colorectal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Keith A. Cengel

    2007-04-01

    Full Text Available Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor o was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFRactivated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

  9. Oncogenic K-Ras Signals through Epidermal Growth Factor Receptor and Wild-Type H-Ras to Promote Radiation Survival in Pancreatic and Colorectal Carcinoma Cells1

    Science.gov (United States)

    Cengel, Keith A.; Voong, K. Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B.; Stanbridge, Eric; Kao, Gary D.; McKenna, W. Gillies; Bernhard, Eric J.

    2007-01-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers. PMID:17460778

  10. Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells.

    Science.gov (United States)

    Cengel, Keith A; Voong, K Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B; Stanbridge, Eric; Kao, Gary D; McKenna, W Gillies; Bernhard, Eric J

    2007-04-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

  11. Differences in protein synthesis between wild type and intracellular growth-deficient strains of Legionella pneumophila in U937 and Acanthamoeba polyphaga.

    Science.gov (United States)

    Miyake, Masaki; Fukui, Takashi; Imai, Yasuyuki

    2006-04-01

    An important aspect of Legionnaires' disease is the growth of the causative agent, Legionella pneumophila, within infected host cells. Many proteins including stress proteins of L. pneumophila were strongly induced in a wild type strain that had been used to infect U937 human macrophage-like cells. In contrast, the expression of the proteins was much weaker within a protozoan host, Acanthamoeba polyphaga. The results suggested that active bacterial protein synthesis is required more within macrophages than within protozoa for adaptation of L. pneumophila to intracellular environments. The synthesis of these proteins was not observed in intracellular growth-deficient strains after infection in either type of host cells. The inability of protein synthesis in these strains is correlated with their inability of intracellular growth. Furthermore, on U937 infection, the synthesis of beta-galactosidase encoded in an inducible reporter construct immediately ceased in the in intracellular growth-deficient strains after infection, while the wild type strain was able to synthesize it during the course of infection. These results suggested that the intracellular growth of Legionella pneumophila within macrophages requires active protein synthesis from an earlier stage of bacterial infection.

  12. Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

    Science.gov (United States)

    Guan, Wenna; Zhao, Hui; Lu, Xuefeng; Wang, Cong; Yang, Menglong; Bai, Fali

    2011-11-11

    Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.

  13. Wild-type Kaposi's sarcoma-associated herpesvirus isolated from the oropharynx of immune-competent individuals has tropism for cultured oral epithelial cells.

    Science.gov (United States)

    Duus, Karen M; Lentchitsky, Vivian; Wagenaar, Timothy; Grose, Charles; Webster-Cyriaque, Jennifer

    2004-04-01

    Based on the observation that wild-type Kaposi's sarcoma-associated herpesvirus (KSHV) DNA can be detected in the oral cavity of healthy, immunocompetent individuals, we hypothesized that epithelial cells could be infected in vitro by wild-type (WT) KSHV isolated from immunocompetent individuals. Primary oral epithelial (P-EPI) cells and telomerase-immortalized oral epithelial cells were generated from human gingival tissue and were then infected in vitro with WT KSHV isolated from throat wash samples. Markers of lytic and latent KSHV infection were detected in cultures by 24 h postinfection by immunofluorescence confocal microscopic assays. The infectivity of the WT and BCBL virus was blocked by neutralizing antibodies against KSHV gB. The presence of KSHV DNA in these cells was confirmed by real-time PCR amplification of different regions of the viral genome. The significant in vitro viral replication that had occurred was inhibited by ganciclovir and by neutralizing antibodies against gB. When infected cultures were examined by scanning electron microscopy, thousands of KSHV particles were clearly visible across the surfaces of P-EPI cells. The detection of enveloped particles indicated that the infectious cycle had proceeded through assembly and egress. We thus demonstrated that oral WT KSHV isolated from immunocompetent individuals was able to infect and replicate in vitro in a relevant primary cell type. Furthermore, our results provide compelling evidence for KSHV transmission within infected oral epithelial cells derived from healthy, immunocompetent populations.

  14. Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice

    Science.gov (United States)

    Cesaroni, Valentina; Gregori, Andrej; Repetti, Margherita; Romano, Chiara; Orrù, Germano; Botta, Laura; Girometta, Carolina; Guglielminetti, Maria Lidia; Savino, Elena

    2017-01-01

    Hericium erinaceus (Bull.) Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

  15. Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells

    Directory of Open Access Journals (Sweden)

    Madhu C Mohanty

    2013-01-01

    Full Text Available Background & objectives: Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV and Sabin attenuated type 1 poliovirus (Sabin PV in cultured human neuronal cells. Methods: By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH infected with Sabin PV and wild PV. Results: Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5 m-RNA in neuronal cells at the beginning of infection (up to 4 h as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Interpretation & conclusions: Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.

  16. Ala67Thr mutation in the poliovirus receptor CD155 is a potential risk factor for vaccine and wild-type paralytic poliomyelitis.

    Science.gov (United States)

    Kindberg, Elin; Ax, Cecilia; Fiore, Lucia; Svensson, Lennart

    2009-05-01

    Poliovirus infections can be asymptomatic or cause severe paralysis. Why some individuals develop paralytic poliomyelitis is unknown, but a role for host genetic factors has been suggested. To investigate if a polymorphism, Ala67Thr, in the poliovirus receptor, which has been found to facilitate increased resistance against poliovirus-induced cell lysis and apoptosis, is associated with increased risk of paralytic poliomyelitis, poliovirus receptor genotyping was undertaken among Italian subjects with vaccine-associated (n = 9), or with wild-type paralytic poliomyelitis (n = 6), and control subjects (n = 71), using RFLP-PCR and pyrosequencing. Heterozygous poliovirus receptor Ala67Thr genotype was found in 13.3% of the patients with paresis and in 8.5% of the controls (Odds Ratio = 1.667). The frequency of Ala67Thr among the controls is in agreement with earlier published data. It is concluded that the Ala67Thr mutation in the poliovirus receptor is a possible risk factor for the development of vaccine-associated or paralytic poliomyelitis associated with wild-type virus.

  17. Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice

    Directory of Open Access Journals (Sweden)

    Federico Brandalise

    2017-01-01

    Full Text Available Hericium erinaceus (Bull. Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

  18. Comparative transcriptome of wild type and selected strains of the microalgae Tisochrysis lutea provides insights into the genetic basis, lipid metabolism and the life cycle.

    Science.gov (United States)

    Carrier, Gregory; Garnier, Matthieu; Le Cunff, Loïc; Bougaran, Gaël; Probert, Ian; De Vargas, Colomban; Corre, Erwan; Cadoret, Jean-Paul; Saint-Jean, Bruno

    2014-01-01

    The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea.

  19. Comparative transcriptome of wild type and selected strains of the microalgae Tisochrysis lutea provides insights into the genetic basis, lipid metabolism and the life cycle.

    Directory of Open Access Journals (Sweden)

    Gregory Carrier

    Full Text Available The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso, we took advantage of the availability of wild type (Tiso-Wt and selected (Tiso-S2M2 strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel. We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea.

  20. Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice.

    Science.gov (United States)

    Brandalise, Federico; Cesaroni, Valentina; Gregori, Andrej; Repetti, Margherita; Romano, Chiara; Orrù, Germano; Botta, Laura; Girometta, Carolina; Guglielminetti, Maria Lidia; Savino, Elena; Rossi, Paola

    2017-01-01

    Hericium erinaceus (Bull.) Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

  1. The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles.

    Science.gov (United States)

    Zagrobelny, Mika; Bak, Søren; Ekstrøm, Claus Thorn; Olsen, Carl Erik; Møller, Birger Lindberg

    2007-01-01

    Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.

  2. MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

    Science.gov (United States)

    Fei, Bing-Yuan; Wang, Xiu-Ying; Fang, Xue-Dong

    2016-05-01

    Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

  3. Comparisons of ectomycorrhizal colonization of transgenic american chestnut with those of the wild type, a conventionally bred hybrid, and related fagaceae species.

    Science.gov (United States)

    D'Amico, Katherine M; Horton, Thomas R; Maynard, Charles A; Stehman, Stephen V; Oakes, Allison D; Powell, William A

    2015-01-01

    American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.

  4. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    Science.gov (United States)

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

  5. Wild-Type MIC Distributions and Epidemiological Cutoff Values for Amphotericin B and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)▿

    Science.gov (United States)

    Espinel-Ingroff, A.; Cuenca-Estrella, M.; Fothergill, A.; Fuller, J.; Ghannoum, M.; Johnson, E.; Pelaez, T.; Pfaller, M. A.; Turnidge, J.

    2011-01-01

    Although clinical breakpoints have not been established for mold testing, epidemiological cutoff values (ECVs) are available for Aspergillus spp. versus the triazoles and caspofungin. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillus spp. and amphotericin B. Two sets (CLSI/EUCAST broth microdilution) of available MICs were evaluated: those for A. fumigatus (3,988/833), A. flavus (793/194), A. nidulans (184/69), A. niger (673/140), A. terreus (545/266), and A. versicolor (135/22). Three sets of data were analyzed: (i) CLSI data gathered in eight independent laboratories in Canada, Europe, and the United States; (ii) EUCAST data from a single laboratory; and (iii) the combined CLSI and EUCAST data. ECVs, expressed in μg/ml, that captured 95%, 97.5%, and 99% of the modeled wild-type population (CLSI and combined data) were as follows: for A. fumigatus, 2, 2, and 4; for A. flavus, 2, 4, and 4; for A. nidulans, 4, 4, and 4; for A. niger, 2, 2, and 2; for A. terreus, 4, 4, and 8; and for A. versicolor, 2, 2, and 2. Similar to the case for the triazoles and caspofungin, amphotericin B ECVs may aid in the detection of strains with acquired mechanisms of resistance to this agent. PMID:21876047

  6. Wild-Type MIC Distributions and Epidemiological Cutoff Values for Caspofungin and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)▿

    Science.gov (United States)

    Espinel-Ingroff, A.; Fothergill, A.; Fuller, J.; Johnson, E.; Pelaez, T.; Turnidge, J.

    2011-01-01

    Clinical breakpoints have not been established for mold testing. Epidemiologic cutoff values (ECVs) are available for six Aspergillus spp. and the triazoles, but not for caspofungin. Wild-type (WT) minimal effective concentration (MEC) distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillus spp. and caspofungin. The number of available isolates was as follows: 1,691 A. fumigatus, 432 A. flavus, 192 A. nidulans, 440 A. niger, 385 A. terreus, and 75 A. versicolor isolates. CLSI broth microdilution MEC data gathered in five independent laboratories in Canada, Europe, and the United States were aggregated for the analyses. ECVs expressed in μg/ml that captured 95% and 99% of the modeled wild-type population were for A. fumigatus 0.5 and 1, A. flavus 0.25 and 0.5, A. nidulans 0.5 and 0.5, A. niger 0.25 and 0.25, A. terreus 0.25 and 0.5, and A. versicolor 0.25 and 0.5. Although caspofungin ECVs are not designed to predict the outcome of therapy, they may aid in the detection of strains with reduced antifungal susceptibility to this agent and acquired resistance mechanisms. PMID:21422219

  7. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B and Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document).

    Science.gov (United States)

    Espinel-Ingroff, A; Cuenca-Estrella, M; Fothergill, A; Fuller, J; Ghannoum, M; Johnson, E; Pelaez, T; Pfaller, M A; Turnidge, J

    2011-11-01

    Although clinical breakpoints have not been established for mold testing, epidemiological cutoff values (ECVs) are available for Aspergillus spp. versus the triazoles and caspofungin. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillus spp. and amphotericin B. Two sets (CLSI/EUCAST broth microdilution) of available MICs were evaluated: those for A. fumigatus (3,988/833), A. flavus (793/194), A. nidulans (184/69), A. niger (673/140), A. terreus (545/266), and A. versicolor (135/22). Three sets of data were analyzed: (i) CLSI data gathered in eight independent laboratories in Canada, Europe, and the United States; (ii) EUCAST data from a single laboratory; and (iii) the combined CLSI and EUCAST data. ECVs, expressed in μg/ml, that captured 95%, 97.5%, and 99% of the modeled wild-type population (CLSI and combined data) were as follows: for A. fumigatus, 2, 2, and 4; for A. flavus, 2, 4, and 4; for A. nidulans, 4, 4, and 4; for A. niger, 2, 2, and 2; for A. terreus, 4, 4, and 8; and for A. versicolor, 2, 2, and 2. Similar to the case for the triazoles and caspofungin, amphotericin B ECVs may aid in the detection of strains with acquired mechanisms of resistance to this agent.

  8. Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex.: Phaffia rhodozyma).

    Science.gov (United States)

    Lodato, Patricia; Alcaíno, Jennifer; Barahona, Salvador; Retamales, Patricio; Jiménez, Antonio; Cifuentes, Víctor

    2004-01-01

    The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

  9. Alteration of leaf metabolism in Bt-transgenic rice (Oryza sativa L.) and its wild type under insecticide stress.

    Science.gov (United States)

    Zhou, Jia; Zhang, Lei; Chang, Yuwei; Lu, Xin; Zhu, Zhen; Xu, Guowang

    2012-08-03

    Insecticide is always used to control the damage from pests, while the potential influence on plants is rarely known. Time-course metabolic changes of wild and Bacillus thuringiensis (Bt) transgenic rice (Oryza sativa L.) plants after insecticide treatment were investigated by using gas chromatography-mass spectrometry (GC-MS). A combined statistical strategy of 2-way ANOVA and multivariate analyses (principal component analysis and hierarchal cluster analysis) was performed to find the stress-associated effects. The results reveal that a wide range of metabolites were dynamically varied in both varieties as a response to insecticide, in multiple metabolic pathways, such as biosynthesis and metabolism of amino acids, carbohydrates, fatty acids, TCA cycle, and the shikimate/phenylpropanoid pathway, and most of the changes were correlated with the exposure time and dependent on the variety. A set of stress defenses were activated, including phytohormone signaling pathway, antioxidant defense system, shikimate-mediated secondary metabolism, and so on. In particular, insecticide led to much stronger regulations of signaling molecules (salicylate and the precursor of jasmonate) and antioxidants (α-tocopherol and dehydroascorbate/ascorbate) in Bt-transgenic variety at the early stage. Our results demonstrated that the Bt-transgenic rice had a more acute and drastic response to insecticide stress than its non-transgenic counterpart in antioxidant system and signaling regulation.

  10. Inactivated Recombinant Rabies Viruses Displaying the Canine Distemper Virus Glycoproteins Induce Protective Immunity Against Both Pathogens.

    Science.gov (United States)

    Budaszewski, Renata da Fontoura; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Xiangping, Yin; Schnell, Matthias J; von Messling, Veronika

    2017-02-01

    The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Canine distemper (CDV) and rabies (RABV) viruses both cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wild life species. In the current study, we have developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near wild type titers and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were only immunized with a RABV expressing the attachment protein of the CDV vaccine strain Onderstepoort succumbed to the infection with a more recent wild type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.

  11. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Science.gov (United States)

    2010-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  12. Long-term effectiveness of canine-to-canine bonded flexible spiral wire lingual retainers

    NARCIS (Netherlands)

    Renkema, Anne-Marie; Renkema, Alianne; Bronkhorst, Ewald; Katsaros, Christos

    2011-01-01

    Introduction: The flexible spiral wire (FSW) canine-to-canine lingual retainer bonded to all 6 anterior teeth is a frequently used type of mandibular fixed retainer. This study aimed to assess the long-term effectiveness of FSW canine-to-canine lingual retainers in maintaining the alignment of the m

  13. Long-term effectiveness of canine-to-canine bonded flexible spiral wire lingual retainers

    NARCIS (Netherlands)

    Renkema, A.M.; Bronkhorst, E.M.; Katsaros, C.

    2011-01-01

    INTRODUCTION: The flexible spiral wire (FSW) canine-to-canine lingual retainer bonded to all 6 anterior teeth is a frequently used type of mandibular fixed retainer. This study aimed to assess the long-term effectiveness of FSW canine-to-canine lingual retainers in maintaining the alignment of the m

  14. A comparative study of age-related hearing loss in wild type and insulin-like growth factor I deficient mice

    Directory of Open Access Journals (Sweden)

    Raquel Riquelme

    2010-06-01

    Full Text Available Insulin-like growth factor-I (IGF-I belongs to the family of insulin-related peptides that fulfils a key role during the late development of the nervous system. Human IGF1 mutations cause profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis (age-related hearing loss is a common disorder associated with aging that causes social and cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this reduction has been related to cognitive and brain alterations, although there is no information as yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture, insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive and non-invasive techniques were used, including auditory brainstem-evoked response (ABR recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly, there was an age-related spiral ganglion degeneration in wild type mice that was not evident in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required for the correct development and maintenance of hearing, supporting the idea that IGF-I-based therapies could contribute to

  15. A Comparative Study of Age-Related Hearing Loss in Wild Type and Insulin-Like Growth Factor I Deficient Mice

    Science.gov (United States)

    Riquelme, Raquel; Cediel, Rafael; Contreras, Julio; Lourdes, Rodriguez-de la Rosa; Murillo-Cuesta, Silvia; Hernandez-Sanchez, Catalina; Zubeldia, Jose M.; Cerdan, Sebastian; Varela-Nieto, Isabel

    2010-01-01

    Insulin-like growth factor-I (IGF-I) belongs to the family of insulin-related peptides that fulfils a key role during the late development of the nervous system. Human IGF1 mutations cause profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis (age-related hearing loss) is a common disorder associated with aging that causes social and cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this reduction has been related to cognitive and brain alterations, although there is no information as yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture, insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive and non-invasive techniques were used, including auditory brainstem-evoked response (ABR) recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly, there was an age-related spiral ganglion degeneration in wild type mice that was not evident in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required for the correct development and maintenance of hearing, supporting the idea that IGF-I-based therapies could contribute to prevent or

  16. Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild-type and BK5.AktWT mice.

    Science.gov (United States)

    Rho, Okkyung; Kiguchi, Kaoru; Jiang, Guiyu; DiGiovanni, John

    2014-11-01

    In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development.

  17. Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N α2A-adrenoceptor transgenic mice

    Science.gov (United States)

    Zhu, Q-M; Lesnick, J D; Jasper, J R; MacLennan, S J; Dillon, M P; Eglen, R M; Blue, D R

    1999-01-01

    We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N α2A-adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) α2-adrenoceptors and at endogenous (dog) α2A-adrenoceptors. In wild-type mice, rilmenidine, moxonidine (100, 300 and 1000 μg kg−1, i.v.) and clonidine (30, 100 and 300 μg kg−1, i.v.) dose-dependently decreased blood pressure and heart rate. In D79N α2A-adrenoceptor mice, responses to rilmenidine and moxonidine did not differ from vehicle control. Clonidine-induced hypotension was absent, but dose-dependent hypertension and bradycardia were observed. In wild-type mice, responses to moxonidine (1 mg kg−1, i.v.) were antagonized by the non-selective, non-imidazoline α2-adrenoceptor antagonist, RS-79948-197 (1 mg kg−1, i.v.). Affinity estimates (pKi) at human α2A-, α2B- and α2C-adrenoceptors, respectively, were: rilmenidine (5.80, 5.76 and 5.33), moxonidine (5.37, <5 and <5) and clonidine (7.21, 7.16 and 6.87). In a [35S]-GTPγS incorporation assay, moxonidine and clonidine were α2A-adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32). In dog saphenous vein, concentration-dependent contractions were observed (pEC50/intrinsic activity relative to noradrenaline): rilmenidine (5.83/0.70), moxonidine (6.48/0.98) and clonidine (7.22/0.83). Agonist-independent affinities were obtained with RS-79948-197. Thus, expression of α2A-adrenoceptors is a prerequisite for the cardiovascular effects of moxonidine and rilmenidine in conscious mice. There was no evidence of I1-imidazoline receptor-mediated effects. The ability of these compounds to act as α2A-adrenoceptor agonists in vitro supports this conclusion. PMID:10217548

  18. Speckle tracking imaging in evaluation of radial strain of subendocardial and epicardial myocardium in canine acute myocardial ischemia and reperfusion%斑点追踪技术评价犬急性心肌缺血及再灌注心内膜下心肌和心外膜下心肌径向应变

    Institute of Scientific and Technical Information of China (English)

    宋平梅; 任卫东; 马春燕; 王秀芹; 乔伟

    2013-01-01

    Objective To evaluate the radial strain function of subendocardial and epicardial myocardium in acute myocardial ischemia and reperfusion canine with speckle tracking imaging. Methods The first diagonal branch of left coronary artery of 20 adult healthy hybrid dogs were ligated. Radial strain of subendocardial and epicardial myocardium at basal, apical and papillary muscle level were compared by speckle tracking imaging before ligation, ligation immediately, 60,120, 180 min after ligation, reperfusion immediately, and 60, 120 min after reperfusion, respectively. Results Radial strain of basal level elevated by compensatory role in canine acute ischemic process. Radial strain of apical and papillary muscle level decreased obviously. Myocardial reverse movement was even found at apical level. In acute ischemia and reperfusion process, subendocardial myocardium was more sensitive to ischemia. After reperfusion, radial strain of subendocardial and epicardial myocardium at apical and papillary muscle level were still lower than that in basic status. Conclusion Speckle tracking imaging can objectively quantify regional and overall heart function and transmural extent of myocardial infarction in canine.%目的 应用斑点追踪技术检测犬急性心肌缺血及再灌注不同时间点心内膜下心肌和心外膜下心肌的径向应变.方法 选取20只健康成年杂种犬,结扎左冠状动脉第一对角支,分别对结扎前、结扎即刻、60、120和180 min及再灌注即刻、60和120 min基底水平、乳头肌水平和心尖水平心内膜下和心外膜下心肌的径向应变进行比较.结果 在急性缺血过程中,基底水平心肌起代偿作用,其径向应变上升;乳头肌水平和心尖水平心肌下降明显,心尖水平心肌甚至出现反向运动.在急性缺血及再灌注过程中,心内膜下心肌对缺血更加敏感.再灌注后,乳头肌水平和心尖水平心内膜下和心外膜下心肌及跨壁的径向应变

  19. Potent graft-versus-leukemia effect after reduced-intensity allogeneic SCT for intermediate-risk AML with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD.

    Science.gov (United States)

    Labouré, Gaëlle; Dulucq, Stéphanie; Labopin, Myriam; Tabrizi, Reza; Guérin, Estelle; Pigneux, Arnaud; Lafarge, Xavier; Leguay, Thibaut; Bouabdallah, Krimo; Dilhuydy, Marie-Sarah; Duclos, Cédric; Lascaux, Axelle; Marit, Gérald; Mahon, François-Xavier; Boiron, Jean-Michel; Milpied, Noël; Vigouroux, Stéphane

    2012-12-01

    To investigate the role of reduced-intensity allogeneic (RIC-allo) stem cell transplant (SCT) as postremission therapy in adult intermediate-risk patients with acute myelogenous leukemia (AML) with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD, we conducted a single-center retrospective study between January 2001 and December 2010. Sixty-six patients were included: 37 treated with RIC-alloSCT and 29 with nonallogeneic SCT therapies. Both groups were comparable concerning age, WBC count at diagnosis, gender, karyotype, genotype, and number of courses of chemotherapy to reach complete remission (CR1). Median follow-up after CR1 was 37 months (range, 11-112 months) and 48 months (range, 9-83 months) in the allo and no-allo groups, respectively. In the allo versus no-allo groups, the 3-year cumulative incidence of relapse (CIR) rates were 25% ± 8% versus 61% ± 9%; P = .005. The 3-year nonrelapse mortality (NRM), overall survival (OS), and relapse-free survival (RFS) were 22% ± 7% versus 4% ± 4% (P = .005), 52% ± 9% versus 44% ± 10% (P = .75), and 53% ± 9% versus 35% ± 9% (P = .28), respectively. Multivariate analysis indicated that CIR was reduced by allo (hazard ratio [HR], 0.32; P = .01). A landmark analysis performed at day 185 after CR1 confirmed a lower CIR after allo. RIC-allo reduces the risk of relapse, suggesting a potent graft-versus-leukemia (GVL) effect in these patients at a high risk of relapse.

  20. Effect on ultraviolet mutagenesis of genes 32, 41, 44, and 45 alleles of phage T4 in the presence of wild-type or antimutator DNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Mufti, S.

    1980-09-01

    Temperature-sensitive alleles of four genes whose products are essential for DNA elongation in phage T4 were tested for their effects on uv mutagenesis in the presence of an antimutator polymerase allele, tsCB87, or the wild-type polymerase allele. The results suggest that the products of genes 32, 41, 44, and 45, in addition to the gene 43 polymerase, influence the accuracy of the DNA synthesis associated with uv mutagenesis. When considered in combination with previous observations, it appears that thymine dimers in phase T4 DNA may cause mutation through an error-prone repair process which employs a multienzyme system involving at least five components in a replication step, and a final ligation step.

  1. Reversibility of dopamine receptor antagonist-induced hyperprolactinemia and associated histological changes in Tg RasH2 wild-type mice.

    Science.gov (United States)

    Krishna, Gopala; Ganiger, Shivaputhrappa; Kannan, Kamala; Gopalakrishnan, Gopa; Goel, Saryu

    2015-12-01

    The purpose of this study was to better understand the biological effects of increased prolactin levels induced in mice by dopamine D2 receptor antagonist molindone treatment. Toxicokinetics, prolactin levels, and reproductive tissue histology were evaluated in Tg rasH2 wild-type mice treated orally with molindone at 0, 5, 15, and 50mg/kg/day for 6 months, followed by a 2-month posttreatment recovery period. A greater than dose-proportional increase in molindone exposure ([AUC]0‒24) was observed on Day 180 for both sexes. Statistically significant (Phyperprolactinemia, including corpora lutea enlargement and interstitial cell atrophy in the ovaries, and atrophy of the uterus and vagina were observed on Day 180. These changes were reversed during the recovery period in the 5- and 15-mg/kg/day treatment groups. Mice receiving molindone at 50mg/kg/day also showed signs of reversal on histologic examination.

  2. Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor beta-induced protein (TGFBIp).

    Science.gov (United States)

    Runager, Kasper; García-Castellanos, Raquel; Valnickova, Zuzana; Kristensen, Torsten; Nielsen, Niels Chr; Klintworth, Gordon K; Gomis-Rüth, F Xavier; Enghild, Jan J

    2009-03-01

    Transforming growth factor beta-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported.

  3. 犬瘟热的诊治%Diagnosis and Treatment of Canine Distemper

    Institute of Scientific and Technical Information of China (English)

    罗厚强; 段龙川; 王清艳; 涂宜强; 涂国众

    2012-01-01

    Canine distemper is an acute infectious disease caused by the canine distemper virus in Canidae. Many symptoms were characterized by infected dogs, such as biphasic fever, rhinitis, severe inflammation of the digestive tract and respiratory inflammation. The pathogen, epidemiology, clinical symptoms and pathological changes of canine distemper were introduced in the paper. Though a typical case of canine distemper, a series of effective measures to treat canine distemper were introduced in order to provide reference for the control of canine distemper.%犬瘟热是一种由犬瘟热病毒引起的犬科动物急性传染病,病犬以双相热、鼻炎、严重的消化障碍和呼吸道炎症为特征。主要对该病的病原、流行病学、临床症状及病理变化等作一阐述,并以一例犬瘟热典型病例的诊治为例,介绍了治疗该病的有效措施,以期为有效防治犬瘟热提供参考。

  4. Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

    Science.gov (United States)

    Fiorentino, Maria; Levine, Myron M; Sztein, Marcelo B; Fasano, Alessio

    2014-01-01

    Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to

  5. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Science.gov (United States)

    Dvorak-Ewell, Melita; Wendt, Dan; Hague, Chuck; Christianson, Terri; Koppaka, Vish; Crippen, Danielle; Kakkis, Emil; Vellard, Michel

    2010-08-16

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg) and pure (>or=97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake) = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

  6. Amplification-free In Situ KRAS Point Mutation Detection at 60 copies/mL in Urine in a Background of 1000-fold Wild Type

    Science.gov (United States)

    KirimLi, Ceyhun E.; Shih, Wei-Heng; Shih, Wan Y.

    2016-01-01

    We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  7. Development of a Rotavirus-Shedding Model in Rhesus Macaques, Using a Homologous Wild-Type Rotavirus of a New P Genotype

    Science.gov (United States)

    McNeal, Monica M.; Sestak, Karol; Choi, Anthony H.-C.; Basu, Mitali; Cole, Michael J.; Aye, Pyone P.; Bohm, Rudolf P.; Ward, Richard L.

    2005-01-01

    Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection. PMID:15613323

  8. Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease.

    Directory of Open Access Journals (Sweden)

    Franklin R Toapanta

    2016-06-01

    Full Text Available A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD- 6-9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ≥38°C for ≥12h and/or microbiological (S. Typhi bacteremia endpoints. Changes in B cell subpopulations following S. Typhi challenge remain undefined. To address this issue, a subset of volunteers (6 TD and 4 who did not develop TD -NoTD- was evaluated. Notable changes included reduction in the frequency of B cells (cells/ml of TD volunteers during disease days and increase in plasmablasts (PB during the recovery phase (>day 14. Additionally, a portion of PB of TD volunteers showed a significant increase in activation (CD40, CD21 and gut homing (integrin α4β7 molecules. Furthermore, all BM subsets of TD volunteers showed changes induced by S. Typhi infections such as a decrease in CD21 in switched memory (Sm CD27+ and Sm CD27- cells as well as upregulation of CD40 in unswitched memory (Um and Naïve cells. Furthermore, changes in the signaling profile of some BM subsets were identified after S. Typhi-LPS stimulation around time of disease. Notably, naïve cells of TD (compared to NoTD volunteers showed a higher percentage of cells phosphorylating Akt suggesting enhanced survival of these cells. Interestingly, most these changes were temporally associated with disease onset. This is the first study to describe differences in B cell subsets directly related to clinical outcome following oral challenge with wild-type S. Typhi in humans.

  9. Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease

    Science.gov (United States)

    Toapanta, Franklin R.; Bernal, Paula J.; Fresnay, Stephanie; Magder, Laurence S.; Darton, Thomas C.; Jones, Claire; Waddington, Claire S.; Blohmke, Christoph J.; Angus, Brian; Levine, Myron M.; Pollard, Andrew J.; Sztein, Marcelo B.

    2016-01-01

    A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD-) 6–9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ≥38°C for ≥12h) and/or microbiological (S. Typhi bacteremia) endpoints. Changes in B cell subpopulations following S. Typhi challenge remain undefined. To address this issue, a subset of volunteers (6 TD and 4 who did not develop TD -NoTD-) was evaluated. Notable changes included reduction in the frequency of B cells (cells/ml) of TD volunteers during disease days and increase in plasmablasts (PB) during the recovery phase (>day 14). Additionally, a portion of PB of TD volunteers showed a significant increase in activation (CD40, CD21) and gut homing (integrin α4β7) molecules. Furthermore, all BM subsets of TD volunteers showed changes induced by S. Typhi infections such as a decrease in CD21 in switched memory (Sm) CD27+ and Sm CD27- cells as well as upregulation of CD40 in unswitched memory (Um) and Naïve cells. Furthermore, changes in the signaling profile of some BM subsets were identified after S. Typhi-LPS stimulation around time of disease. Notably, naïve cells of TD (compared to NoTD) volunteers showed a higher percentage of cells phosphorylating Akt suggesting enhanced survival of these cells. Interestingly, most these changes were temporally associated with disease onset. This is the first study to describe differences in B cell subsets directly related to clinical outcome following oral challenge with wild-type S. Typhi in humans. PMID:27300136

  10. A Tool for Investigating Asthma and COPD Exacerbations: A Newly Manufactured and Well Characterised GMP Wild-Type Human Rhinovirus for Use in the Human Viral Challenge Model

    Science.gov (United States)

    Fullen, Daniel J.; Murray, Bryan; Mori, Julie; Catchpole, Andrew; Borley, Daryl W.; Murray, Edward J.; Balaratnam, Ganesh; Gilbert, Anthony; Mann, Alex; Hughes, Fiona; Lambkin-Williams, Rob

    2016-01-01

    Background Human Rhinovirus infection is an important precursor to asthma and chronic obstructive pulmonary disease exacerbations and the Human Viral Challenge model may provide a powerful tool in studying these and other chronic respiratory diseases. In this study we have reported the production and human characterisation of a new Wild-Type HRV-16 challenge virus produced specifically for this purpose. Methods and Stock Development A HRV-16 isolate from an 18 year old experimentally infected healthy female volunteer (University of Virginia Children’s Hospital, USA) was obtained with appropriate medical history and consent. We manufactured a new HRV-16 stock by minimal passage in a WI-38 cell line under Good Manufacturing Practice conditions. Having first subjected the stock to rigorous adventitious agent testing and determining the virus suitability for human use, we conducted an initial safety and pathogenicity clinical study in adult volunteers in our dedicated clinical quarantine facility in London. Human Challenge and Conclusions In this study we have demonstrated the new Wild-Type HRV-16 Challenge Virus to be both safe and pathogenic, causing an appropriate level of disease in experimentally inoculated healthy adult volunteers. Furthermore, by inoculating volunteers with a range of different inoculum titres, we have established the minimum inoculum titre required to achieve reproducible disease. We have demonstrated that although inoculation titres as low as 1 TCID50 can produce relatively high infection rates, the optimal titre for progression with future HRV challenge model development with this virus stock was 10 TCID50. Studies currently underway are evaluating the use of this virus as a challenge agent in asthmatics. Trial Registration ClinicalTrials.gov NCT02522832 PMID:27936016

  11. Estimation of the receptor-state affinity constants of ligands in functional studies using wild type and constitutively active mutant receptors: Implications for estimation of agonist bias.

    Science.gov (United States)

    Ehlert, Frederick J; Stein, Richard S L

    We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.

  12. The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2014-02-01

    Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

  13. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

    Science.gov (United States)

    Xie, Changyan; Cao, Xu; Chen, Xibing; Wang, Dong; Zhang, Wei Kevin; Sun, Ying; Hu, Wenbao; Zhou, Zijing; Wang, Yan; Huang, Pingbo

    2016-04-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

  14. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Liu, J; Lombardo, M; Bolstad, D; Wright, D; Anderson, A

    2009-01-01

    Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.

  15. Synthesis, 2D-NMR and molecular modelling studies of pentacycloundecane lactam-peptides and peptoids as potential HIV-1 wild type C-SA protease inhibitors.

    Science.gov (United States)

    Makatini, Maya M; Petzold, Katja; Alves, Cláudio Nahum; Arvidsson, Per I; Honarparvar, Bahareh; Govender, Patrick; Govender, Thavendran; Kruger, Hendrik G; Sayed, Yasien; JerônimoLameira; Maguire, Glenn E M; Soliman, Mahmoud E S

    2013-02-01

    In this study, eight non-natural peptides and peptoids incorporating the pentacycloundecane (PCU) lactam were designed and synthesized as potential inhibitors of the wild type C-SA HIV-protease. Five of these inhibitors gave IC(50) values ranging from 0.5 up to 0.75 µM against the resistance-prone wild type C-South African HIV-protease. NMR EASY-ROESY studies enabled us to describe the secondary structure of three of these compounds in solution. The 3D structures of the selected cage peptides were also modelled in solution using QM/MM/MD simulations. Satisfactory agreement between the NMR observations and the low energy calculated structures exists. Only one of these inhibitors (11 peptoid), which showed the best IC(50)(0.5 µM), exhibited a definable 3-D structure in solution. Autodock4 and AutodockVina were used to model the potential interaction between these inhibitors and the HIV-PR. It appears that the docking results are too crude to be correlated with the relative narrow range of experimental IC(50) values (0.5-10 µM). The PCU-peptides and peptoides were several orders less toxic (145 μM for 11 and 102 μM for 11 peptoid) to human MT-4 cells than lopinavir (0.025 μM). This is the first example of a polycyclic cage framework to be employed as an HIV-PR transition state analogue inhibitor and can potentially be utilized for other diseases related proteases. [Figure: see text].

  16. Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC lines from Rett syndrome patients as in vitro disease model.

    Directory of Open Access Journals (Sweden)

    Gene Ananiev

    Full Text Available Rett syndrome (RTT is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2 gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC, we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon is a common mutation carried by 5-6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.

  17. Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

    Directory of Open Access Journals (Sweden)

    Maria Fiorentino

    Full Text Available Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa and might contribute (along with enterotoxins to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them

  18. Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

    Science.gov (United States)

    Laulumaa, Saara; Nieminen, Tuomo; Lehtimäki, Mari; Aggarwal, Shweta; Simons, Mikael; Koza, Michael M; Vattulainen, Ilpo; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

  19. Comparison of wild-type and UV-mutant beta-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications.

    Science.gov (United States)

    McCarthy, Tracey C; Lalor, Eoin; Hanniffy, Orla; Savage, Angela V; Tuohy, Maria G

    2005-04-01

    A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal beta-glucans. Activity against beta-(1, 3)(1, 4)-D: -glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less beta-glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley beta-glucan (13.0-16.9%), but were more active against crude beta-glucan from barley (16.0-24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash beta-glucan. Finally, TC2 and TC5 produce more efficient beta-glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.

  20. MicroRNA expression in BRAF-mutated and wild-type metastatic melanoma and its correlation with response duration to BRAF inhibitors.

    Science.gov (United States)

    Pinto, Rosamaria; Strippoli, Sabino; De Summa, Simona; Albano, Anna; Azzariti, Amalia; Guida, Gabriella; Popescu, Ondina; Lorusso, Vito; Guida, Michele; Tommasi, Stefania

    2015-01-01

    Objective: Currently, the treatment of BRAF V600-mutated metastatic melanoma with BRAF inhibitors gives a response rate of ~ 50% with a progression-free survival of ~ 6 -- 7 months. In order to identify predictive biomarkers capable of stratifying BRAF-mutated patients at high risk of shorter response duration to anti-BRAF therapy, the authors analyzed the expression of 15 microRNAs (miRNAs) targeting crucial genes involved in melanoma biology and drug response.Research design and methods: A total of 15 miRNAs and target gene expression were investigated in 43 patients (30 BRAF-mutated, and 13 BRAF wild-type). Moreover, 20 BRAF-mutated patients treated with vemurafenib were analyzed for miRNA expression in respect to time-to-progression.Results: All miRNAs except miR-192 showed low expression in BRAF-mutated as compared with BRAF wild-type patients. In particular, miR-101, miR-221,miR-21, miR-338-3p and miR-191 resulted in significant downregulation inBRAF-mutated patients. Moreover, high expression of miR-192 and miR-193b* and low expression of miR-132 resulted in significant association with shorter progression.Conclusion: Three miRNAs were significantly associated with clinical outcome in metastatic melanoma patients. An increased understanding of the molecular assessment of BRAF-mutated melanomas could allow development of specific molecular tests able to predict response duration.

  1. MicroRNA profiling predicts survival in anti-EGFR treated chemorefractory metastatic colorectal cancer patients with wild-type KRAS and BRAF.

    Science.gov (United States)

    Mosakhani, Neda; Lahti, Leo; Borze, Ioana; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Jari; Ristamäki, Raija; Osterlund, Pia; Knuutila, Sakari; Sarhadi, Virinder Kaur

    2012-11-01

    Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers.

  2. Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant.

    Directory of Open Access Journals (Sweden)

    Khaled Barakat

    Full Text Available BACKGROUND: The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298-306 K. METHODOLOGY/PRINCIPAL FINDINGS: This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately. CONCLUSIONS: The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.

  3. Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.

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    Khalid Arhzaouy

    Full Text Available Heterozygous mutations in the human VCP (p97 gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia, ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia and HSP (hereditary spastic paraplegia. Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97 or mutant p97 (p97(R155C fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO cells. Native gel electrophoresis showed that both p97 and p97(R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3 indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO/p97(R155C-RFP and ATG9(KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO mutant. A major finding is that the expression of p97(R155C-RFP in the ATG9(KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.

  4. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Mezard, C.; Nicolas, A. [Universite Paris-Sud, Orsay (France)

    1994-02-01

    Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions; (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product. 67 refs., 4 figs., 5 tabs.

  5. Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease.

    Science.gov (United States)

    Toapanta, Franklin R; Bernal, Paula J; Fresnay, Stephanie; Darton, Thomas C; Jones, Claire; Waddington, Claire S; Blohmke, Christoph J; Dougan, Gordon; Angus, Brian; Levine, Myron M; Pollard, Andrew J; Sztein, Marcelo B

    2015-06-01

    A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD-) 5-10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12 h) and/or microbiological (S. Typhi bacteremia) endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e.g., monocytes, dendritic cells -DCs-). Various changes in circulating monocytes and DCs have been described in the murine S. Typhimurium model; however, whether similar changes are present in humans remains to be explored. To address these questions, a subset of volunteers (5 TD and 3 who did not develop typhoid despite oral challenge -NoTD-) were evaluated for changes in circulating monocytes and DCs. Expression of CD38 and CD40 were upregulated in monocytes and DCs in TD volunteers during the disease days (TD-0h to TD-96h). Moreover, integrin α4β7, a gut homing molecule, was upregulated on monocytes but not DCs. CD21 upregulation was only identified in DCs. These changes were not observed among NoTD volunteers despite the same oral challenge. Moreover, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi one day after challenge. These monocytes showed phosphorylation of p38MAPK, NFkB and Erk1/2 upon stimulation with S. Typhi-LPS-QDot micelles. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48 h and 96 h post-TD, and only Erk1/2 phosphorylation. This is the first study to describe different activation and migration profiles, as well as differential signaling patterns, in monocytes and DCs which relate directly to the clinical outcome following oral challenge with wild type S. Typhi.

  6. Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease.

    Science.gov (United States)

    Toapanta, Franklin R; Bernal, Paula J; Fresnay, Stephanie; Magder, Laurence S; Darton, Thomas C; Jones, Claire; Waddington, Claire S; Blohmke, Christoph J; Angus, Brian; Levine, Myron M; Pollard, Andrew J; Sztein, Marcelo B

    2016-06-01

    A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD-) 6-9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ≥38°C for ≥12h) and/or microbiological (S. Typhi bacteremia) endpoints. Changes in B cell subpopulations following S. Typhi challenge remain undefined. To address this issue, a subset of volunteers (6 TD and 4 who did not develop TD -NoTD-) was evaluated. Notable changes included reduction in the frequency of B cells (cells/ml) of TD volunteers during disease days and increase in plasmablasts (PB) during the recovery phase (>day 14). Additionally, a portion of PB of TD volunteers showed a significant increase in activation (CD40, CD21) and gut homing (integrin α4β7) molecules. Furthermore, all BM subsets of TD volunteers showed changes induced by S. Typhi infections such as a decrease in CD21 in switched memory (Sm) CD27+ and Sm CD27- cells as well as upregulation of CD40 in unswitched memory (Um) and Naïve cells. Furthermore, changes in the signaling profile of some BM subsets were identified after S. Typhi-LPS stimulation around time of disease. Notably, naïve cells of TD (compared to NoTD) volunteers showed a higher percentage of cells phosphorylating Akt suggesting enhanced survival of these cells. Interestingly, most these changes were temporally associated with disease onset. This is the first study to describe differences in B cell subsets directly related to clinical outcome following oral challenge with wild-type S. Typhi in humans.

  7. Hepatitis B virus genotype C isolates with wild-type core promoter sequence replicate less efficiently than genotype B isolates but possess higher virion secretion capacity.

    Science.gov (United States)

    Qin, Yanli; Tang, Xiaoli; Garcia, Tamako; Hussain, Munira; Zhang, Jiming; Lok, Anna; Wands, Jack; Li, Jisu; Tong, Shuping

    2011-10-01

    Infection by hepatitis B virus (HBV) genotype C is associated with a prolonged viremic phase, delayed hepatitis B e antigen (HBeAg) seroconversion, and an increased incidence of liver cirrhosis and hepatocellular carcinoma compared with genotype B infection. Genotype C is also associated with the more frequent emergence of core promoter mutations, which increase genome replication and are independently associated with poor clinical outcomes. We amplified full-length HBV genomes from serum samples from Chinese and U. S. patients with chronic HBV infection and transfected circularized genome pools or dimeric constructs of individual clones into Huh7 cells. The two genotypes could be differentiated by Western blot analysis due to the reactivities of M and L proteins toward a monoclonal pre-S2 antibody and slightly different S-protein mobilities. Great variability in replication capacity was observed for both genotypes. The A1762T/G1764A core promoter mutations were prevalent in genotype C isolates and correlated with increased replication capacity, while the A1752G/T mutation frequently found in genotype B isolates correlated with a low replication capacity. Importantly, most genotype C isolates with wild-type core promoter sequence replicated less efficiently than the corresponding genotype B isolates due to less efficient transcription of the 3.5-kb RNA. However, genotype C isolates often displayed more efficient virion secretion. We propose that the low intracellular levels of viral DNA and core protein of wild-type genotype C delay immune clearance and trigger the subsequent emergence of A1762T/G1764A core promoter mutations to upregulate replication; efficient virion secretion compensates for the low replication capacity to ensure the establishment of persistent infection by genotype C.

  8. Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodeling by p53 aggregation.

    Science.gov (United States)

    De Smet, Frederik; Saiz Rubio, Mirian; Hompes, Daphne; Naus, Evelyne; De Baets, Greet; Langenberg, Tobias; Hipp, Mark S; Houben, Bert; Claes, Filip; Charbonneau, Sarah; Blanco, Javier Delgado; Plaisance, Stephane; Ramkissoon, Shakti; Ramkissoon, Lori; Simons, Colinda; van den Brandt, Piet; Weijenberg, Matty; Van England, Manon; Lambrechts, Sandrina; Amant, Frederic; D'Hoore, André; Ligon, Keith L; Sagaert, Xavier; Schymkowitz, Joost; Rousseau, Frederic

    2016-12-30

    Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nuclear inclusion body formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines for which nIB formation correlated to the loss of p53s transcriptional activity. Importantly, protein aggregation also fueled the dysregulation of the proteostasis network in the tumour cell by inducing a hyper-activated, oncogenic heat-shock response to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients exhibiting tumours with p53-positive nIBs suffered from a poor clinical outcome similar to loss-of-p53-expression, and tumour biopsies displayed a differential proteostatic expression profile associated to p53-nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (i) the functional inactivation of p53 through mutation and/or aggregation and (ii) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration.

  9. The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor

    Directory of Open Access Journals (Sweden)

    Xiaobing Liang

    2011-01-01

    Full Text Available The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742 increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy.

  10. The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor.

    Science.gov (United States)

    Liang, Xiaobing; Guo, Yi; Figg, William Douglas; Fojo, Antonio Tito; Mueller, Michael D; Yu, Jing Jie

    2011-01-01

    The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient) cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742) increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy.

  11. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Directory of Open Access Journals (Sweden)

    Melita Dvorak-Ewell

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS, an enzyme that degrades keratan sulfate (KS. Currently no therapy for MPS IVA is available. We produced recombinant human (rhGALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg and pure (>or=97% rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake = 2.5 nM, thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for

  12. The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type

    Science.gov (United States)

    Ceruso, Marina; Fratamico, Pina; Chirollo, Claudia; Taglialatela, Rosanna; Cortesi, Maria Luisa

    2014-01-01

    Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment. PMID:27800311

  13. Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

    Science.gov (United States)

    Johnen, G; Kowlessur, D; Citron, B A; Kaufman, S

    1995-01-01

    The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels. PMID:8618906

  14. Expanded and Wild-type Ataxin-3 Modify the Redox Status of SH-SY5Y Cells Overexpressing α-Synuclein.

    Science.gov (United States)

    Noronha, Carolina; Perfeito, Rita; Laço, Mário; Wüllner, Ullrich; Rego, A Cristina

    2017-02-25

    Neurodegenerative diseases are considered to be distinct clinical entities, although they share the formation of proteinaceous aggregates and several neuropathological mechanisms. Increasing evidence suggest a possible interaction between proteins that have been classically associated to distinct neurodegenerative diseases. Thus, common molecular and cellular pathways might explain similarities between disease phenotypes. Interestingly, the characteristic Parkinson's disease (PD) phenotype linked to bradykinesia is also a clinical presentation of other neurodegenerative diseases. An example is Machado-Joseph disease (MJD), with some patients presenting parkinsonism and a positive response to levodopa (L-DOPA). Protein aggregates positive for α-synuclein (α-Syn), a protein associated with PD, in the substantia nigra of MJD models made us hypothesize a putative additive biological effect induced by expression of α-Syn and ataxin-3 (Atx3), the protein affected in MJD. Hence, in this study we analysed the influence of these two proteins (α-Syn and wild-type or mutant Atx3) on modified redox signaling, a pathological process potentially linked to both diseases, and also the impact of exposure to iron and rotenone in SH-SY5Y neuroblastoma cells. Our results show that both α-Syn and mutant Atx3 overexpression per se increased oxidation of dichlorodihydrofluorescein (DCFH2), and co-expression of these proteins exhibited additive effect on intracellular oxidation, with no correlation with apoptotic features. Mutant Atx3 and α-Syn also potentiated altered redox status induced by iron and rotenone, a hint to how these proteins might influence neuronal dysfunction under pro-oxidant conditions. We further show that overexpression of wild-type Atx3 decreased intracellular DCFH2 oxidation, possibly exerting a neuroprotective role.

  15. A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid.

    Science.gov (United States)

    Alves, Cláudio Nahum; Martí, Sergio; Castillo, Raquel; Andrés, Juan; Moliner, Vicent; Tuñón, Iñaki; Silla, Estanislao

    2008-04-01

    Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg(2+) cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg(2+) cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.

  16. Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy.

    Science.gov (United States)

    Müller, Pavel; Brettel, Klaus; Grama, Laszlo; Nyitrai, Miklos; Lukacs, Andras

    2016-05-04

    DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH(-) (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD(.-) and FADH(.) formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD(.-) (formed by ET from the tryptophan chain) in plant CRYs in about 1.5 μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 μs into a state with spectral features that are different from both FADH(.) and FAD(.-) . Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

  17. Variations in the mycorrhization characteristics in roots of wild-type and ABA-deficient tomato are accompanied by specific transcriptomic alterations.

    Science.gov (United States)

    Garrido, José Manuel García; Morcillo, Rafael Jorge León; Rodríguez, José Angel Martín; Bote, Juan Antonio Ocampo

    2010-05-01

    Abscissic acid (ABA) determines mycorrhiza functionality and arbuscule development. In this study, we performed transcriptome analysis in response to different mycorrhization status according to the ABA content in the root to identify genes that may play a role in arbuscule functionality. Affymetrix Tomato GeneChip (approximately 10,000 probes) allowed us to detect and compare the transcriptional root profiling of tomato (Solanum lycopersicum) wild-type and ABA-deficient sitiens plants colonized by Glomus intraradices. A number of identified genes in tomato belong to a category of genes already described as "mycorrhizal core-set" in other host plants. The impairment in arbuscular mycorrhiza (AM) formation in ABA-deficient mutants was associated with upregulation of genes related to defense and cell wall modification, whereas functional mycorrhization in wild-type plants was associated with activation of genes related to isoprenoid metabolism. The oxylipin pathway was activated in tomato mycorrhizal roots at late stages of interaction, and was related to the control of fungal spread in roots, not with the establishment of the symbiosis. Induction of selected genes, representing a range of biological functions and representative of the three sets of genes specifically upregulated in the different plant phenotype, was confirmed by quantitative reverse-transcription polymerase chain reaction, and their response to phythohormone treatment was tested, showing that ethylene and jasmonic acid are key regulators of gene expression during AM development. Comparative analysis of mycorrhiza upregulated functional categories revealed significant changes in gene expression associated with the different mycorrhization status according to the ABA content in the roots.

  18. Podoplanin Expression in Canine Melanoma.

    Science.gov (United States)

    Ogasawara, Satoshi; Honma, Ryusuke; Kaneko, Mika K; Fujii, Yuki; Kagawa, Yumiko; Konnai, Satoru; Kato, Yukinari

    2016-12-01

    A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not react with lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma. PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistry. Of interest, PMab-38 stained the lymphatic endothelial cells and cancer-associated fibroblasts in melanoma tissues, althoug