Regulation of homocysteine metabolism and methylation in human and mouse tissues

Science.gov (United States)

Chen, Natalie C.; Yang, Fan; Capecci, Louis M.; Gu, Ziyu; Schafer, Andrew I.; Durante, William; Yang, Xiao-Feng; Wang, Hong

2010-01-01

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine β-synthase, cystathionine-γ-lyase, paraxonase 1, 5,10-methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyltransferase, phosphatidylethanolamine N-methyltransferases and glycine N-methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl-tRNA synthase, 5-methyltetrahydrofolate:Hcy methyltransferase, S-adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone-lysine N-methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.—Chen, N. C., Yang, F., Capecci, L. M., Gu, Z., Schafer, A. I., Durante, W., Yang, X.-F., Wang, H. Regulation of homocysteine metabolism and methylation in human and mouse tissues. PMID:20305127

Adipose tissue deficiency of hormone-sensitive lipase causes fatty liver in mice.

Science.gov (United States)

Xia, Bo; Cai, Guo He; Yang, Hao; Wang, Shu Pei; Mitchell, Grant A; Wu, Jiang Wei

2017-12-01

Fatty liver is a major health problem worldwide. People with hereditary deficiency of hormone-sensitive lipase (HSL) are reported to develop fatty liver. In this study, systemic and tissue-specific HSL-deficient mice were used as models to explore the underlying mechanism of this association. We found that systemic HSL deficient mice developed fatty liver in an age-dependent fashion between 3 and 8 months of age. To further explore the mechanism of fatty liver in HSL deficiency, liver-specific HSL knockout mice were created. Surprisingly, liver HSL deficiency did not influence liver fat content, suggesting that fatty liver in HSL deficiency is not liver autonomous. Given the importance of adipose tissue in systemic triglyceride metabolism, we created adipose-specific HSL knockout mice and found that adipose HSL deficiency, to a similar extent as systemic HSL deficiency, causes age-dependent fatty liver in mice. Mechanistic study revealed that deficiency of HSL in adipose tissue caused inflammatory macrophage infiltrates, progressive lipodystrophy, abnormal adipokine secretion and systemic insulin resistance. These changes in adipose tissue were associated with a constellation of changes in liver: low levels of fatty acid oxidation, of very low density lipoprotein secretion and of triglyceride hydrolase activity, each favoring the development of hepatic steatosis. In conclusion, HSL-deficient mice revealed a complex interorgan interaction between adipose tissue and liver: the role of HSL in the liver is minimal but adipose tissue deficiency of HSL can cause age-dependent hepatic steatosis. Adipose tissue is a potential target for treating the hepatic steatosis of HSL deficiency.

Triose phosphate isomerase deficiency is caused by altered dimerization--not catalytic inactivity--of the mutant enzymes.

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Markus Ralser

Full Text Available Triosephosphate isomerase (TPI deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations.

Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer

Science.gov (United States)

2012-01-01

Background Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. Purpose To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. Results Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. Conclusions The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million

Kinetic compartmental analysis of carnitine metabolism in the human carnitine deficiency syndromes. Evidence for alterations in tissue carnitine transport

International Nuclear Information System (INIS)

Rebouche, C.J.; Engel, A.G.

1984-01-01

The human primary carnitine deficiency syndromes are potentially fatal disorders affecting children and adults. The molecular etiologies of these syndromes have not been determined. In this investigation, we considered the hypothesis that these syndromes result from defective transport of carnitine into tissues, particularly skeletal muscle. The problem was approached by mathematical modeling, by using the technique of kinetic compartmental analysis. A tracer dose of L-[methyl-3H]carnitine was administered intravenously to six normal subjects, one patient with primary muscle carnitine deficiency (MCD), and four patients with primary systemic carnitine deficiency (SCD). Specific radioactivity was followed in plasma for 28 d. A three-compartment model (extracellular fluid, muscle, and ''other tissues'') was adopted. Rate constants, fluxes, pool sizes, and turnover times were calculated. Results of these calculations indicated reduced transport of carnitine into muscle in both forms of primary carnitine deficiency. However, in SCD, the reduced rate of carnitine transport was attributed to reduced plasma carnitine concentration. In MCD, the results are consistent with an intrinsic defect in the transport process. Abnormal fluctuations of the plasma carnitine, but of a different form, occurred in MCD and SCD. The significance of these are unclear, but in SCD they suggest abnormal regulation of the muscle/plasma carnitine concentration gradient. In 8 of 11 subjects, carnitine excretion was less than dietary carnitine intake. Carnitine excretion rates calculated by kinetic compartmental analysis were higher than corresponding rates measured directly, indicating degradation of carnitine. However, we found no radioactive metabolites of L-[methyl-3H]carnitine in urine. These observations suggest that dietary carnitine was metabolized in the gastrointestinal tract

Biochemical Characterization of Porphobilinogen Deaminase–Deficient Mice During Phenobarbital Induction of Heme Synthesis and the Effect of Enzyme Replacement

Science.gov (United States)

Johansson, Annika; Möller, Christer; Fogh, Jens; Harper, Pauline

2003-01-01

Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-dependent manner with maximal effect seen after 30 min and 2 h, respectively. Injection of rhPBGD subcutaneously twice daily during a 4-d phenobarbital induction reduced urinary PBG excretion to 25% of the levels found in PBGD-deficient mice administered with only phenobarbital. This study points to the liver as the main producer of PBG and ALA in the phenobarbital-induced PBGD-deficient mice and demonstrates efficient removal of accumulated PBG in plasma and urine by enzyme-replacement therapy. PMID:15208740

Kininase enzymes of cat eye tissues

International Nuclear Information System (INIS)

Ryan, J.W.; Anderson, D.R.

1986-01-01

Eye tissues contain kininase activities, including an angiotensin converting enzyme (ACE)-like activity. The authors have begun further to characterize the ACE-like activity and to examine for another reputed kininase, carboxypeptidase N (CPN). Homogenates of tissues of 6 cat eyes and paired plasmas were assayed for ACE using 3 acyl-tripeptide substrates, 3 H-benzoylated F-A-P, F-G-P and A-G-P (respectively, BFAP, BFGP and BAGP). CPN was assayed using 3 H-benzoyl-A-R. All eye tissues and fluids contained ACE- and CPN-like activities. The ACE activity was clearly owing to ACE: relative values of Kc/Km for BFAP, BFGP and BAGP were those for pure ACE (2.213, 1.751 and 1.0); reactivities with inhibitors were as expected (Ki for captopril, MK 422 and RAC-X-65: 2.7, 0.62 and 0.31 nM). EDTA inhibited both ACE and CPN (I 50 's: 43 and 47 μM). CPN activity was inhibited by 2-mercaptomethyl-3-guanidinoethylthiopropionate (Ki 2.4 nM). However, distributions of the two enzymes differed markedly. Virtually all tissues contained ACE at specific activities higher than that of plasma. Specific activities appeared to be a function of tissue vascularity (for choroid, ciliary body, iris, retina and plasma: 7.31, 2.57, 1.98, 1.53 and 0.21 pmol/mg protein). Only iris contained more CPN that did plasma (23.0 v. 7.21 pmol/mg protein). The tissue distribution of ACE is that expected for an endothelial-associated enzyme. Plasma may be the major source of CPN in eye tissues other than iris

Modulation of low dose radiation effect on pentose phosphate pathway enzymes by B-multivitamin deficiency

International Nuclear Information System (INIS)

Zimatkina, T.I.; Lashak, L.K.; Moiseenok, A.G.

1997-01-01

Blood, liver, thymus and spleen of albino rats injected subcutaneously with antivitamins (othythiamine and methotrexate) and subjected to prolonger γ-irradiation in the overall dose of 0.75 Gy were assayed for transketolase and glucose-6-phosphate dehydrogenase after 12h, 1, 2, 5 and 40 days from the last radiation dose. High transketolase sensitivity was found both to radiation (activation) and the combined effects of vitamin deficiency and radiation (potentiation of antivitamin inhibitory action) in all the tissues studied. The activity of glucose-6-phosphate dehydrogenase was little changed under the given experimental manipulations, but the combined effect of the factors considerably inhibited the enzyme activities in the organs of the immune system. Consequently, in B-multivitamin deficiency the effect of low radiation doses was subjected to a considerable modulation resulting in profound inhibition of the oxidation and nonoxidative branches of the pentose phosphate pathway. (author). 9 refs, 2 tabs

Connective tissue integrity is lost in vitamin B-6-deficient chicks

Science.gov (United States)

Masse, P. G.; Yamauchi, M.; Mahuren, J. D.; Coburn, S. P.; Muniz, O. E.; Howell, D. S.

1995-01-01

The objective of the present investigation was to characterize further the connective tissue disorder produced by pyridoxine (vitamin B-6) deficiency, as previously evidenced by electron microscopy. Following the second post-natal week, fast growing male chicks were deprived of pyridoxine for a 1-mo period. Six weeks post-natally, blood concentrations in the experimental deficiency group had declined to deficiency levels as registered by low concentrations of pyridoxal phosphate (coenzyme form) in erythrocytes, but did not reach levels associated with neurological symptoms. Light microscopic study showed abnormalities in the extracellular matrix of the connective tissues. Collagen cross-links and the aldehyde contents were not significantly lower in cartilage and tendon collagens of vitamin B-6-deficient animals than in age-matched controls; also, their proteoglycan degrading protease and collagenase activities measured in articular cartilages were not greater. Thus, proteolysis was an unlikely alternative mechanism to account for the loss of connective tissue integrity. These results point to the need for further investigation into adhesive properties of collagen associated proteoglycans or other proteins in vitamin B-6-deficient connective tissue.

Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

Energy Technology Data Exchange (ETDEWEB)

Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

2006-01-09

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

Science.gov (United States)

Nichols, Buford L; Avery, Stephen E; Quezada-Calvillo, Roberto; Kilani, Shadi B; Lin, Amy Hui-Mei; Burrin, Douglas G; Hodges, Benjamin E; Chacko, Shaji K; Opekun, Antone R; Hindawy, Marwa El; Hamaker, Bruce R; Oda, Sen-Ichi

2017-08-01

Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

NARCIS (Netherlands)

Blakely, E.L.; Mitchell, A.L.; Fisher, N.; Meunier, B.; Nijtmans, L.G.J.; Schaefer, A.M.; Jackson, M.J.; Turnbull, D.M.; Taylor, R.W.

2005-01-01

Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical

Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

NARCIS (Netherlands)

A.G.A. Bijvoet (Agnes); M.A. Kroos (Marian); F.R. Pieper (Frank); M. Van der Vliet (Martin); H.A. de Boer (Herman); A.T. van der Ploeg (Ans); M.Ph. Verbeet (Martin); A.J.J. Reuser (Arnold)

1998-01-01

textabstractGlycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective

Enzyme Replacement Therapy for Fabry Disease

Directory of Open Access Journals (Sweden)

Maria Dolores Sanchez-Niño PhD

2016-11-01

Full Text Available Fabry disease is a rare X-linked disease caused by the deficiency of α-galactosidase that leads to the accumulation of abnormal glycolipid. Untreated patients develop potentially lethal complications by age 30 to 50 years. Enzyme replacement therapy is the current standard of therapy for Fabry disease. Two formulations of recombinant human α-galactosidase A (agalsidase are available in most markets: agalsidase-α and agalsidase-β, allowing a choice of therapy. However, the US Food and Drug Administration rejected the application for commercialization of agalsidase-α. The main difference between the 2 enzymes is the dose. The label dose for agalsidase-α is 0.2 mg/kg/2 weeks, while the dose for agalsidase-β is 1.0 mg/kg/2 weeks. Recent evidence suggests a dose-dependent effect of enzyme replacement therapy and agalsidase-β is 1.0 mg/kg/2 weeks, which has been shown to reduce the occurrence of hard end points (severe renal and cardiac events, stroke, and death. In addition, patients with Fabry disease who have developed tissue injury should receive coadjuvant tissue protective therapy, together with enzyme replacement therapy, to limit nonspecific progression of the tissue injury. It is likely that in the near future, additional oral drugs become available to treat Fabry disease, such as chaperones or substrate reduction therapy.

L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome.

Science.gov (United States)

Choe, Chi-un; Nabuurs, Christine; Stockebrand, Malte C; Neu, Axel; Nunes, Patricia; Morellini, Fabio; Sauter, Kathrin; Schillemeit, Stefan; Hermans-Borgmeyer, Irm; Marescau, Bart; Heerschap, Arend; Isbrandt, Dirk

2013-01-01

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

DEFF Research Database (Denmark)

Blomberg Jensen, Martin; Nielsen, John E; Jørgensen, Anne

2010-01-01

, since it is not solely dependent on VDR expression, but also on cellular uptake of circulating VD and presence and activity of VD metabolizing enzymes. Expression of VD metabolizing enzymes has not previously been investigated in human testis and male reproductive tract. Therefore, we performed......The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex...

Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases.

Science.gov (United States)

Joachims, Michelle L; Marble, Patrick A; Laurent, Aletha B; Pastuszko, Peter; Paliotta, Marco; Blackburn, Michael R; Thompson, Linda F

2008-12-01

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

International Nuclear Information System (INIS)

Tang, J.C.T.; Li, S.H.

1984-01-01

Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

First contiguous gene deletion causing biotinidase deficiency: The enzyme deficiency in three Sri Lankan children

Directory of Open Access Journals (Sweden)

Danika Nadeen Senanayake

2015-03-01

Full Text Available We report three symptomatic children with profound biotinidase deficiency from Sri Lanka. All three children presented with typical clinical features of the disorder. The first is homozygous for a missense mutation in the BTD gene (c.98_104 del7insTCC; p.Cys33PhefsX36 that is commonly seen in the western countries, the second is homozygous for a novel missense mutation (p.Ala439Asp, and the third is the first reported instance of a contiguous gene deletion causing the enzyme deficiency. In addition, this latter finding exemplifies the importance of considering a deletion within the BTD gene for reconciling enzymatic activity with genotype, which can occur in asymptomatic children who are identified by newborn screening.

Mechanized syringe homogenization of human and animal tissues.

Science.gov (United States)

Kurien, Biji T; Porter, Andrew C; Patel, Nisha C; Kurono, Sadamu; Matsumoto, Hiroyuki; Scofield, R Hal

2004-06-01

Tissue homogenization is a prerequisite to any fractionation schedule. A plethora of hands-on methods are available to homogenize tissues. Here we report a mechanized method for homogenizing animal and human tissues rapidly and easily. The Bio-Mixer 1200 (manufactured by Innovative Products, Inc., Oklahoma City, OK) utilizes the back-and-forth movement of two motor-driven disposable syringes, connected to each other through a three-way stopcock, to homogenize animal or human tissue. Using this method, we were able to homogenize human or mouse tissues (brain, liver, heart, and salivary glands) in 5 min. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric enzyme assay for prolidase, we have found that the homogenates obtained were as good or even better than that obtained used a manual glass-on-Teflon (DuPont, Wilmington, DE) homogenization protocol (all-glass tube and Teflon pestle). Use of the Bio-Mixer 1200 to homogenize animal or human tissue precludes the need to stay in the cold room as is the case with the other hands-on homogenization methods available, in addition to freeing up time for other experiments.

The molecular basis of aminoacylase 1 deficiency

DEFF Research Database (Denmark)

Sommer, Anke; Christensen, Ernst; Schwenger, Susanne

2011-01-01

deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted...... using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase...

Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

LENUS (Irish Health Repository)

Krijt, Jakub

2011-02-01

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

Are brain and heart tissue prone to the development of thiamine deficiency?

NARCIS (Netherlands)

Klooster, Astrid; Larkin, James R.; Wiersema-Buist, Janneke; Gans, Reinold O. B.; Thornalley, Paul J.; Navis, Gerjan; van Goor, Harry; Leuvenink, Henri G. D.; Bakker, Stephan J. L.

Thiamine deficiency is a continuing problem leading to beriberi and Wernicke's encephalopathy. The symptoms of thiamine deficiency develop in the heart, brain and neuronal tissue. Yet, it is unclear how rapid thiamine deficiency develops and which organs are prone to development of thiamine

Zinc Deficiency in Humans and its Amelioration

OpenAIRE

Yashbir Singh Shivay

2015-01-01

Zinc (Zn) deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in ...

Deficiency of the Chemotactic Factor Inactivator in Human Sera with α1-Antitrypsin Deficiency

Science.gov (United States)

Ward, Peter A.; Talamo, Richard C.

1973-01-01

As revealed by appropriate fractionation procedures, human serum deficient in α1-antitrypsin (α1-AT) is also deficient in the naturally occurring chemotactic factor inactivator. These serum donors had severe pulmonary emphysema. Serum from patients with clinically similar pulmonary disease, but with presence of α1-AT in the serum, showed no such deficiency of the chemotactic factor inactivator. When normal human serum and α1-AT-deficient human sera are chemotactically activated by incubation with immune precipitates, substantially more chemotactic activity is generated in α1-AT-deficient serum. These data indicate that in α1-AT-deficient serum there is an imbalance in the generation and control of chemotactic factors. It is suggested that the theory regarding development of pulmonary emphysema in patients lacking the α1-antitrypsin in their serum should be modified to take into account a deficiency of the chemotactic factor inactivator. PMID:4683887

Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction.

Science.gov (United States)

Carbonaro, Denise A; Jin, Xiangyang; Wang, Xingchao; Yu, Xiao-Jin; Rozengurt, Nora; Kaufman, Michael L; Wang, Xiaoyan; Gjertson, David; Zhou, Yang; Blackburn, Michael R; Kohn, Donald B

2012-11-01

Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

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Shuji Mizumoto

2014-01-01

Full Text Available Glycosaminoglycans (GAGs are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs.

Human tissue models in cancer research: looking beyond the mouse.

Science.gov (United States)

Jackson, Samuel J; Thomas, Gareth J

2017-08-01

Mouse models, including patient-derived xenograft mice, are widely used to address questions in cancer research. However, there are documented flaws in these models that can result in the misrepresentation of human tumour biology and limit the suitability of the model for translational research. A coordinated effort to promote the more widespread development and use of 'non-animal human tissue' models could provide a clinically relevant platform for many cancer studies, maximising the opportunities presented by human tissue resources such as biobanks. A number of key factors limit the wide adoption of non-animal human tissue models in cancer research, including deficiencies in the infrastructure and the technical tools required to collect, transport, store and maintain human tissue for lab use. Another obstacle is the long-standing cultural reliance on animal models, which can make researchers resistant to change, often because of concerns about historical data compatibility and losing ground in a competitive environment while new approaches are embedded in lab practice. There are a wide range of initiatives that aim to address these issues by facilitating data sharing and promoting collaborations between organisations and researchers who work with human tissue. The importance of coordinating biobanks and introducing quality standards is gaining momentum. There is an exciting opportunity to transform cancer drug discovery by optimising the use of human tissue and reducing the reliance on potentially less predictive animal models. © 2017. Published by The Company of Biologists Ltd.

Current concepts on the physiology and genetics of neurotransmitters-mediating enzyme-aromatic L-amino acid decarboxylase

International Nuclear Information System (INIS)

Rahman, M.K.

1993-03-01

Two most important neurotransmitters, dopamine and serotonin are mediated by the enzyme aromatic L-amino acid decarboxylase (AADC). Because of their importance in the regulation of neuronal functions, behaviour and emotion of higher animals, many researchers are working on this enzyme to elucidate its physiological properties, structure and genetic aspects. We have discovered this enzyme in the mammalian blood, we established sensitive assay methods for the assay of the activities of this enzyme. We have made systematic studies on this enzyme in the tissues and brains of rats, and human subjects. We have found an endogenous inhibitor of this enzyme in the monkey's blood. The amino acid sequences of human AADC has been compared to rat or bovine. A full-length cDNA clone encoding human AADC has been isolated. Very recently the structure of human AADC gene including 5'-flaking region has been characterized and the transcriptional starting point has been determined. The human AADC gene assigned to chromosome 7. Up-to-date research data have shown that AADC is encoded by a single gene. Recently two patients with AADC deficiency were reported. This paper describes the systematic up-to-date review studies on AADC. (author). 62 refs, 5 figs, 8 tabs

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

Science.gov (United States)

Roldán-Arjona, Teresa; Wei, Ying-Fei; Carter, Kenneth C.; Klungland, Arne; Anselmino, Catherine; Wang, Rui-Ping; Augustus, Meena; Lindahl, Tomas

1997-01-01

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen. PMID:9223306

Improved starch digestion of sucrase deficient shrews treated with oral glucoamylase enzyme supplements

Science.gov (United States)

Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with Congenita...

The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue

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Rebecca C. Schugar

2017-06-01

Full Text Available Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3 conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

Human tissue models in cancer research: looking beyond the mouse

Directory of Open Access Journals (Sweden)

Samuel J. Jackson

2017-08-01

Full Text Available Mouse models, including patient-derived xenograft mice, are widely used to address questions in cancer research. However, there are documented flaws in these models that can result in the misrepresentation of human tumour biology and limit the suitability of the model for translational research. A coordinated effort to promote the more widespread development and use of ‘non-animal human tissue’ models could provide a clinically relevant platform for many cancer studies, maximising the opportunities presented by human tissue resources such as biobanks. A number of key factors limit the wide adoption of non-animal human tissue models in cancer research, including deficiencies in the infrastructure and the technical tools required to collect, transport, store and maintain human tissue for lab use. Another obstacle is the long-standing cultural reliance on animal models, which can make researchers resistant to change, often because of concerns about historical data compatibility and losing ground in a competitive environment while new approaches are embedded in lab practice. There are a wide range of initiatives that aim to address these issues by facilitating data sharing and promoting collaborations between organisations and researchers who work with human tissue. The importance of coordinating biobanks and introducing quality standards is gaining momentum. There is an exciting opportunity to transform cancer drug discovery by optimising the use of human tissue and reducing the reliance on potentially less predictive animal models.

Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction

Science.gov (United States)

Jin, Xiangyang; Wang, Xingchao; Yu, Xiao-Jin; Rozengurt, Nora; Kaufman, Michael L.; Wang, Xiaoyan; Gjertson, David; Zhou, Yang; Blackburn, Michael R.; Kohn, Donald B.

2012-01-01

Gene therapy (GT) for adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada−/−). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist. PMID:22833548

Zinc Deficiency in Humans and its Amelioration

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Yashbir Singh Shivay

2015-01-01

Full Text Available Zinc (Zn deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in blood does not decrease in proportion of the Zn deficiency. Adverse effects of Zn deficiency vary with age: low weight gain, diarrhoea, aneroxia and neurobehavioral disturbances are observed in infants, while skin changes and dwarfism are frequent in toddlers and adolescents. Common manifestations of Zn deficiency among elderly include hypogeusia, chronic non-healing ulcers and recurrent infections.Ameliorative measures of Zn deficiency in humans can be classified in two groups, namely, nutraceutical and biofortification of food grains. Nutraceutical interventions include pharmaceutical supplements, dietary supplements and dietary diversification, while biofortification of food grains can be achieved by genetic modification (GM of crops or by agronomic techniques that include soil or/and foliar fertilization of crops.The major disadvantage of nutraceutical approaches is that the major beneficiaries are urban people and the poor rural masses that need adequate Zn nutrition most are left out. Genetic biofortification of food grains requires large amounts of funds and a fairly long-period of time. Further, a large number of countries have not yet accepted genetically modified (GM foods. On the other hand agronomic biofortification of food grains yields immediate effects and rural and urban people are equally benefitted. Our studies have shown that Zn concentration in cereals (rice, wheat etc and pulses can be considerably increased by soil or/and foliar

Zinc Deficiency in Humans and its Amelioration

Directory of Open Access Journals (Sweden)

Yashbir Singh Shivay

2015-12-01

Full Text Available Zinc (Zn deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in blood does not decrease in proportion of the Zn deficiency. Adverse effects of Zn deficiency vary with age: low weight gain, diarrhoea, aneroxia and neurobehavioral disturbances are observed in infants, while skin changes and dwarfism are frequent in toddlers and adolescents. Common manifestations of Zn deficiency among elderly include hypogeusia, chronic non-healing ulcers and recurrent infections. Ameliorative measures of Zn deficiency in humans can be classified in two groups, namely, nutraceutical and biofortification of food grains. Nutraceutical interventions include pharmaceutical supplements, dietary supplements and dietary diversification, while biofortification of food grains can be achieved by genetic modification (GM of crops or by agronomic techniques that include soil or/and foliar fertilization of crops. The major disadvantage of nutraceutical approaches is that the major beneficiaries are urban people and the poor rural masses that need adequate Zn nutrition most are left out. Genetic biofortification of food grains requires large amounts of funds and a fairly long-period of time. Further, a large number of countries have not yet accepted genetically modified (GM foods. On the other hand agronomic biofortification of food grains yields immediate effects and rural and urban people are equally benefitted. Our studies have shown that Zn concentration in cereals (rice, wheat etc and pulses can be considerably increased by soil or/and foliar

N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment

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Caldovic L

2011-08-01

Full Text Available Nicholas Ah Mew, Ljubica CaldovicCenter for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington DC, USAAbstract: The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1, requires an allosteric activator, N-acetylglutamate (NAG. The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS. An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.Keywords: urea cycle, urea cycle disorder, N-acetyl-L-glutamate, N-acetylglutamate synthase, hyperammonemia, N-carbamyl-L-glutamate

Tissue and plasma enzyme activities in juvenile green iguanas.

Science.gov (United States)

Wagner, R A; Wetzel, R

1999-02-01

To determine activities of intracellular enzymes in 8 major organs in juvenile green iguanas and to compare tissue and plasma activities. 6 green iguanas iguanas, but high values may not always indicate overt muscle disease. The AMS activity may be specific for the pancreas, but the wide range of plasma activity would likely limit its diagnostic usefulness. Activities of AST and LDH may reflect tissue damage or inflammation, but probably do not reflect damage to specific tissues or organs.

Recombinant human G6PD for quality control and quality assurance of novel point-of-care diagnostics for G6PD deficiency.

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Maria Kahn

Full Text Available A large gap for the support of point-of-care testing is the availability of reagents to support quality control (QC of diagnostic assays along the supply chain from the manufacturer to the end user. While reagents and systems exist to support QC of laboratory screening tests for glucose-6-phosphate dehydrogenase (G6PD deficiency, they are not configured appropriately to support point-of-care testing. The feasibility of using lyophilized recombinant human G6PD as a QC reagent in novel point-of-care tests for G6PD deficiency is demonstrated.Human recombinant G6PD (r-G6PD was expressed in Escherichia coli and purified. Aliquots were stored at -80°C. Prior to lyophilization, aliquots were thawed, and three concentrations of r-G6PD (representing normal, intermediate, and deficient clinical G6PD levels were prepared and mixed with a protective formulation, which protects the enzyme activity against degradation from denaturation during the lyophilization process. Following lyophilization, individual single-use tubes of lyophilized r-G6PD were placed in individual packs with desiccants and stored at five temperatures for one year. An enzyme assay for G6PD activity was used to ascertain the stability of r-G6PD activity while stored at different temperatures.Lyophilized r-G6PD is stable and can be used as a control indicator. Results presented here show that G6PD activity is stable for at least 365 days when stored at -80°C, 4°C, 30°C, and 45°C. When stored at 55°C, enzyme activity was found to be stable only through day 28.Lyophilized r-G6PD enzyme is stable and can be used as a control for point-of-care tests for G6PD deficiency.

Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations.

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Bradford, Kathryn L; Moretti, Federico A; Carbonaro-Sarracino, Denise A; Gaspar, Hubert B; Kohn, Donald B

2017-10-01

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T - B - NK - ), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

METABOLIC MAPPING BY ENZYME HISTOCHEMISTRY IN LIVING ANIMALS, TISSUES AND CELLS

NARCIS (Netherlands)

van Noorden, C. J. F.

2009-01-01

Imaging of reporter molecules such as fluorescent proteins in intact animals, tissue and cells has become an indispensable tool in cell biology Imaging activity of enzymes, which is called metabolic mapping, provides information on subcellular localisation in combination with function of the enzymes

DGAT enzymes and triacylglycerol biosynthesis

Science.gov (United States)

Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

2008-01-01

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

Toll-like receptor 4 (TLR4) deficient mice are protected from adipose tissue inflammation in aging.

Science.gov (United States)

Ghosh, Amiya K; O'Brien, Martin; Mau, Theresa; Yung, Raymond

2017-09-07

Adipose tissue (AT) inflammation is a central mechanism for metabolic dysfunction in both diet-induced obesity and age-associated obesity. Studies in diet-induced obesity have characterized the role of Fetuin A (Fet A) in Free Fatty Acids (FFA)-mediated TLR4 activation and adipose tissue inflammation. However, the role of Fet A & TLR4 in aging-related adipose tissue inflammation is unknown. In the current study, analysis of epidymymal fat pads of C57/Bl6 male mice, we found that, in contrast to data from diet-induced obesity models, adipose tissue from aged mice have normal Fet A and TLR4 expression. Interestingly, aged TLR4-deficient mice have diminished adipose tissue inflammation compared to normal controls. We further demonstrated that reduced AT inflammation in old TLR4-deficient mice is linked to impaired ER stress, augmented autophagy activity, and diminished senescence phenomenon. Importantly, old TLR4-deficient mice have improved glucose tolerance compared to age-matched wild type mice, suggesting that the observed reduced AT inflammation in aged TLR4-deficient mice has important physiological consequences. Taken together, our present study establishes novel aspect of aging-associated AT inflammation that is distinct from diet-induced AT inflammation. Our results also provide strong evidence that TLR4 plays a significant role in promoting aging adipose tissue inflammation.

Cobalt deficiency effects on trace elements, hormones and enzymes involved in energy metabolism of cattle.

Science.gov (United States)

Stangl, G I; Schwarz, F J; Kirchgessner, M

1999-03-01

This study was conducted to investigate the physiological consequences of long-term moderate cobalt deficiency in beef cattle, which have not hitherto been studied in detail. Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal corn silage-based diet that was moderately low in cobalt (83 micrograms Co/kg), or the same diet supplemented with cobalt to a total of 200 micrograms per kg, for 43 weeks. Cobalt deficiency was induced, as judged by inappetance, diminished growth gain and a markedly reduced vitamin B12 status in serum and liver. The long-term cobalt deprivation which was primarily a combination of reduced feed intake and a tissue vitamin B12 deficiency did not show evidence of a significant dysfunction of energy metabolism. The activities of glucose-6-phosphate dehydrogenase and cytochrome oxidase in liver remained unaffected by cobalt deficiency, nor was there a significant change in serum glucose level of cattle on the cobalt-deprived diet. However, analysis of thyroid hormone status indicated a slight reduction of type I thyroxine monodeiodinase activity in liver accompanied by a significant reduction of the triiodothyronine level in serum. The diminished liver vitamin B12 level resulted in significantly reduced folate level in this tissue, reduced concentrations of heme-depending blood parameters. Moreover cobalt deficiency or rather vitamin B12 deficiency was accompanied by a dramatic accumulation of the trace elements iron and nickel in liver. These results indicate that long-term moderate cobalt deficiency may induce a number of physiological changes in cattle, but a follow-up study, which excluded different feed levels by including a pair-fed control group, will be necessary to actually obtain the single effect of cobalt deficiency in cattle.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Science.gov (United States)

Boedeker, J C; Doolittle, M H; White, A L

2001-11-01

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.

The PAXgene(® tissue system preserves phosphoproteins in human tissue specimens and enables comprehensive protein biomarker research.

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Sibylle Gündisch

Full Text Available Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE and enzyme-linked immunosorbent assay (ELISA to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.

Sucrose-Metabolizing Enzymes in Transport Tissues and Adjacent Sink Structures in Developing Citrus Fruit 1

Science.gov (United States)

Lowell, Cadance A.; Tomlinson, Patricia T.; Koch, Karen E.

1989-01-01

Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import

A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

Science.gov (United States)

Hantzis, Laura J; Kroh, Gretchen E; Jahn, Courtney E; Cantrell, Michael; Peers, Graham; Pilon, Marinus; Ravet, Karl

2018-01-01

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. © 2018 American Society of Plant Biologists. All Rights Reserved.

Impact of androgenic/antiandrogenic compounds (AAC) on human sex steroid metabolizing key enzymes

International Nuclear Information System (INIS)

Allera, A.; Lo, S.; King, I.; Steglich, F.; Klingmueller, D.

2004-01-01

Various pesticides, industrial pollutants and synthetic compounds, to which human populations are exposed, are known or suspected to interfere with endogenous sex hormone functions. Such interference potentially affect the development and expression of the male and female reproductive system or both. Chemicals in this class are thus referred to as endocrine disruptors (ED). This emphazises on the relevance of screening ED for a wide range of sex hormone-mimicking effects. These compounds are believed to exert influence on hormonal actions predominantly by (i) interfering with endogenous steroids in that they functionally interact with plasma membrane-located receptors as well as with nuclear receptors both for estrogens and androgens or (ii) affecting the levels of sex hormones as a result of their impact on steroid metabolizing key enzymes. Essential sex hormone-related enzymes within the endocrine system of humans are aromatase, 5α-reductase 2 as well as specific sulfotransferases and sulfatases (so-called phase I and phase II enzymes, respectively). Using suitable human tissues and human cancer cell lines (placenta, prostate, liver and JEG-3, lymph node carcinoma of prostate (LnCaP) cells) we investigated the impact of 10 widely used chemicals suspected of acting as ED with androgenic or antiandrogenic activity (so-called AAC) on the activity of these sex hormone metabolizing key enzymes in humans. In addition, the respective effects of six substances were also studied as positive controls due to their well-known specific hormonal agonistic/antagonistic activities. The aim of this report and subsequent investigations is to improve human health risk assessment for AAC and other ED

Fucosylation Deficiency in Mice Leads to Colitis and Adenocarcinoma.

Science.gov (United States)

Wang, Yiwei; Huang, Dan; Chen, Kai-Yuan; Cui, Min; Wang, Weihuan; Huang, Xiaoran; Awadellah, Amad; Li, Qing; Friedman, Ann; Xin, William W; Di Martino, Luca; Cominelli, Fabio; Miron, Alex; Chan, Ricky; Fox, James G; Xu, Yan; Shen, Xiling; Kalady, Mathew F; Markowitz, Sanford; Maillard, Ivan; Lowe, John B; Xin, Wei; Zhou, Lan

2017-01-01

De novo synthesis of guanosine diphosphate (GDP)-fucose, a substrate for fucosylglycans, requires sequential reactions mediated by GDP-mannose 4,6-dehydratase (GMDS) and GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase (FX or tissue specific transplantation antigen P35B [TSTA3]). GMDS deletions and mutations are found in 6%-13% of colorectal cancers; these mostly affect the ascending and transverse colon. We investigated whether a lack of fucosylation consequent to loss of GDP-fucose synthesis contributes to colon carcinogenesis. FX deficiency and GMDS deletion produce the same biochemical phenotype of GDP-fucose deficiency. We studied a mouse model of fucosylation deficiency (Fx-/- mice) and mice with the full-length Fx gene (controls). Mice were placed on standard chow or fucose-containing diet (equivalent to a control fucosylglycan phenotype). Colon tissues were collected and analyzed histologically or by enzyme-linked immunosorbent assays to measure cytokine levels; T cells also were collected and analyzed. Fecal samples were analyzed by 16s ribosomal RNA sequencing. Mucosal barrier function was measured by uptake of fluorescent dextran. We transplanted bone marrow cells from Fx-/- or control mice (Ly5.2) into irradiated 8-week-old Fx-/- or control mice (Ly5.1). We performed immunohistochemical analyses for expression of Notch and the hes family bHLH transcription factor (HES1) in colon tissues from mice and a panel of 60 human colorectal cancer specimens (27 left-sided, 33 right-sided). Fx-/- mice developed colitis and serrated-like lesions. The intestinal pathology of Fx-/- mice was reversed by addition of fucose to the diet, which restored fucosylation via a salvage pathway. In the absence of fucosylation, dysplasia appeared and progressed to adenocarcinoma in up to 40% of mice, affecting mainly the right colon and cecum. Notch was not activated in Fx-/- mice fed standard chow, leading to decreased expression of its target Hes1. Fucosylation deficiency

Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

Science.gov (United States)

Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

2015-01-01

Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

International Nuclear Information System (INIS)

Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy

2015-01-01

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

Energy Technology Data Exchange (ETDEWEB)

Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy, E-mail: shivanna@bcm.edu

2015-07-15

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

[PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

Science.gov (United States)

Sudarkina, O Yu; Dergunova, L V

2015-01-01

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

Ultraviolet light induction of diphtheria toxin-resistant mutations in normal and DNA repair-deficient human and Chinese hamster fibroblasts

International Nuclear Information System (INIS)

Trosko, J.E.; Schultz, R.S.; Chang, C.C.; Glover, T.

1980-01-01

The role on unrepaired DNA lesions in the production of mutations is suspected of contributing to the initiation phase of carcinogenesis. Since the molecular basis of mutagenesis is not understood in eukaryotic cells, development of new genetic markers for quantitative in vitro measurement of mutations for mammalian cells is needed. Furthermore, mammalian cells, genetically deficient for various DNA repair enzymes, will be needed to study the role of unrepaired DNA lesions in mutagenesis. The results in this report relate to preliminary attempts to characterize the diphtheria toxin resistance marker as a useful quantitative genetic marker in human cells and to isolate and characterize various DNA repair-deficient Chinese hamster cells

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

Science.gov (United States)

Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A

2013-02-15

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

Antioxidant enzymes activity in embryogenic and non-embryogenic tissues in Sugarcane

International Nuclear Information System (INIS)

Marina Medeiros de Araujo Silva; Ulisses, Claudia; Lacerda E Medeiros, Maria Jaislanny; Cavalcante Granja, Manuela Maria; Willadino, Lilia; Camara, Terezinha

2014-01-01

The objective of this work was to induce direct somatic embryogenesis from segments of immature leaves of the RB872552 variety of sugarcane and to correlate this morphogenic event with oxidative stress. Two previously described protocols were utilized for the induction of somatic embryogenesis in sugarcane with different supplementations of the culture medium and different incubation conditions. For the conversion of embryos into plants was used ms medium without phytoregulators. Histological analyses and activity of antioxidant enzymes were also conducted for the embryogenic and non-embryogenic tissues. The formation of somatic embryos was obtained in 81 % of the explants with the combination of regulators 2,4-D (2,4-dichlorophenoxyacetic acid)and BAP (6-benzylaminopurine) when incubated under 16 h photoperiod. With regards to the antioxidant enzymes, there was increased activity of peroxidase and an increase in the soluble protein content in embryogenic tissues, whereas lower activities of polyphenol oxidase and catalase appeared in these tissues compared to nonembryogenic tissues. It could be inferred that oxidative stress plays an important role in the induction of somatic embryogenesis in sugarcane.

Biochemical assessement of liver enzymes in immunocompromised ...

African Journals Online (AJOL)

Aim: This study aims at the estimation of serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and glutmyltransferase GGT (Liver enzymes) in Human immunodeficiency virus(HIV) and/or Acquired immune deficiency syndrome(AIDS) patients in parts of Edo State, Nigeria.

Matrix Metalloproteinase Enzyme Family

Directory of Open Access Journals (Sweden)

Ozlem Goruroglu Ozturk

2013-04-01

Full Text Available Matrix metalloproteinases play an important role in many biological processes such as embriogenesis, tissue remodeling, wound healing, and angiogenesis, and in some pathological conditions such as atherosclerosis, arthritis and cancer. Currently, 24 genes have been identified in humans that encode different groups of matrix metalloproteinase enzymes. This review discuss the members of the matrix metalloproteinase family and their substrate specificity, structure, function and the regulation of their enzyme activity by tissue inhibitors. [Archives Medical Review Journal 2013; 22(2.000: 209-220

Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

Directory of Open Access Journals (Sweden)

Joan Villarroya

Full Text Available Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT and brown (BAT adipose tissues in thymidine kinase 2 (Tk2 H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues.

Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

Science.gov (United States)

Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

2011-01-01

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues. © 2011 Villarroya et al.

Increased activities of mitochondrial enzymes in white adipose tissue in trained rats

DEFF Research Database (Denmark)

Stallknecht, B; Vinten, J; Ploug, T

1991-01-01

of 8-12 rats were swim trained for 10 wk or served as either sedentary, sham swim-trained, or cold-stressed controls. White adipose tissue was removed, and the activities of the respiratory chain enzyme cytochrome-c oxidase (CCO) and of the enzyme malate dehydrogenase (MDH), which participates...... 0.05). In female rats the CCO activity expressed per milligram protein was increased 4.5-fold in the trained compared with the sedentary control rats (P less than 0.01). Neither cold stress nor sham swim training increased CCO or MDH activities in white adipose tissue (P greater than 0...

Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.

Science.gov (United States)

Hadler-Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan-Olof; Uhlin-Hansen, Lars

2010-01-01

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

Phenolsulphotransferase in human tissue: radiochemical enzymatic assay and biochemical properties

International Nuclear Information System (INIS)

Anderson, R.J.; Weinshilboum, R.M.

1980-01-01

Phenolsulphotransferase (EC 2.8.2.1) (PST) is an important catecholamine and drug metabolizing enzyme. Optimal conditions have been determined for the accurate measurement of PST activity in the human platelet, human renal cortex, and human jejunum with a radiochemical microassay. 3-Methoxy-4-hydroxyphenylglycol (MHPG) and 35 S-3'-phosphoadenosine-5'-phosphosulfate ( 35 S-PAPS) were the substrates for the reaction. The apparent Michaelis-Menten (Ksub(m)) values for MHPG with platelet, renal cortex, and jejunum were 1.09, 0.46 and 1.16 mmol/l, respectively. Apparent Ksub(m) values for PAPS in the same tissues were 0.14, 0.13 and 0.21 μmol/l. The pH optimum of the reacton in all three tissues was approximately 6.2-6.8 with three different buffer systems. The coefficients of variation for the assay of platelet, renal cortex, and jejunal activities were 6.2%, 3.4% and 4.4%, respectively. Mean platelet PST activity in blood samples from 75 randomly selected adult subjects was 5.0 +- 1.72 mmol of MHPG sulfate formed per hour per mg of platelet protein (8.3 X 10 -5 +- 2.9 X 10 -5 μmol min -1 mg -1 , mean +- S.D.). There was a 5-fold intersubject variation in platelet PST activity within two standard deviations of the mean value. Experiments in which partially purified human erythrocyte PST was added to platelet, kidney and gut homogenates under these assay conditions provided evidence that endogenous PST inhibitors did not affect the observed enzyme activity. (Auth.)

Glucose-6-phosphate dehydrogenase deficiency in Singapore.

Science.gov (United States)

Quak, S H; Saha, N; Tay, J S

1996-01-01

Glucose-6-phosphate dehydrogenase (G6PD) in man is an X-linked enzyme. The deficiency of this enzyme is one of the most common inherited metabolic disorders in man. In Singapore, three clinical syndromes associated with G6PD deficiency had been described: severe haemolysis in neonates with kernicterus, haemoglobinuria and "viral hepatitis"-like syndrome. The human G6PD monomer consists of 515 amino acids. Only the tetrameric or dimeric forms composed of a single type subunit are catylitically active. The complete amino acid sequence of G6PD had been elucidated in man and various other animals. The region of high homology among the enzymes of various animals is presumably functionally active. Among the Chinese in Singapore, three common molecular variants had been identified: Canton (nt 1376 G --> T), Kaiping (nt 1388 G --> A) and Mediterranean (nt 563 C --> T) in frequencies of 24%, 21% and 10% respectively. In addition, two common mutants (Gaozhou, nt 95 A --> G and Chinese 5, nt 1024 C --> T) have been detected in Singapore Chinese in low frequencies. In Malays, 6 different deficient variants are known in Singapore (3 new, 1 Mahidol, 1 Indonesian and 1 Mediterranean).

In vitro tissue-digesting properties of krill enzymes compared with fibrinolysin/DNAse, papain and placebo

NARCIS (Netherlands)

Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Kammeyer, A.; Westerhof, W.

1997-01-01

Wound debridement, the removal of necrotic tissue, can be achieved with proteolytic enzymes. Recently, a new multi-enzyme preparation, krill enzyme, isolated from Antarctic shrimp-like organisms (Euphausia superba), was reported to possess powerful proteolytic activity towards protein substrates. In

Hypocretin deficiency develops during onset of human narcolepsy with cataplexy

DEFF Research Database (Denmark)

Savvidou, Andri; Knudsen, Stine; Olsson-Engman, Mia

2013-01-01

Although hypothesized through animal studies, a temporal and causal association between hypocretin deficiency and the onset of narcolepsy with cataplexy (NC) has never been proven in humans.......Although hypothesized through animal studies, a temporal and causal association between hypocretin deficiency and the onset of narcolepsy with cataplexy (NC) has never been proven in humans....

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue

OpenAIRE

Nanduri, Bindu; Shack, Leslie A.; Rai, Aswathy N.; Epperson, William B.; Baumgartner, Wes; Schmidt, Ty B.; Edelmann, Mariola J.

2016-01-01

To develop a reproducible tissue-lysis method that retains enzyme function for activity-based protein profiling, we compared four different tissue lysis methods of bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue and focused ultrasonication had also the fastest pr...

Analysis of human error and organizational deficiency in events considering risk significance

International Nuclear Information System (INIS)

Lee, Yong Suk; Kim, Yoonik; Kim, Say Hyung; Kim, Chansoo; Chung, Chang Hyun; Jung, Won Dea

2004-01-01

In this study, we analyzed human and organizational deficiencies in the trip events of Korean nuclear power plants. K-HPES items were used in human error analysis, and the organizational factors by Jacobs and Haber were used for organizational deficiency analysis. We proposed the use of CCDP as a risk measure to consider risk information in prioritizing K-HPES items and organizational factors. Until now, the risk significance of events has not been considered in human error and organizational deficiency analysis. Considering the risk significance of events in the process of analysis is necessary for effective enhancement of nuclear power plant safety by focusing on causes of human error and organizational deficiencies that are associated with significant risk

Molecular mechanisms of mitochondrial DNA depletion diseases caused by deficiencies in enzymes in purine and pyrimidine metabolism.

Science.gov (United States)

Eriksson, Staffan; Wang, Liya

2008-06-01

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.

Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.

Science.gov (United States)

Yen, Chi-Liang Eric; Stone, Scot J; Koliwad, Suneil; Harris, Charles; Farese, Robert V

2008-11-01

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases.

Enzyme alterations in mediastine during and after radiotherapy. 2

International Nuclear Information System (INIS)

Alheit, H.D.; Alheit, C.; Herrmann, T.

1986-01-01

Results are presented estimating the serum activity of transaminases (ASAT and ALAT) in 72 patients after mediastinal irradiation. During and after mediastinal irradiation both enzymes showed essentially a parallel reaction. One day after irradiation a decrease of enzymes in patients who were irradiated with high single dosis (5 Gy) was observed, while patients irradiated with low or middle single dosis showed an increase of enzyme activity. A different temporal enzyme reaction is discussed to be the cause for this reaction in dependence on the applied single dose so that in patients with high single doses an initial enzyme increase caused by the radiation insult has changed into a following decrease under the starting level at the first control 24 hours later. Because patients without mediastinal tumors react in the same manner, the normal tissue surrounding the tumor is discussed to be the original place of enzyme secretion. Up to the end of irradiation a decrease of enzymes was observed in patients with high single dose or with high total dose (60 Gy) which is interpreted as an enzyme deficiency in tissue in consequence of destruction in formation places. In patients with middle total and low single doses an enzyme increase is registered with a still sufficient restoration capacity of the tissue discussed to be the cause of it. An enzyme increase, observed from the end of irradiation to the control date 3 to 6 months after irradiation, is mainly caused by a tumor progression (increased rate of liver metastases, especially in bronchial carcinoma) and can still be intensified by occurrence of pulmonal or cardiac radioreactions. (author)

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

Directory of Open Access Journals (Sweden)

Korson Mark

2007-04-01

Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

Science.gov (United States)

... 5-fluorouracil and capecitabine. These drugs are not broken down efficiently by people with dihydropyrimidine dehydrogenase deficiency ... of this enzyme. Because fluoropyrimidine drugs are also broken down by the dihydropyrimidine dehydrogenase enzyme, deficiency of ...

Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

Science.gov (United States)

Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Zhang, Nan; Szweda, Luke I.; Griffin, Timothy M.; Barlic-Dicen, Jana

2014-01-01

The chemokine receptor CXCR4 is expressed on adipocytes and macrophages in adipose tissue, but its role in this tissue remains unknown. We evaluated whether deficiency in either adipocyte or myeloid leukocyte CXCR4 affects body weight (BW) and adiposity in a mouse model of high-fat-diet (HFD)-induced obesity. We found that ablation of adipocyte, but not myeloid leukocyte, CXCR4 exacerbated obesity. The HFD-fed adipocyte-specific CXCR4-knockout (AdCXCR4ko) mice, compared to wild-type C57BL/6 control mice, had increased BW (average: 52.0 g vs. 35.5 g), adiposity (average: 49.3 vs. 21.0% of total BW), and inflammatory leukocyte content in white adipose tissue (WAT), despite comparable food intake. As previously reported, HFD feeding increased uncoupling protein 1 (UCP1) expression (fold increase: 3.5) in brown adipose tissue (BAT) of the C57BL/6 control mice. However, no HFD-induced increase in UCP1 expression was observed in the AdCXCR4ko mice, which were cold sensitive. Thus, our study suggests that adipocyte CXCR4 limits development of obesity by preventing excessive inflammatory cell recruitment into WAT and by supporting thermogenic activity of BAT. Since CXCR4 is conserved between mouse and human, the newfound role of CXCR4 in mouse adipose tissue may parallel the role of this chemokine receptor in human adipose tissue.—Yao, L., Heuser-Baker, J., Herlea-Pana, O., Zhang, N., Szweda, L. I., Griffin, T. M., Barlic-Dicen, J. Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity. PMID:25016030

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Science.gov (United States)

Pandey, Manoj K; Burrow, Thomas A; Rani, Reena; Martin, Lisa J; Witte, David; Setchell, Kenneth D; Mckay, Mary A; Magnusen, Albert F; Zhang, Wujuan; Liou, Benjamin; Köhl, Jörg; Grabowski, Gregory A

2017-03-02

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes.

Science.gov (United States)

Miners, J Scott; Palmer, Jennifer C; Tayler, Hannah; Palmer, Laura E; Ashby, Emma; Kehoe, Patrick G; Love, Seth

2014-01-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.

Science.gov (United States)

Shi, Yuguang; Cheng, Dong

2009-07-01

Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.

Deficiency of acyl-CoA: Dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebro-hepato-renal) syndrome

NARCIS (Netherlands)

Bosch, H. van den; Schutgens, R.B.H.; Romeyn, G.J.; Wanders, R.J.A.; Schrakamp, G.; Heymans, H.S.A.

1984-01-01

We have recently reported on plasmalogen deficiency in tissues and fibroblasts from patients with Zellweger syndrome. In this paper we have analyzed the activity of the first enzyme in the pathway leading to plasmalogen biosynthesis, i.e. acyl-CoA: dihydroxyacetone phosphate acyltransferase in

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

Directory of Open Access Journals (Sweden)

Mogilevski Grigori

2004-09-01

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

Superoxide Dismutase (SOD Enzyme Activity Assay in Fasciola spp. Para-sites and Liver Tissue Extract

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M Assady

2011-09-01

Full Text Available Background: The purpose of this comparative study was to detect superoxide dismutase (SOD activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level.Methods: Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues, 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference.Results: Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass. Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05.Conclusion: Fasciola species and liver infection are effective causes on SOD enzyme activity level.

Interaction between cytotoxic effects of γ-radiation and folate deficiency in relation to choline reserves

International Nuclear Information System (INIS)

Batra, Vipen; Devasagayam, Thomas Paul Asir

2009-01-01

The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. Recent studies have indicated that bio-molecules such as folate and choline might be of radio-protective value as they are, within broad dose ranges, non-toxic to humans and experimental animals. The objective of the present study was to investigate choline dependent adaptive response to potential synergistic cytotoxic effect of folate deficiency and γ-radiation. Male Swiss mice maintained on folate sufficient diet (FSD) and folate free diet (FFD) based on AIN-93 M formula, were subjected to 1-4 Gy total body γ-irradiation. To investigate liver DNA damage, apurinic/apyrimidinic sites (AP sites) were quantified. A significant increase in liver DNA AP sites with concomitant depletion of liver choline reserves was observed when γ-radiation was combined with folate deficiency. Further work in this direction suggested that cytotoxic interaction between folate deficiency and gamma radiation might induce utilization of choline and choline containing moieties by modifying levels of key regulatory enzymes dihydrofolate reductase (DHFR) and choline oxidase (ChoOx). Another major finding of these studies is that significant liver damage at higher doses of radiation (3-4 Gy), might release considerable amounts of choline reserves to serum. In conclusion, a plausible interpretation of the present studies is that folate deprivation and γ-radiation interact to mobilize additional choline reserves of hepatic tissue, for redistribution to other organs, which could not be utilized by folate deficiency alone. Present results clearly indicated a distinct choline pool in liver and kidney tissues that could be utilized by folate deficient animals only under radiation stress conditions

Partial resolution of bone lesions. A child with severe combined immunodeficiency disease and adenosine deaminase deficiency after enzyme-replacement therapy

International Nuclear Information System (INIS)

Yulish, B.S.; Stern, R.C.; Polmar, S.H.

1980-01-01

A child with severe combined immunodeficiency disease and adenosine deaminase deficiency, with characteristic bone dysplasia, was treated with transfusions of frozen irradiated RBCs as a means of enzyme replacement. This therapy resulted in restoration of immunologic competence and partial resolution of the bone lesions. Although the natural history of these lesions without therapy is not known, enzyme-replacement therapy may have played a role in the resolution of this patient's bone lesions

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

Science.gov (United States)

Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

1999-01-01

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

High predictive value of brain MRI imaging in primary mitochondrial respiratory chain deficiency.

Science.gov (United States)

de Beaurepaire, Isaure; Grévent, David; Rio, Marlène; Desguerre, Isabelle; de Lonlay, Pascale; Levy, Raphaël; Dangouloff-Ros, Volodia; Bonnefont, Jean-Paul; Barcia, Giulia; Funalot, Benoit; Besmond, Claude; Metodiev, Metodi D; Ruzzenente, Benedetta; Assouline, Zahra; Munnich, Arnold; Rötig, Agnès; Boddaert, Nathalie

2018-06-01

Because the mitochondrial respiratory chain (RC) is ubiquitous, its deficiency can theoretically give rise to any symptom in any organ or tissue at any age with any mode of inheritance, owing to the twofold genetic origin of respiratory enzyme machinery, that is, nuclear and mitochondrial. Not all respiratory enzyme deficiencies are primary and secondary or artefactual deficiency is frequently observed, leading to a number of misleading conclusions and inappropriate investigations in clinical practice. This study is aimed at investigating the potential role of brain MRI in distinguishing primary RC deficiency from phenocopies and other aetiologies. Starting from a large series of 189 patients (median age: 3.5 years (8 days-56 years), 58% males) showing signs of RC enzyme deficiency, for whom both brain MRIs and disease-causing mutations were available, we retrospectively studied the positive predictive value (PPV) and the positive likelihood ratio (LR+) of brain MRI imaging and its ability to discriminate between two groups: primary deficiency of the mitochondrial RC machinery and phenocopies. Detection of (1) brainstem hyperintensity with basal ganglia involvement (P≤0.001) and (2) lactate peak with either brainstem or basal ganglia hyperintensity was highly suggestive of primary RC deficiency (P≤0.01). Fourteen items had a PPV>95% and LR+ was greater than 9 for seven signs. Biallelic SLC19A3 mutations represented the main differential diagnosis. Non-significant differences between the two groups were found for cortical/subcortical atrophy, leucoencephalopathy and involvement of caudate nuclei, spinothalamic tract and corpus callosum. Based on these results and owing to invasiveness of skeletal muscle biopsies and cost of high-throughput DNA sequencing, we suggest giving consideration to brain MRI imaging as a diagnostic marker and an informative investigation to be performed in patients showing signs of RC enzyme deficiency. © Article author(s) (or their

Erythrocyte pyruvate kinase deficiency in the Ohio Amish: origin and characterization of the mutant enzyme.

OpenAIRE

Muir, W A; Beutler, E; Wasson, C

1984-01-01

We have identified eight individuals in an Amish population in Geauga County, Ohio, who have a congenital hemolytic anemia and red cell pyruvate kinase (PK) deficiency. The mutant enzyme is a low Km phosphoenolpyruvate (PEP) variant associated with a slower (77.5% of normal) electrophoretic mobility in starch gel. Because of the high consanguinity in this population, we assume the affected individuals are homozygous for the mutant gene. Genealogical records allow us to trace all eight cases b...

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

OpenAIRE

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-01-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally...

Creatine synthesis and exchanges between brain cells: What can be learned from human creatine deficiencies and various experimental models?

Science.gov (United States)

Hanna-El-Daher, Layane; Braissant, Olivier

2016-08-01

While it has long been thought that most of cerebral creatine is of peripheral origin, the last 20 years has provided evidence that the creatine synthetic pathway (AGAT and GAMT enzymes) is expressed in the brain together with the creatine transporter (SLC6A8). It has also been shown that SLC6A8 is expressed by microcapillary endothelial cells at the blood-brain barrier, but is absent from surrounding astrocytes, raising the concept that the blood-brain barrier has a limited permeability for peripheral creatine. The first creatine deficiency syndrome in humans was also discovered 20 years ago (GAMT deficiency), followed later by AGAT and SLC6A8 deficiencies, all three diseases being characterized by creatine deficiency in the CNS and essentially affecting the brain. By reviewing the numerous and latest experimental studies addressing creatine transport and synthesis in the CNS, as well as the clinical and biochemical characteristics of creatine-deficient patients, our aim was to delineate a clearer view of the roles of the blood-brain and blood-cerebrospinal fluid barriers in the transport of creatine and guanidinoacetate between periphery and CNS, and on the intracerebral synthesis and transport of creatine. This review also addresses the question of guanidinoacetate toxicity for brain cells, as probably found under GAMT deficiency.

P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

Science.gov (United States)

Miller, Walter L

2012-10-23

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

Single administration of recombinant IL-6 restores the gene expression of lipogenic enzymes in liver of fasting IL-6-deficient mice

DEFF Research Database (Denmark)

Gavito, A L; Cabello, R; Suarez, J

2016-01-01

BACKGROUND AND PURPOSE: Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL-6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL-6 in mediating fasting/re-feeding changes in the expression of hepatic...... lipogenic enzymes. EXPERIMENTAL APPROACH: Gene and protein expression of lipogenic enzymes were examined in livers of wild-type (WT) and IL-6-deficient (IL-6(-/-) ) mice during fasting and re-feeding conditions. Effects of exogenous IL-6 administration on gene expression of these enzymes were evaluated...

Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

Science.gov (United States)

Goodwin, T. J.; McCarthy, M.; Lin, Y-H

2006-01-01

In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissues

DEFF Research Database (Denmark)

Covington, Elizabeth Dunn; Roitsch, Thomas Georg; Dermastia, Marina

2016-01-01

Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity...... assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf...

Congenital leptin deficiency and thyroid function

Directory of Open Access Journals (Sweden)

Paz-Filho Gilberto

2009-11-01

Full Text Available Abstract Thyroid function is closely related to leptin's secretion by the adipose tissue. In states of leptin-deficiency, the circadian rhythm of TSH is altered, leading to central hypothyroidism in animal models. In humans, central hypothyroidism has also been described in rare cases of congenital leptin deficiency. However, the thyroid phenotype in these cases is heterogeneous, with the occurrence of central hypothyroidism in a minority of cases. Here we describe thyroid function in four leptin-deficient humans (2 males aged 5 and 27, and 2 females aged 35 and 40, before and during leptin replacement with recombinant human methionyl leptin (r-metHuLeptin. The child was evaluated for four years, and the adults, for eight years. In addition, the adults were submitted to a brief withdrawal of leptin during six weeks in the sixth year. Our results show that, regardless of leptin replacement, our leptin-deficient patients have normal thyroid function. In spite of having an important role in regulating the hypothalamic-pituitary-thyroidal axis, leptin is not required for normal thyroid function. Trial Registration ClinicalTrials.gov Identifiers: NCT00659828 and NCT00657605

Human BLCAP transcript: new editing events in normal and cancerous tissues.

Science.gov (United States)

Galeano, Federica; Leroy, Anne; Rossetti, Claudia; Gromova, Irina; Gautier, Philippe; Keegan, Liam P; Massimi, Luca; Di Rocco, Concezio; O'Connell, Mary A; Gallo, Angela

2010-07-01

Bladder cancer-associated protein (BLCAP) is a highly conserved protein among species, and it is considered a novel candidate tumor suppressor gene originally identified from human bladder carcinoma. However, little is known about the regulation or the function of this protein. Here, we show that the human BLCAP transcript undergoes multiple A-to-I editing events. Some of the new editing events alter the highly conserved amino terminus of the protein creating alternative protein isoforms by changing the genetically coded amino acids. We found that both ADAR1 and ADAR2-editing enzymes cooperate to edit this transcript and that different tissues displayed distinctive ratios of edited and unedited BLCAP transcripts. Moreover, we observed a general decrease in BLCAP-editing level in astrocytomas, bladder cancer and colorectal cancer when compared with the related normal tissues. The newly identified editing events, found to be downregulated in cancers, could be useful for future studies as a diagnostic tool to distinguish malignancies or epigenetic changes in different tumors.

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

Science.gov (United States)

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

2016-02-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

Clinical significance of enzymatic deficiencies in the gastrointestinal tract with particular reference to lactase deficiency.

Science.gov (United States)

Rossi, E; Lentze, M J

1984-12-01

The study of deficiencies of small intestinal brush-border hydrolases increased our knowledge about the specific functions of hydrolases in the digestion of smaller molecules on the microvillus surface of the absorptive cells. The sucrase-isomaltase (SI) complex has been shown to be synthesized as a precursor (pro-sucrase-isomaltase) which is then incorporated into the membrane. The hydrophobic N-terminal end of the molecule is anchored in the lipid bilayer. In SI deficiency the molecular base of the disease is still not clear. Absence of SI activity could be due to complete lack of precursor synthesis or to structural changes within the N-terminal end of the SI-complex. Deficiencies of peptide hydrolases have not been reported with the exception of enteropeptidase (EP). Here a congenital deficiency of the enzyme was observed as the primary defect in enzyme synthesis within the enterocytes and as a secondary defect due to exocrine pancreatic insufficiency. In contrast to the primary EP deficiency, the activity of EP can be restored in the cases of exocrine pancreatic insufficiency by treatment with pancreatic extracts. Primary lactase deficiency exists in various forms. Besides congenital lactase deficiency, the late onset or adult type of lactase deficiency has been observed. The latter occurs in many different ethnic groups around the world. Here, using gel electrophoresis and immunoelectrophoresis, the lack of enzyme activity could be shown to be a primary defect in enzyme protein synthesis. In man and in the rat, two different lactases have been identified. In contrast to adult lactase, fetal lactase contains sialic acid at the end of carbohydrate side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethical tissue: a not-for-profit model for human tissue supply.

Science.gov (United States)

Adams, Kevin; Martin, Sandie

2011-02-01

Following legislative changes in 2004 and the establishment of the Human Tissue Authority, access to human tissues for biomedical research became a more onerous and tightly regulated process. Ethical Tissue was established to meet the growing demand for human tissues, using a process that provided ease of access by researchers whilst maintaining the highest ethical and regulatory standards. The establishment of a licensed research tissue bank entailed several key criteria covering ethical, legal, financial and logistical issues being met. A wide range of stakeholders, including the HTA, University of Bradford, flagged LREC, hospital trusts and clinical groups were also integral to the process.

Release of Zn from maternal tissues in pregnant rats deficient in Zn or Zn and Ca

International Nuclear Information System (INIS)

Hurley, L.S.; Masters, D.G.; Lonnerdal, B.; Keen, C.L.

1986-01-01

Earlier studies have shown that diets that increase tissue catabolism reduce the teratogenic effects of Zn deficiency. The hypothesis that Zn may be released from body tissues when the metabolic state is altered was further tested. Nonpregnant Sprague Dawley females were injected with Zn-65; after equilibration, the two major pools of Zn, bone and muscle, had different specific activities (SA), muscle being much higher. Females were mated and fed diets adequate in Zn and Ca (C) or deficient in Zn (ZnD) or deficient in both Zn and Ca (ZnCaD). Calculations using weight loss in ZnD and ZnCaD rats, Zn content of maternal bone and muscle, and total fetal Zn at term indicated that in ZnCaD rats a relatively small amount of Zn from bone early in pregnancy was sufficient to prevent abnormal organogenesis, but most fetal Zn came from breakdown of maternal muscle in the last 3 days of pregnancy. Isotope data supported this conclusion. SA of Zn in ZnD fetuses was equal and high, indicating that most Zn came from the same maternal tissue. High muscle SA prior to mating, and increased SA in tibia and liver during pregnancy suggest that muscle provided Zn for other maternal tissues as well as fetuses. In contrast, SA in C fetuses was less than 30% of that of the D groups, consistent with the earlier hypothesis that most fetal Zn in C rats is accrued directly from the diet

Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease.

Directory of Open Access Journals (Sweden)

Min Peng

2008-04-01

Full Text Available Coenzyme Q (CoQ is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2(kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2(kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2(loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2(loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment.

Strategies to rescue the consequences of inducible arginase-1 deficiency in mice.

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Laurel L Ballantyne

Full Text Available Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation either failed to extend lifespan (ornithine or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug. A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.

Arginase-1 deficiency.

Science.gov (United States)

Sin, Yuan Yan; Baron, Garrett; Schulze, Andreas; Funk, Colin D

2015-12-01

Arginase-1 (ARG1) deficiency is a rare autosomal recessive disorder that affects the liver-based urea cycle, leading to impaired ureagenesis. This genetic disorder is caused by 40+ mutations found fairly uniformly spread throughout the ARG1 gene, resulting in partial or complete loss of enzyme function, which catalyzes the hydrolysis of arginine to ornithine and urea. ARG1-deficient patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation, and infrequent episodes of hyperammonemia, a clinical pattern that differs strikingly from other urea cycle disorders. This review briefly highlights the current understanding of the etiology and pathophysiology of ARG1 deficiency derived from clinical case reports and therapeutic strategies stretching over several decades and reports on several exciting new developments regarding the pathophysiology of the disorder using ARG1 global and inducible knockout mouse models. Gene transfer studies in these mice are revealing potential therapeutic options that can be exploited in the future. However, caution is advised in extrapolating results since the lethal disease phenotype in mice is much more severe than in humans indicating that the mouse models may not precisely recapitulate human disease etiology. Finally, some of the functions and implications of ARG1 in non-urea cycle activities are considered. Lingering questions and future areas to be addressed relating to the clinical manifestations of ARG1 deficiency in liver and brain are also presented. Hopefully, this review will spark invigorated research efforts that lead to treatments with better clinical outcomes.

How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

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Kohn, Donald B; Gaspar, H Bobby

2017-05-01

Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

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Shimojo, M; Ricketts, M L; Petrelli, M D; Moradi, P; Johnson, G D; Bradwell, A R; Hewison, M; Howie, A J; Stewart, P M

1997-03-01

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the

The use of animal tissues alongside human tissue: Cultural and ethical considerations.

Science.gov (United States)

Kaw, Anu; Jones, D Gareth; Zhang, Ming

2016-01-01

Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

Science.gov (United States)

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

DEFF Research Database (Denmark)

Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

2002-01-01

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...... procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit approximately 0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated...... as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore...

Thioredoxin 1 in Prostate Tissue Is Associated with Gleason Score, Erythrocyte Antioxidant Enzyme Activity, and Dietary Antioxidants

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Terrence M. Vance

2015-01-01

Full Text Available Background. Prostate cancer is the most common noncutaneous cancer and second leading cause of cancer-related mortality in men in the US. Growing evidence suggests that oxidative stress is involved in prostate cancer. Methods. In this study, thioredoxin 1 (Trx 1, an enzyme and subcellular indicator of redox status, was measured in prostate biopsy tissue from 55 men from the North Carolina-Louisiana Prostate Cancer Project. A pathologist blindly scored levels of Trx 1. The association between Trx 1 and the Gleason score, erythrocyte antioxidant enzyme activity, and dietary antioxidant intake was determined using Fisher’s exact test. Results. Trx 1 levels in benign prostate tissue in men with incident prostate cancer were positively associated with the Gleason score (P=0.01 and inversely associated with dietary antioxidant intake (P=0.03. In prostate cancer tissue, Trx 1 levels were associated with erythrocyte glutathione peroxidase activity (P=0.01. No association was found for other erythrocyte enzymes. Greater Gleason score of malignant tissue corresponds to a greater difference in Trx 1 levels between malignant and benign tissue (P=0.04. Conclusion. These results suggest that the redox status of prostate tissue is associated with prostate cancer grade and both endogenous and exogenous antioxidants.

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

Science.gov (United States)

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

2016-01-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

Glutathione system in Wolfram syndrome 1‑deficient mice.

Science.gov (United States)

Porosk, Rando; Kilk, Kalle; Mahlapuu, Riina; Terasmaa, Anton; Soomets, Ursel

2017-11-01

Wolfram syndrome 1 (WS) is a rare neurodegenerative disease that is caused by mutations in the Wolfram syndrome 1 (WFS1) gene, which encodes the endoplasmic reticulum (ER) glycoprotein wolframin. The pathophysiology of WS is ER stress, which is generally considered to induce oxidative stress. As WS has a well‑defined monogenetic origin and a model for chronic ER stress, the present study aimed to characterize how glutathione (GSH), a major intracellular antioxidant, was related to the disease and its progression. The concentration of GSH and the activities of reduction/oxidation system enzymes GSH peroxidase and GSH reductase were measured in Wfs1‑deficient mice. The GSH content was lower in most of the studied tissues, and the activities of antioxidative enzymes varied between the heart, kidneys and liver tissues. The results indicated that GSH may be needed for ER stress control; however, chronic ER stress from the genetic syndrome eventually depletes the cellular GSH pool and leads to increased oxidative stress.

Antepartum Ornithine Transcarbamylase Deficiency

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Hitoshi Nakajima

2014-11-01

Full Text Available Ornithine transcarbamylase deficiency (OTCD is the most common type urea cycle enzyme deficiencies. This syndrome results from a deficiency of the mitochondrial enzyme ornithine transcarbamylase, which catalyzes the conversion of ornithine and carbamoyl phosphate to citrullin. Our case was a 28-year-old female diagnosed with OTCD following neurocognitive deficit during her first pregnancy. Although hyperammonemia was suspected as the cause of the patient's mental changes, there was no evidence of chronic liver disease. Plasma amino acid and urine organic acid analysis revealed OTCD. After combined modality treatment with arginine, sodium benzoate and hemodialysis, the patient's plasma ammonia level stabilized and her mental status returned to normal. At last she recovered without any damage left.

Marine Enzymes: Production and Applications for Human Health.

Science.gov (United States)

Rao, T Eswara; Imchen, M; Kumavath, R

Marine microbial enzymes have wide applications in bioindustries. Selection of microorganisms for enzyme production at the industrial level requires good yield and high production rate. A number of enzymes such as amylase, caseinase, lipase, gelatinase, and DNases have been discovered from microbes isolated from extreme marine environments. Such enzymes are thermostable, tolerant to a varied range of pH and other harsh conditions required in industrial applications. Novelty in their structure and characteristics has shown promising scope to the researchers in academia and industry. In this chapter, we present a bird's eye view on recent research works in the field of enzyme production from marine origin as well as their potential biological applications relevant to human health. © 2017 Elsevier Inc. All rights reserved.

Targeting Poxvirus Decapping Enzymes and mRNA Decay to Generate an Effective Oncolytic Virus

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Hannah Burgess

2018-03-01

Full Text Available Through the action of two virus-encoded decapping enzymes (D9 and D10 that remove protective caps from mRNA 5′-termini, Vaccinia virus (VACV accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy. Keywords: oncolytic virus, mRNA decay, decapping

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

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Thai Q Tran

2017-11-01

Full Text Available Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

Science.gov (United States)

Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

2017-11-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Drug repositioning for enzyme modulator based on human metabolite-likeness.

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Lee, Yoon Hyeok; Choi, Hojae; Park, Seongyong; Lee, Boah; Yi, Gwan-Su

2017-05-31

Recently, the metabolite-likeness of the drug space has emerged and has opened a new possibility for exploring human metabolite-like candidates in drug discovery. However, the applicability of metabolite-likeness in drug discovery has been largely unexplored. Moreover, there are no reports on its applications for the repositioning of drugs to possible enzyme modulators, although enzyme-drug relations could be directly inferred from the similarity relationships between enzyme's metabolites and drugs. We constructed a drug-metabolite structural similarity matrix, which contains 1,861 FDA-approved drugs and 1,110 human intermediary metabolites scored with the Tanimoto similarity. To verify the metabolite-likeness measure for drug repositioning, we analyzed 17 known antimetabolite drugs that resemble the innate metabolites of their eleven target enzymes as the gold standard positives. Highly scored drugs were selected as possible modulators of enzymes for their corresponding metabolites. Then, we assessed the performance of metabolite-likeness with a receiver operating characteristic analysis and compared it with other drug-target prediction methods. We set the similarity threshold for drug repositioning candidates of new enzyme modulators based on maximization of the Youden's index. We also carried out literature surveys for supporting the drug repositioning results based on the metabolite-likeness. In this paper, we applied metabolite-likeness to repurpose FDA-approved drugs to disease-associated enzyme modulators that resemble human innate metabolites. All antimetabolite drugs were mapped with their known 11 target enzymes with statistically significant similarity values to the corresponding metabolites. The comparison with other drug-target prediction methods showed the higher performance of metabolite-likeness for predicting enzyme modulators. After that, the drugs scored higher than similarity score of 0.654 were selected as possible modulators of enzymes for

Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.

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Sandra L Diaz

Full Text Available Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc, but can metabolically incorporate it from dietary sources (particularly red meat and milk into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac.We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA, Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell

Inhibition of tissue angiotensin converting enzyme. Quantitation by autoradiography

International Nuclear Information System (INIS)

Sakaguchi, K.; Chai, S.Y.; Jackson, B.; Johnston, C.I.; Mendelsohn, F.A.

1988-01-01

Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after administration of lisinopril, an ACE inhibitor. Tissue ACE was assessed by quantitative in vitro autoradiography using the ACE inhibitor [ 125 I]351A, as a ligand, and serum ACE was measured by a fluorimetric method. Following oral administration of lisinopril (10 mg/kg), serum ACE activity was acutely reduced but recovered gradually over 24 hours. Four hours after lisinopril administration, ACE activity was markedly inhibited in kidney (11% of control level), adrenal (8%), duodenum (8%), and lung (33%; p less than 0.05). In contrast, ACE in testis was little altered by lisinopril (96%). In brain, ACE activity was markedly reduced 4 hours after lisinopril administration in the circumventricular organs, including the subfornical organ (16-22%) and organum vasculosum of the lamina terminalis (7%; p less than 0.05). In other areas of the brain, including the choroid plexus and caudate putamen, ACE activity was unchanged. Twenty-four hours after administration, ACE activity in peripheral tissues and the circumventricular organs of the brain had only partially recovered toward control levels, as it was still below 50% of control activity levels. These results establish that lisinopril has differential effects on inhibiting ACE in different tissues and suggest that the prolonged tissue ACE inhibition after a single oral dose of lisinopril may reflect targets involved in the hypotensive action of ACE inhibitors

On the molecular basis of D-bifunctional protein deficiency type III.

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Maija L Mehtälä

Full Text Available Molecular basis of D-bifunctional protein (D-BP deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2 protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m, V(max and k(cat of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

Science.gov (United States)

Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

The "multiple hormone deficiency" theory of aging: is human senescence caused mainly by multiple hormone deficiencies?

Science.gov (United States)

Hertoghe, T

2005-12-01

In the human body, the productions, levels and cell receptors of most hormones progressively decline with age, gradually putting the body into various states of endocrine deficiency. The circadian cycles of these hormones also change, sometimes profoundly, with time. In aging individuals, the well-balanced endocrine system can fall into a chaotic condition with losses, phase-advancements, phase delays, unpredictable irregularities of nycthemeral hormone cycles, in particular in very old or sick individuals. The desynchronization makes hormone activities peak at the wrong times and become inefficient, and in certain cases health threatening. The occurrence of multiple hormone deficits and spilling through desynchronization may constitute the major causes of human senescence, and they are treatable causes. Several arguments can be put forward to support the view that senescence is mainly a multiple hormone deficiency syndrome: First, many if not most of the signs, symptoms and diseases (including cardiovascular diseases, cancer, obesity, diabetes, osteoporosis, dementia) of senescence are similar to physical consequences of hormone deficiencies and may be caused by hormone deficiencies. Second, most of the presumed causes of senescence such as excessive free radical formation, glycation, cross-linking of proteins, imbalanced apoptosis system, accumulation of waste products, failure of repair systems, deficient immune system, may be caused or favored by hormone deficiencies. Even genetic causes such as limits to cell proliferation (such as the Hayflick limit of cell division), poor gene polymorphisms, premature telomere shortening and activation of possible genetic "dead programs" may have links with hormone deficiencies, being either the consequence, the cause, or the major favoring factor of hormone deficiencies. Third, well-dosed and -balanced hormone supplements may slow down or stop the progression of signs, symptoms, or diseases of senescence and may often

Familial lipoprotein lipase deficiency

Science.gov (United States)

... lack an enzyme called lipoprotein lipase. Without this enzyme, the body cannot break down fat from digested food. Fat particles called chylomicrons build up in the blood. Risk factors include a family history of lipoprotein lipase deficiency. The condition is usually ...

A novel method to depurate β-lactam antibiotic residues by administration of a broad-spectrum β-lactamase enzyme in fish tissues

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Young-Sik Choe

2016-12-01

Full Text Available Abstract As a novel strategy to remove β-lactam antibiotic residues from fish tissues, utilization of β-lactamase, enzyme that normally degrades β-lactam structure-containing drugs, was explored. The enzyme (TEM-52 selectively degraded β-lactam antibiotics but was completely inactive against tetracycline-, quinolone-, macrolide-, or aminoglycoside-structured antibacterials. After simultaneous administration of the enzyme with cefazolin (a β-lactam antibiotic to the carp, significantly lowered tissue cefazolin levels were observed. It was confirmed that the enzyme successfully reached the general circulation after intraperitoneal administration, as the carp serum obtained after enzyme injection could also degrade cefazolin ex vivo. These results suggest that antibiotics-degrading enzymes can be good candidates for antibiotic residue depuration.

Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

Science.gov (United States)

Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

1995-10-01

Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of

Dihydropyrimidine Dehydrogenase Deficiency in Two Malaysian Siblings with Abnormal MRI Findings

NARCIS (Netherlands)

Chen, Bee Chin; Mohd Rawi, Rowani; Meinsma, Rutger; Meijer, Judith; Hennekam, Raoul C. M.; van Kuilenburg, André B. P.

2014-01-01

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine metabolism. Deficiency of this enzyme leads to an accumulation of thymine and uracil and a deficiency of metabolites distal to the catabolic enzyme. The disorder presents with a wide clinical

Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

Science.gov (United States)

Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

2010-08-01

Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

Science.gov (United States)

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Kinetic and structural evidences on human prolidase pathological mutants suggest strategies for enzyme functional rescue.

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Roberta Besio

Full Text Available Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells.

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Marjorie Gangolf

Full Text Available BACKGROUND: Thiamine (vitamin B1 is an essential molecule for all life forms because thiamine diphosphate (ThDP is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP, thiamine triphosphate (ThTP and adenosine thiamine triphosphate (AThTP, present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. METHODOLOGY AND PRINCIPAL FINDINGS: Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP]/([Thiamine]+[ThMP] ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. CONCLUSIONS AND SIGNIFICANCE: The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells

Comparative tissue distribution profiles of five major bio-active components in normal and blood deficiency rats after oral administration of Danggui Buxue Decoction by UPLC-TQ/MS.

Science.gov (United States)

Shi, Xuqin; Tang, Yuping; Zhu, Huaxu; Li, Weixia; Li, Zhenhao; Li, Wei; Duan, Jin-ao

2014-01-01

Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) were frequently combined and used in China as herbal pair called as Danggui Buxue Decoction (DBD) for treatment of blood deficiency syndrome, such as women's ailments. This study is to investigate the tissue distribution profiles of five major bio-active constituents (ferulic acid, caffeic acid, calycosin-7-O-β-glucoside, ononin and astragaloside IV) in DBD after oral administration of DBD in blood deficiency rats, and to compare the difference between normal and blood deficiency rats. The blood deficiency rats were induced by bleeding from orbit at the dosages of 5.0mLkg(-1) every day, and the experimental period was 12 days. At the finally day of experimental period, both normal and blood deficiency rats were orally administrated with DBD, and then the tissues samples were collected at different time points. Ferulic acid, caffeic acid, calycosin-7-O-β-glucoside, ononin and astragaloside IV in different tissues were detected simultaneously by UPLC-TQ/MS, and the histograms were drawn. The results showed that the overall trend was CLiver>CKidney>CHeart>CSpleen>CLung, CC-30min>CM-30min>CM-60min>CC-5min>CM-5min>CC-60min>CM-240min>CC-240min. The contents of the detected compounds in liver were more than that in other tissues no matter in normal or blood deficiency rats. Compared to normal rats, partial contents of the compounds in blood deficiency rats' tissues at different time points had significant difference (Pdistribution investigation in blood deficiency animals which is conducted by bleeding. And the results demonstrated that the five DBD components in normal and blood deficiency rats had obvious differences in some organs and time points, suggesting that the blood flow and perfusion rate of the organ were altered in blood deficiency animals. Copyright © 2013 Elsevier B.V. All rights reserved.

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

NARCIS (Netherlands)

Hogenboom, Sietske; Romeijn, Gerrit Jan; Houten, Sander M.; Baes, Myriam; Wanders, Ronald J. A.; Waterham, Hans R.

2002-01-01

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger

Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues.

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Gregory Lacraz

Full Text Available IL-15 is an inflammatory cytokine secreted by many cell types. IL-15 is also produced during physical exercise by skeletal muscle and has been reported to reduce weight gain in mice. Contrarily, our findings on IL-15 knockout (KO mice indicate that IL-15 promotes obesity. The aim of this study is to investigate the mechanisms underlying the pro-obesity role of IL-15 in adipose tissues.Control and IL-15 KO mice were maintained on high fat diet (HFD or normal control diet. After 16 weeks, body weight, adipose tissue and skeletal mass, serum lipid levels and gene/protein expression in the adipose tissues were evaluated. The effect of IL-15 on thermogenesis and oxygen consumption was also studied in primary cultures of adipocytes differentiated from mouse preadipocyte and human stem cells.Our results show that IL-15 deficiency prevents diet-induced weight gain and accumulation of lipids in visceral and subcutaneous white and brown adipose tissues. Gene expression analysis also revealed elevated expression of genes associated with adaptive thermogenesis in the brown and subcutaneous adipose tissues of IL-15 KO mice. Accordingly, oxygen consumption was increased in the brown adipocytes from IL-15 KO mice. In addition, IL-15 KO mice showed decreased expression of pro-inflammatory mediators in their adipose tissues.Absence of IL-15 results in decreased accumulation of fat in the white adipose tissues and increased lipid utilization via adaptive thermogenesis. IL-15 also promotes inflammation in adipose tissues that could sustain chronic inflammation leading to obesity-associated metabolic syndrome.

[Distributions of H3K27me3 and its modification enzymes in different tissues of mice].

Science.gov (United States)

Wang, Yuying; Wang, Xinli; Zhang, Ran; Zhang, Zhiyan; Wang, Yu; Yang, Bo; Wang, Guanjie; Zhang, Xin; Ma, Fuhao; Xu, Hongye; Wu, Xiaohui; Zhang, Feng; Li, Qing

2017-11-01

Objective To investigate the levels of trimethylated histone 3 at lysine residue 27 (H3K27me3) and its modification enzymes Zeste gene enhancer homolog 2 (EZH2), lysine-specific demethylase 6B (Kdm6B/JMJD3) and lysine-specific demethylase 6A (Kdm6A/UTX) in tissues and organs of 7-day and 2-month postnatal mice. Methods Immunohistochemistry was used to detect the expressions of H3K27me3 and its modification enzymes EZH2, JMJD3 and UTX in the brain, salivary glands, back fat, thymus, lung, heart, stomach, intestines, liver, testes, and skin of 7-day and 2-month mice. Real-time quantitative PCR was used to confirm the results. The relationships between H3K27me3 and its modification enzymes were analyzed statistically. Results Immunohistochemistry showed H3K27me3 persistently present in all examined tissues of 7-day and 2-month mice. EZH2 was persistently expressed in the brain, heart, liver, and skin of 7-day and 2-month mice, but only expressed in the salivary glands, adipose tissues, thymus, lung, intestines, and testes of 2-month mice. JMJD3 was expressed in the brain, salivary glands, adipose tissues, lung, heart, stomach, intestines, testes, skin of 7-day mice, but was not expressed in the lung, adipose tissues and stomach of 2-month mice. UTX was expressed in the brain, salivary glands, adipose tissues, lung, heart, testes, skin of 7-day mice, but only expressed in the testes of 2-month mice. Most mRNA of H3K27 modification enzymes were moderately or highly expressed as their immunohistochemical results were positive. Conclusion There was H3K27me3 persistently present in the all examined tissues at different stages. EZH2 was mostly expressed in the brain, salivary glands, adipose tissues, thymus, lung, heart, intestines, liver, testes and skin of 2-month-old mice. JMJD3 and UTX were mostly expressed in the brain, salivary glands, adipose tissues, lung, heart, skin and testes of 7-day-old mice. No significant association was found between the distribution of H3K

Transcriptomics resources of human tissues and organs

DEFF Research Database (Denmark)

Uhlén, Mathias; Hallström, Björn M.; Lindskog, Cecilia

2016-01-01

a framework for defining the molecular constituents of the human body as well as for generating comprehensive lists of proteins expressed across tissues or in a tissue-restricted manner. Here, we review publicly available human transcriptome resources and discuss body-wide data from independent genome......Quantifying the differential expression of genes in various human organs, tissues, and cell types is vital to understand human physiology and disease. Recently, several large-scale transcriptomics studies have analyzed the expression of protein-coding genes across tissues. These datasets provide...

Tissue-based map of the human proteome

DEFF Research Database (Denmark)

Uhlén, Mathias; Fagerberg, Linn; Hallström, Björn M.

2015-01-01

Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transc...

Biotin deficiency enhances the inflammatory response of human dendritic cells.

Science.gov (United States)

Agrawal, Sudhanshu; Agrawal, Anshu; Said, Hamid M

2016-09-01

The water-soluble biotin (vitamin B7) is indispensable for normal human health. The vitamin acts as a cofactor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte-derived DCs. Culture of DCs in a biotin-deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of the proinflammatory cytokines TNF-α, IL-12p40, IL-23, and IL-1β compared with LPS-activated DCs cultured in biotin-sufficient (control) and biotin-oversupplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN-γ and IL-17 indicating Th1/Th17 bias in T cells compared with cells maintained in biotin control or biotin-oversupplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency.

Chapter 8. Ionisation radiation and human organism. Radioactivity of human tissues

International Nuclear Information System (INIS)

Toelgyessy, J.; Harangozo, M.

2000-01-01

This is a chapter of textbook of radioecology for university students. In this chapter authors deal with ionisation radiation and human organism as well as with radioactivity of human tissues. Chapter consists of next parts: (1) Radiation stress of human organism; (2) Radioactivity of human tissues and the factors influencing radioactive contamination; (3) Possibilities of decreasing of radiation stress

Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

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Ana Rivera-Barahona

Full Text Available The spf/ash mouse model of ornithine transcarbamylase (OTC deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations.

Mining the human tissue proteome for protein citrullination.

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Lee, Chien-Yun; Wang, Dongxue; Wilhelm, Mathias; Zolg, Daniel Paul; Schmidt, Tobias; Schnatbaum, Karsten; Reimer, Ulf; Pontén, Fredrik; Uhlén, Mathias; Hahne, Hannes; Kuster, Bernhard

2018-04-02

Citrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ~2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins arguing that the development of specific enrichment methods would be required in order

Activities of asymmetric dimethylarginine-related enzymes in white adipose tissue are associated with circulating lipid biomarkers

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Iwasaki Hiroaki

2012-04-01

Full Text Available Abstract Background Asymmetric NG,NG-dimethylarginine (ADMA, an endogenous inhibitor of nitric oxide synthase, is regulated by the enzymatic participants of synthetic and metabolic processes, i.e., type I protein N-arginine methyltransferase (PRMT and dimethylarginine dimethylaminohydrolase (DDAH. Previous reports have demonstrated that circulating ADMA levels can vary in patients with type 1 and type 2 diabetes mellitus (T2DM. White adipose tissue expresses the full enzymatic machinery necessary for ADMA production and metabolism; however, modulation of the activities of adipose ADMA-related enzymes in T2DM remains to be determined. Methods A rodent model of T2DM using 11- and 20-week old Goto-Kakizaki (GK rats was used. The expression and catalytic activity of PRMT1 and DDAH1 and 2 in the white adipose tissues (periepididymal, visceral and subcutaneous fats and femur skeletal muscle tissue were determined by immunoblotting, in vitro methyltransferase and in vitro citrulline assays. Results Non-obese diabetic GK rats showed low expression and activity of adipose PRMT1 compared to age-matched Wistar controls. Adipose tissues from the periepididymal, visceral and subcutaneous fats of GK rats had high DDAH1 expression and total DDAH activity, whereas the DDAH2 expression was lowered below the control value. This dynamic of ADMA-related enzymes in white adipose tissues was distinct from that of skeletal muscle tissue. GK rats had lower levels of serum non-esterified fatty acids (NEFA and triglycerides (TG than the control rats. In all subjects the adipose PRMT1 and DDAH activities were statistically correlated with the levels of serum NEFA and TG. Conclusion Activities of PRMT1 and DDAH in white adipose tissues were altered in diabetic GK rats in an organ-specific manner, which was reflected in the serum levels of NEFA and TG. Changes in adipose ADMA-related enzymes might play a part in the function of white adipose tissue.

Rapid release of tissue enzymes into blood after blast exposure: potential use as biological dosimeters.

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Peethambaran Arun

Full Text Available Explosive blast results in multiple organ injury and polytrauma, the intensity of which varies with the nature of the exposure, orientation, environment and individual resilience. Blast overpressure alone may not precisely indicate the level of body or brain injury after blast exposure. Assessment of the extent of body injury after blast exposure is important, since polytrauma and systemic factors significantly contribute to blast-induced traumatic brain injury. We evaluated the activity of plasma enzymes including aspartate aminotransferase (AST, alanine aminotransferase (ALT, lactate dehydrogenase (LDH and creatine kinase (CK at different time points after blast exposure using a mouse model of single and repeated blast exposures to assess the severity of injury. Our data show that activities of all the enzymes in the plasma were significantly increased as early as 1 h after blast exposure. The elevated enzyme activity remained up to 6 h in an overpressure dose-dependent manner and returned close to normal levels at 24 h. Head-only blast exposure with body protection showed no increase in the enzyme activities suggesting that brain injury alone does not contribute to the systemic increase. In contrast to plasma increase, AST, ALT and LDH activity in the liver and CK in the skeletal muscle showed drastic decrease at 6 h after blast exposures. Histopathology showed mild necrosis at 6 h and severe necrosis at 24 h after blast exposures in liver and no changes in the skeletal muscle suggesting that the enzyme release from the tissue to plasma is probably triggered by transient cell membrane disruption from shockwave and not due to necrosis. Overpressure dependent transient release of tissue enzymes and elevation in the plasma after blast exposure suggest that elevated enzyme activities in the blood can be potentially used as a biological dosimeter to assess the severity of blast injury.

Clinical Aspects of Trace Elements: Zinc in Human Nutrition – Zinc Deficiency and Toxicity

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Michelle M Pluhator

1996-01-01

Full Text Available Available evidence suggests that trace elements, such as zinc, once thought to have no nutritional relevance, are possibly deficient in large sections of the human population. Conditioned deficiencies have been reported to result from malabsorption syndromes, acrodermatitis enteropathica, alcoholism, gastrointestinal disease, thermal injury, chronic diseases (eg, diabetes, sickle cell anemia, and in total parenteral nutrition therapy. Awareness that patients with these problems are at risk has led health professionals to focus increasingly on the importance of zinc therapy in the prevention and treatment of deficiency. More recently zinc toxicity and its role in human nutrition and well-being have come under investigation. Reports have focused on the role of zinc toxicity in causes of copper deficiency, changes in the immune system and alterations in blood lipids. As the numerous challenges presented by the study of zinc in human nutrition are met, more appropriate recommendations for dietary and therapeutic zinc intake are being made.

Peroxisome proliferator-activated receptor α (PPARα mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue

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Kurokawa Tsuyoshi

2011-12-01

Full Text Available Abstract Background Peroxisome proliferator-activated receptor α (PPARα regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer. Methods The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system. Results The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections. Conclusion The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

Contrasting Features of Urea Cycle Disorders in Human Patients and Knockout Mouse Models

OpenAIRE

Deignan, Joshua L.; Cederbaum, Stephen D.; Grody, Wayne W.

2007-01-01

The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a cha...

mtDNA depletion myopathy: elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency.

Science.gov (United States)

Saada, Ann; Shaag, Avraham; Elpeleg, Orly

2003-05-01

Decreased mitochondrial thymidine kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency.

MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells.

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Christoph Campregher

Full Text Available BACKGROUND/AIM: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. METHODS: HCT116 and HCT116+chr3 (both MSH3-deficient and primary human colon epithelial cells (HCEC, MSH3-wildtype were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs were assessed by Comet assay. RESULTS: Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4 at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50, apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. CONCLUSIONS: MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon

Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration

International Nuclear Information System (INIS)

Chowdhury, Uttam K.; Zakharyan, Robert A.; Hernandez, Alba; Avram, Mihaela D.; Kopplin, Michael J.; Aposhian, H. Vasken

2006-01-01

Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic + 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested. The livers of male knockout (KO) mice, in which 222 bp of Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate

Random lasing in human tissues

International Nuclear Information System (INIS)

Polson, Randal C.; Vardeny, Z. Valy

2004-01-01

A random collection of scatterers in a gain medium can produce coherent laser emission lines dubbed 'random lasing'. We show that biological tissues, including human tissues, can support coherent random lasing when infiltrated with a concentrated laser dye solution. To extract a typical random resonator size within the tissue we average the power Fourier transform of random laser spectra collected from many excitation locations in the tissue; we verified this procedure by a computer simulation. Surprisingly, we found that malignant tissues show many more laser lines compared to healthy tissues taken from the same organ. Consequently, the obtained typical random resonator was found to be different for healthy and cancerous tissues, and this may lead to a technique for separating malignant from healthy tissues for diagnostic imaging

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

Science.gov (United States)

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

Cartilage oligomeric matrix protein deficiency promotes early onset and the chronic development of collagen-induced arthritis

DEFF Research Database (Denmark)

Geng, Hui; Carlsen, Stefan; Nandakumar, Kutty

2008-01-01

ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a homopentameric protein in cartilage. The development of arthritis, like collagen-induced arthritis (CIA), involves cartilage as a target tissue. We have investigated the development of CIA in COMP-deficient mice. METHODS: COMP......-deficient mice in the 129/Sv background were backcrossed for 10 generations against B10.Q mice, which are susceptible to chronic CIA. COMP-deficient and wild-type mice were tested for onset, incidence, and severity of arthritis in both the collagen and collagen antibody-induced arthritis models. Serum anti......-collagen II and anti-COMP antibodies as well as serum COMP levels in arthritic and wild-type mice were measured by enzyme-linked immunosorbent assay. RESULTS: COMP-deficient mice showed a significant early onset and increase in the severity of CIA in the chronic phase, whereas collagen II-antibody titers were...

Functional characterization of genetic enzyme variations in human lipoxygenases

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Thomas Horn

2013-01-01

Full Text Available Mammalian lipoxygenases play a role in normal cell development and differentiation but they have also been implicated in the pathogenesis of cardiovascular, hyperproliferative and neurodegenerative diseases. As lipid peroxidizing enzymes they are involved in the regulation of cellular redox homeostasis since they produce lipid hydroperoxides, which serve as an efficient source for free radicals. There are various epidemiological correlation studies relating naturally occurring variations in the six human lipoxygenase genes (SNPs or rare mutations to the frequency for various diseases in these individuals, but for most of the described variations no functional data are available. Employing a combined bioinformatical and enzymological strategy, which included structural modeling and experimental site-directed mutagenesis, we systematically explored the structural and functional consequences of non-synonymous genetic variations in four different human lipoxygenase genes (ALOX5, ALOX12, ALOX15, and ALOX15B that have been identified in the human 1000 genome project. Due to a lack of a functional expression system we resigned to analyze the functionality of genetic variations in the hALOX12B and hALOXE3 gene. We found that most of the frequent non-synonymous coding SNPs are located at the enzyme surface and hardly alter the enzyme functionality. In contrast, genetic variations which affect functional important amino acid residues or lead to truncated enzyme variations (nonsense mutations are usually rare with a global allele frequency<0.1%. This data suggest that there appears to be an evolutionary pressure on the coding regions of the lipoxygenase genes preventing the accumulation of loss-of-function variations in the human population.

Peripheral kynurenine-3-monooxygenase deficiency as a potential risk factor for metabolic syndrome in schizophrenia patients.

Science.gov (United States)

Oxenkrug, Gregory; van der Hart, Marieke; Roeser, Julien; Summergrad, Paul

2017-01-01

Increased predisposition of schizophrenia patients (SP) to development of obesity and insulin resistance suggested common signaling pathway between metabolic syndrome (MetS) and schizophrenia. Deficiency of kynurenine-3-monooxygenase (KMO), enzyme catalyzing formation of 3-hydroxykynurenine (3-HK) from kynurenine (Kyn), a tryptophan (Trp) metabolite, might contribute to development of MetS as suggested by non-expression of KMO genes in human fat tissue and elevated serum concentrations of Kyn and its metabolites, kynurenic (KYNA) and anthranilic (ANA) acids, in diabetic patients and Zucker fatty rats (ZFR). Markers of KMO deficiency: decreased 3-HK and elevated Kyn, KYNA and ANA, were observed in brains and spinal fluids of SP, and in brains and serum of experimental animals with genetically- or pharmacologically-induced KMO deficiency. However, elevated concentrations of ANA and decreased 3-HK were reported in serum of SP without concurrent increase of Kyn and KYNA. Present study aimed to re-assess serum Kyn metabolites (HPLC-MS) in a sub-group of SP with elevated KYNA. We found increased Kyn concentrations (by 30%) and Kyn:Trp ratio (by 20%) in serum of SP with elevated KYNA concentrations (by 40%). Obtained results and our previous data suggest that peripheral KMO deficiency might be manifested by, at least, two different patterns: elevated ANA with decreased 3-HK; and elevated KYNA and KYN. The latter pattern was previously described in type 2 diabetes patients and might underline increased predisposition of SP to development of MetS. Assessment of peripheral KMO deficiency might identify SP predisposed to MetS. Attenuation of the consequences of peripheral KMO deficiency might be a new target for prevention/treatment of obesity and diabetes in SP.

Leukocyte adhesion deficiencies

NARCIS (Netherlands)

van de Vijver, Edith; van den Berg, Timo K.; Kuijpers, Taco W.

2013-01-01

During inflammation, leukocytes play a key role in maintaining tissue homeostasis through elimination of pathogens and removal of damaged tissue. Leukocytes migrate to the site of inflammation by crawling over and through the blood vessel wall, into the tissue. Leukocyte adhesion deficiencies (ie,

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Science.gov (United States)

Tong, Wenyong; Dwyer, Chrissa A; Thacker, Bryan E; Glass, Charles A; Brown, Jillian R; Hamill, Kristina; Moremen, Kelley W; Sarrazin, Stéphane; Gordts, Philip L S M; Dozier, Lara E; Patrick, Gentry N; Tor, Yitzhak; Esko, Jeffrey D

2017-12-06

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Micronuclei Formation and 8-Hydroxy-2-Deoxyguanosine Enzyme Detection in Ovarian Tissues After Radiofrequency Exposure at 1800 MHz in Adult Sprague–Dawley Rats

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Ali Saeed Hammoodi Alchalabi

2017-04-01

Full Text Available Human fertility and its correlation to ovarian function and cytological changes are linked to ever-increasing use of mobile phones. Wireless communications have become a critical topic of concern because of an increasing number of studies in this field with controversial outcomes. The aim of this study was to assess the genotoxic effect of GSM frequency at 1800 MHz on ovarian function. Sixty female Sprague–Dawley rats were distributed over six groups (control group and the exposure groups with whole-body exposure for 2 h/day, 7 days/week for 15, 30 and 60 continuous days. The study investigated the oxidative stress, 8-hydroxy-2-deoxyguanosine enzyme, micronuclei formation and histopathological changes in ovarian tissue. The results showed an induced oxidative stress via an increase in lipid peroxidation and decreased antioxidant enzyme activity. There was also an elevation in the 8-hydroxy-2-deoxyguanosine enzyme and an increased rate of micronuclei formation in ovarian tissues of exposed animals with 60-day exposure compared with control animals. Cytological changes were recorded such as micronuclei formation, vacuolation, degeneration and impaired folliculogenesis. The study suggests that GSM frequency at 1800 MHz was negatively impacted on female reproductive performances mediated by oxidative stress induction and 8-hydroxy-2-deoxyguanosine formation leading to overall impaired ovarian function.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

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Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

2016-01-01

Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

Leptin Deficiency: Clinical Implications and Opportunities for Therapeutic Interventions

OpenAIRE

Bl?her, Susan; Shah, Sunali; Mantzoros, Christos S.

2009-01-01

The discovery of leptin has significantly advanced our understanding of the metabolic importance of adipose tissue and has revealed that both leptin deficiency and leptin excess are associated with severe metabolic, endocrine, and immunological consequences. We and others have shown that a prominent role of leptin in humans is to mediate the neuroendocrine adaptation to energy deprivation. Humans with genetic mutations in the leptin and leptin receptor genes have deregulated food intake and e...

[Significant decrease in factor VII activity by tissue thromboplastin derived from rabbit brain in a patient with congenital factor VII deficiency (FVII Padua)].

Science.gov (United States)

Sekiya, Akiko; Morishita, Eriko; Maruyama, Keiko; Asakura, Hidesaku; Nakao, Shinji; Ohtake, Shigeki

2012-03-01

Congenital factor VII (FVII) deficiency is a bleeding disorder that requires optimal hemostatic management for each case due to its wide variety of bleeding symptoms. We experienced a patient with inherited FVII deficiency who demonstrated different FVII activities depending on tissue thromboplastins used for assays. An 82-year-old woman without any episodes of abnormal bleeding was found to have different FVII activities of 1.4% and 32% when assayed using thromboplastins from rabbit brain and human placenta, respectively. DNA sequencing analysis revealed a homozygous missense mutation of G10828A (FVII Padua) that caused an amino acid substitution of Arg304 to Gln (R304Q). Carriers of 304Q alleles are usually clinically asymptomatic and do not require FVII replacement therapies even in cases of homozygotes. In case a prolonged prothrombin time or reduced FVII activity is detected, re-examination using thromboplastins of other sources can be helpful for preliminary diagnosis of R304Q, in order to prevent unnecessary FVII replacement therapies.

Chymase-dependent generation of angiotensin II from angiotensin-(1-12 in human atrial tissue.

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Sarfaraz Ahmad

Full Text Available Since angiotensin-(1-12 [Ang-(1-12] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12 by plasma membranes (PM isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure. PM was incubated with highly purified ¹²⁵I-Ang-(1-12 at 37°C for 1 h with or without renin-angiotensin system (RAS inhibitors [lisinopril for angiotensin converting enzyme (ACE, SCH39370 for neprilysin (NEP, MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. ¹²⁵I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, ¹²⁵I-Ang-(1-12 was converted into Ang I (2±2%, Ang II (69±21%, Ang-(1-7 (5±2%, and Ang-(1-4 (2±1%. In the absence of all RAS inhibitor, only 22±10% of ¹²⁵I-Ang-(1-12 was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of ¹²⁵I-Ang-(1-12 remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that ¹²⁵I-Ang-(1-12 was primarily converted into Ang II (65±18% by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol × min⁻¹ × mg⁻¹, n = 9 from ¹²⁵I-Ang-(1-12 and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min⁻¹ × mg⁻¹. Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12.

Normal mast cell numbers in the tissues of AhR-deficient mice.

Science.gov (United States)

Pilz, Caroline; Feyerabend, Thorsten; Sonner, Jana; Redaelli, Chiara; Peter, Katharina; Kunze, Anja; Haas, Katharina; Esser, Charlotte; Schäkel, Knut; Wick, Wolfgang; Rodewald, Hans-Reimer; Lanz, Tobias V; Platten, Michael

2016-01-01

The transcription factor aryl hydrocarbon receptor (AhR) acts as an immunomodulatory molecule in several immune cell lineages. Recently, it has been implicated in development and maintenance of immune cells in barrier tissues such as skin and mucosa. To investigate its role on mast cell development and maintenance in skin, peritoneal exudate cells (PECs) and lymph nodes, we studied in depth their phenotype in AhR-deficient mice. Our findings do not provide any evidence for a suspected role of the AhR in mast cell homeostasis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Humanized Mouse Model Generated Using Surplus Neonatal Tissue

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Matthew E. Brown

2018-04-01

Full Text Available Summary: Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs. Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue. : Corresponding author William Burlingham and colleagues created a humanized mouse model called the NeoThy. The NeoThy uses human neonatal, rather than fetal, tissue sources for generating a human immune system within immunocompromised mouse hosts. NeoThy mice are an attractive alternative to conventional humanized mouse models, as they enable robust and reproducible iPSC immunogenicity experiments in vivo. Keywords: NeoThy, humanized mouse, iPSC, PSC, immunogenicity, transplantation, immunology, hematopoietic stem cells, induced pluripotent stem cells, thymus

Contrasting features of urea cycle disorders in human patients and knockout mouse models.

Science.gov (United States)

Deignan, Joshua L; Cederbaum, Stephen D; Grody, Wayne W

2008-01-01

The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders.

31P-NMR study of human pyrimidine 5'-nucleotidase deficient erythrocytes

International Nuclear Information System (INIS)

Higaki, Tsuyoshi; Kagimoto, Tadashi; Nagata, Koichi; Tanase, Sumio; Morino, Yoshimasa; Takatsuki, Kiyoshi

1982-01-01

Metabolic disorder of nucleotides in human pyrimidine 5'-nucleotidase (P5N) deficient erythrocytes was studied by 31 P-NMR with high resolution. Identification by combination of high-speed liquid chromatography revealed two-fold increases from the normal in the spectra in the α-, β- and γ-zones of nucleoside triphosphates of P5N deficient erythrocytes, 2,3-diphosphoglycerate shifted to the 0.3 ppm low magnetic field and signals of NAD and UDP-sugars(s) in the diphosphodiester zone. These results were obtained from the 31 P-NMR spectrum about one hour after blood sampling, indicating the high utility of this NMR for the diagnosis of P5N deficiency. (Chiba, N.)

High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue

International Nuclear Information System (INIS)

Magnusson, Lisa U.; Lundqvist, Annika; Asp, Julia; Synnergren, Jane; Johansson, Cecilia Thalén; Palmqvist, Lars; Jeppsson, Anders; Hultén, Lillemor Mattsson

2012-01-01

Highlights: ► We found a 17-fold upregulation of ALOX15 in the ischemic heart. ► Incubation of human muscle cells in hypoxia showed a 22-fold upregulation of ALOX15. ► We observed increased levels of proinflammatory markers in ischemic heart tissue. ► Suggesting a link between ischemia and inflammation in ischemic heart biopsies. -- Abstract: A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1α (HIF-1α) regulates adaptive responses to low concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression of ALOX15, and found an almost 2-fold increase in HIF-1α mRNA expression and a 17-fold upregulation of ALOX15 mRNA expression in the ischemic heart biopsies from patients undergoing coronary bypass surgery compared with non ischemic heart tissue. To investigate the effect of low oxygen concentration on ALOX15 we incubated human vascular muscle cells in hypoxia and showed that expression of ALOX15 increased 22-fold compared with cells incubated in normoxic conditions. We also observed increased mRNA levels of proinflammatory markers in ischemic heart tissue compared with non-ischemic controls. In summary, we demonstrate increased ALOX15 in human ischemic heart biopsies. Furthermore we demonstrate that hypoxia increases ALOX15 in human muscle cells. Our results yield important insights into the underlying association between hypoxia and inflammation in the human ischemic heart disease.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

Science.gov (United States)

Shi, Hongjun; Enriquez, Annabelle; Rapadas, Melissa; Martin, Ella M M A; Wang, Roni; Moreau, Julie; Lim, Chai K; Szot, Justin O; Ip, Eddie; Hughes, James N; Sugimoto, Kotaro; Humphreys, David T; McInerney-Leo, Aideen M; Leo, Paul J; Maghzal, Ghassan J; Halliday, Jake; Smith, Janine; Colley, Alison; Mark, Paul R; Collins, Felicity; Sillence, David O; Winlaw, David S; Ho, Joshua W K; Guillemin, Gilles J; Brown, Matthew A; Kikuchi, Kazu; Thomas, Paul Q; Stocker, Roland; Giannoulatou, Eleni; Chapman, Gavin; Duncan, Emma L; Sparrow, Duncan B; Dunwoodie, Sally L

2017-08-10

Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Dietary Components Affect the Plasma and Tissue Levels of Lutein in Aged Rats with Lutein Deficiency--A Repeated Gavage and Dietary Study.

Science.gov (United States)

Sheshappa, Mamatha Bangera; Ranganathan, Arunkumar; Bhatiwada, Nidhi; Talahalli, Ramprasad Ravichandra; Vallikannan, Baskaran

2015-10-01

The aim of this study was to find out the influence of selected dietary components on plasma and tissue response of repeated micellar and dietary lutein in aged rats with lutein deficiency. In repeated (16 d) gavage study, micellar lutein was co-ingested with either phosphatidylcholine (PC), lyso-phosphatidylcholine (lysoPC), β-carotene, dietary fiber or vegetable fat (3% soybean oil). In dietary study, rats were fed (4 wk) semi-synthetic diet either with lutein + PC, lutein + dietary fiber or B. alba (lutein source) + PC. The post-prandial plasma and tissue response of lutein was measured by HPLC. Results showed that micellar fat, PC and lysoPC significantly (P ≤ 0.05) increased the lutein levels in plasma (31.1%, 26.8%, and 34.9%), liver (27.4%, 29.5%, and 8.6%), and eyes (63.5%, 90.2%, and 86%) compared to the control group (group gavaged micelles with no dietary components studied). Similarly, dietary study showed an enhanced plasma, liver, and eye lutein levels by 44.8%, 24.1%, and 42.0% (lutein + PC group) and 51.7%, 39.8%, and 31.7% (B.alba + PC group), respectively compared to control. The activity of antioxidant enzymes in plasma and liver of both the studies were also affected compared to control. Result reveals, that PC enhance the intestinal absorption of both micellar and dietary lutein which is either in free or bound form with food matrices in aged rats with lutein deficiency. Hence, PC at a concentration used in this study can be considered to improve the lutein bioavailability in lutein deficiency. Lutein and zeaxanthin are macular pigments acquired mostly from greens, that play an significant role in protecting vision from Age related macular degeneration (AMD). However, their biological availability is poor and affected by dietary components. This study demonstrates the positive influence of dietary PC and lyso PC in improving intestinal uptake of lutein. Our previous and present finding shows there is a possibility of developing functional

Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

Science.gov (United States)

Sun, Ren; Wang, Liya

2014-10-07

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

DEFF Research Database (Denmark)

Selman, L; Henriksen, M L; Brandt, J

2012-01-01

-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies....... The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined...... and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes....

Expression of human DNA polymerase β in Escherichia coli and characterization of the recombinant enzyme

International Nuclear Information System (INIS)

Abbotts, J.; SenGupta, D.N.; Zmudzka, B.; Widen, S.G.; Notario, V.; Wilson, S.H.

1988-01-01

The coding region of a human β-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural β-polymerases. The purified enzyme was free of nuclease activity. The authors studied detailed catalytic properties of the recombinant β-polymerase using defined template-primer systems. The results indicate that this β-polymerase is essentially identical with natural β-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N 3 -methyl-dT or O 6 -methyl-dG

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism.

Science.gov (United States)

Mohammed, Akram; Guda, Chittibabu

2015-01-01

Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

Science.gov (United States)

2015-01-01

Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency

DEFF Research Database (Denmark)

Jackson, Robert S; Creemers, John W M; Farooqi, I Sadaf

2003-01-01

. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A's negligible PC1 activity......We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound...

Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis.

Science.gov (United States)

Hsieh, En-Jung; Waters, Brian M

2016-10-01

Iron (Fe) is an essential mineral that has low solubility in alkaline soils, where its deficiency results in chlorosis. Whether low Fe supply and alkaline pH stress are equivalent is unclear, as they have not been treated as separate variables in molecular physiological studies. Additionally, molecular responses to these stresses have not been studied in leaf and root tissues simultaneously. We tested how plants with the Strategy I Fe uptake system respond to Fe deficiency at mildly acidic and alkaline pH by measuring root ferric chelate reductase (FCR) activity and expression of selected Fe uptake genes and riboflavin synthesis genes. Alkaline pH increased cucumber (Cucumis sativus L.) root FCR activity at full Fe supply, but alkaline stress abolished FCR response to low Fe supply. Alkaline pH or low Fe supply resulted in increased expression of Fe uptake genes, but riboflavin synthesis genes responded to Fe deficiency but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but alkaline stress blocked up-regulation of these genes in Fe-deficient leaves. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These results suggest a model implicating shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

TLR4 deficiency promotes autophagy during cigarette smoke-induced pulmonary emphysema.

Science.gov (United States)

An, Chang Hyeok; Wang, Xiao Mei; Lam, Hilaire C; Ifedigbo, Emeka; Washko, George R; Ryter, Stefan W; Choi, Augustine M K

2012-11-01

Toll-like receptors (TLRs) exert important nonimmune functions in lung homeostasis. TLR4 deficiency promotes pulmonary emphysema. We examined the role of TLR4 in regulating cigarette smoke (CS)-induced autophagy, apoptosis, and emphysema. Lung tissue was obtained from chronic obstructive lung disease (COPD) patients. C3H/HeJ (Tlr4-mutated) mice and C57BL/10ScNJ (Tlr4-deficient) mice and their respective control strains were exposed to chronic CS or air. Human or mouse epithelial cells (wild-type, Tlr4-knockdown, and Tlr4-deficient) were exposed to CS-extract (CSE). Samples were analyzed for TLR4 expression, and for autophagic or apoptotic proteins by Western blot analysis or confocal imaging. Chronic obstructive lung disease lung tissues and human pulmonary epithelial cells exposed to CSE displayed increased TLR4 expression, and increased autophagic [microtubule-associated protein-1 light-chain-3B (LC3B)] and apoptotic (cleaved caspase-3) markers. Beas-2B cells transfected with TLR4 siRNA displayed increased expression of LC3B relative to control cells, basally and after exposure to CSE. The basal and CSE-inducible expression of LC3B and cleaved caspase-3 were elevated in pulmonary alveolar type II cells from Tlr4-deficient mice. Wild-type mice subjected to chronic CS-exposure displayed airspace enlargement;, however, the Tlr4-mutated or Tlr4-deficient mice exhibited a marked increase in airspace relative to wild-type mice after CS-exposure. The Tlr4-mutated or Tlr4-deficient mice showed higher levels of LC3B under basal conditions and after CS exposure. The expression of cleaved caspase-3 was markedly increased in Tlr4-deficient mice exposed to CS. We describe a protective regulatory function of TLR4 against emphysematous changes of the lung in response to CS.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

Science.gov (United States)

Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

Glucose-6-phosphate dehydrogenase deficiency: an unusual cause of acute jaundice after paracetamol overdose.

Science.gov (United States)

Phillpotts, Simon; Tash, Elliot; Sen, Sambit

2014-11-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest human enzyme defect causing haemolytic anaemia after exposure to specific triggers. Paracetamol-induced haemolysis in G6PD deficiency is a rare complication and mostly reported in children. We report the first case (to the best of our knowledge) of acute jaundice without overt clinical features of a haemolytic crisis, in an otherwise healthy adult female following paracetamol overdose, due to previously undiagnosed G6PD deficiency. It is important that clinicians consider this condition when a patient presents following a paracetamol overdose with significant and disproportionate jaundice, without transaminitis or coagulopathy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A rare case of Addison's disease, hepatitis, thyreoiditis, positive IgG anti-tissue transglutaminase antibodies and partial IgA deficiency.

Science.gov (United States)

Baleva, Marta P; Mihaylova, Snejina; Yankova, Petja; Atanasova, Iliana; Nikolova-Vlahova, Milena; Naumova, Elissaveta

2016-01-01

Selective IgA deficiency (IgAD) is the most prevalent type of primary immune deficiencies, but partial IgA deficiency is even more common. Addison's disease is a rare condition associated with primary adrenal insufficiency due to infection or autoimmune destruction of the adrenals. The association between IgA deficiency and Addison's disease is very rare. We observed a 22-year-old male patient with marked darkening of the skin, especially on the palms and areolae, jaundice on the skin and sclera, astheno-adynamia, hypotension (80/50 mm Hg), and pain in the right hypochondrium. The laboratory investigations revealed increased serum levels of total and indirect bilirubin, AST, ALT, GGT and LDH, negative HBsAg, anti-HBc IgM, anti-HCV and anti-HAV IgM, very low serum IgA levels (0.16 g/l) with normal IgG and IgM, negative ANA, ANCA, AMA, LKM-1, anti-GAD-60, anti-IA-2, anti-thyroglobulin antibodies, a mild increase in anti-TPO antibodies titer, a marked increase in IgG anti-tissue transglutaminase antibodies, with no typical changes in cellular immunity, negative T-SPOT-TB test, HLA - A*01; B*08; DRB1*03; DQB1*02, karyotype - 46, XY. We present a rare case of partial IgA deficiency with Addison's disease, hepatitis, thyroiditis and positive anti-tissue transglutaminase antibodies. IgAD and some autoimmune disorders share several predisposing HLA genes, thus explaining the increased prevalence of IgAD in certain patient groups.

Molecular diagnosis and characterization of medium-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Andresen, B S; Bross, P; Jensen, T G

1995-01-01

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans. It is an autosomal recessive disorder which usually presents in infancy. The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may...... of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous...

Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

Science.gov (United States)

Nagatsu, Toshiharu; Nagatsu, Ikuko

2016-11-01

Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

Leptin deficiency: clinical implications and opportunities for therapeutic interventions.

Science.gov (United States)

Blüher, Susan; Shah, Sunali; Mantzoros, Christos S

2009-10-01

The discovery of leptin has significantly advanced our understanding of the metabolic importance of adipose tissue and has revealed that both leptin deficiency and leptin excess are associated with severe metabolic, endocrine, and immunological consequences. We and others have shown that a prominent role of leptin in humans is to mediate the neuroendocrine adaptation to energy deprivation. Humans with genetic mutations in the leptin and leptin receptor genes have deregulated food intake and energy expenditure leading to a morbidly obese phenotype and a disrupted regulation in neuroendocrine and immune function and in glucose and fat metabolism. Observational and interventional studies in humans with (complete) congenital leptin deficiency caused by mutations in the leptin gene or with relative leptin deficiency as seen in states of negative energy balance such as lipoatrophy, anorexia nervosa, or exercise-induced hypothalamic and neuroendocrine dysfunction have contributed to the elucidation of the pathophysiological role of leptin in these conditions and of the clinical significance of leptin administration in these subjects. More specifically, interventional studies have demonstrated that several neuroendocrine, metabolic, or immune disturbances in these states could be restored by leptin administration. Leptin replacement therapy is currently available through a compassionate use program for congenital complete leptin deficiency and under an expanded access program to subjects with leptin deficiency associated with congenital or acquired lipoatrophy. In addition, leptin remains a potentially forthcoming treatment for several other states of energy deprivation including anorexia nervosa or milder forms of hypothalamic amenorrhea pending appropriate clinical trials.

Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Science.gov (United States)

Solleti, Siva Kumar; Srisuma, Sorachai; Bhattacharya, Soumyaroop; Rangel-Moreno, Javier; Bijli, Kaiser M; Randall, Troy D; Rahman, Arshad; Mariani, Thomas J

2016-07-01

Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2(-/-) mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2(-/-) mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.-Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel-Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation. © FASEB.

Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations

Directory of Open Access Journals (Sweden)

Timothy J. Bauler

2011-03-01

SHP-2 (encoded by PTPN11 is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities. To study how SHP-2 regulates tissue homeostasis in normal adults, we used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality. Most strikingly, induced SHP-2-deficient mice developed severe skeletal abnormalities, including kyphoses and scolioses of the spine. Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass. Osteoclasts were essentially absent from the bones of SHP-2-deficient mice, thus accounting for the osteopetrotic phenotype. Studies in vitro revealed that osteoclastogenesis that was stimulated by macrophage colony-stimulating factor (M-CSF and receptor activator of nuclear factor kappa B ligand (RANKL was defective in SHP-2-deficient mice. At least in part, this was explained by a requirement for SHP-2 in M-CSF-induced activation of the pro-survival protein kinase AKT in hematopoietic precursor cells. These findings illustrate an essential role for SHP-2 in skeletal growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

Polarized spectral features of human breast tissues through wavelet ...

Indian Academy of Sciences (India)

Abstract. Fluorescence characteristics of human breast tissues are investigated through wavelet transform and principal component analysis (PCA). Wavelet transform of polar- ized fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate different tissue types.

Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase and accumulation of gamma-hydroxybutyrate associated with its deficiency

Directory of Open Access Journals (Sweden)

Malaspina Patrizia

2009-01-01

Full Text Available Abstract Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22 occupies a central position in central nervous system (CNS neurotransmitter metabolism as one of two enzymes necessary for γ-aminobutyric acid (GABA recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1-/- knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2, represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH or γ-hydroxybutyrate (GHB; AKR7A2. A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE, an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE. Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.

Estimation of Tissue Distribution of mRNA Transcripts for Desaturase and Elongase Enzymes in Channa striata (Bloch, 1793 Fingerlings using PCR Technique

Directory of Open Access Journals (Sweden)

M. Aliyu-Paiko

2013-06-01

Full Text Available Fish species are varied in their capacity to biosynthesize n-3 highlyunsaturated fatty acids (HUFA such as eicosapentaenoic and docosahexaenoic acids (EPA & DHA that are crucial to the health and well-being of all higher vertebrates. Experts report that HUFA metabolism involves enzyme-mediated fatty acyl desaturation (FAD and elongation (FAE processes. In previous studies, different workers cloned, characterized, identified and reported several genes for FAD and FAE enzymes in different fish species such as Atlantic salmon, gilthead seabream, rainbow trout and zebrafish, and also demonstrated the up- and down-regulation in the activity of these enzymes in response to fluctuations in dietary HUFA. In this paper, we report on the expression of genes (mRNA transcripts for FAD and FAE enzymes in different tissues of Channa striata (Bloch, 1793 fingerling, to evaluate the tissues of the fish in which activity of both enzymes are high. To achieve this objective, we used conventional polymerase chain reaction (PCR technique to isolate and quantify the absolute copy number for each gene transcripts from 8 different tissues of the fish (reared with a commercial feed. Our estimate show that the distribution of the 2 enzyme transcripts were significantly (P < 0.05 higher in the liver and brain of C. striata than detected in the 6 other tissues evaluated (muscle, ovary, testis, intestine, kidney and skin. Subsequently, we discuss here extensively, the implication of this observation with respect to the use of vegetable oils (VO as substitute to fish oil (FO in diets for freshwater fish species.

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

International Nuclear Information System (INIS)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2007-01-01

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are ∼ 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (∼ 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected

Anti-human tissue factor antibody ameliorated intestinal ischemia reperfusion-induced acute lung injury in human tissue factor knock-in mice.

Directory of Open Access Journals (Sweden)

Xiaolin He

Full Text Available BACKGROUND: Interaction between the coagulation and inflammation systems plays an important role in the development of acute respiratory distress syndrome (ARDS. Anti-coagulation is an attractive option for ARDS treatment, and this has promoted development of new antibodies. However, preclinical trials for these antibodies are often limited by the high cost and availability of non-human primates. In the present study, we developed a novel alternative method to test the role of a humanized anti-tissue factor mAb in acute lung injury with transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: Human tissue factor knock-in (hTF-KI transgenic mice and a novel humanized anti-human tissue factor mAb (anti-hTF mAb, CNTO859 were developed. The hTF-KI mice showed a normal and functional expression of hTF. The anti-hTF mAb specifically blocked the pro-coagulation activity of brain extracts from the hTF-KI mice and human, but not from wild type mice. An extrapulmonary ARDS model was used by intestinal ischemia-reperfusion. Significant lung tissue damage in hTF-KI mice was observed after 2 h reperfusion. Administration of CNTO859 (5 mg/kg, i.v. attenuated the severity of lung tissue injury, decreased the total cell counts and protein concentration in bronchoalveolar lavage fluid, and reduced Evans blue leakage. In addition, the treatment significantly reduced alveolar fibrin deposition, and decreased tissue factor and plasminogen activator inhibitor-1 activity in the serum. This treatment also down-regulated cytokine expression and reduced cell death in the lung. CONCLUSIONS: This novel anti-hTF antibody showed beneficial effects on intestinal ischemia-reperfusion induced acute lung injury, which merits further investigation for clinical usage. In addition, the use of knock-in transgenic mice to test the efficacy of antibodies against human-specific proteins is a novel strategy for preclinical studies.

Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

Science.gov (United States)

Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

2009-07-01

Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

Tissue engineering for lateral ridge augmentation with recombinant human bone morphogenetic protein 2 combination therapy: a case report.

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Mandelaris, George A; Spagnoli, Daniel B; Rosenfeld, Alan L; McKee, James; Lu, Mei

2015-01-01

This case report describes a tissue-engineered reconstruction with recombinant human bone morphogenetic protein 2/acellular collagen sponge (rhBMP-2/ ACS) + cancellous allograft and space maintenance via Medpor Contain mesh in the treatment of a patient requiring maxillary and mandibular horizontal ridge augmentation to enable implant placement. The patient underwent a previously unsuccessful corticocancellous bone graft at these sites. Multiple and contiguous sites in the maxilla and in the mandibular anterior, demonstrating advanced lateral ridge deficiencies, were managed using a tissue engineering approach as an alternative to autogenous bone harvesting. Four maxillary and three mandibular implants were placed 9 and 10 months, respectively, after tissue engineering reconstruction, and all were functioning successfully after 24 months of follow-up. Histomorphometric analysis of a bone core obtained at the time of the maxillary implant placement demonstrated a mean of 76.1% new vital bone formation, 22.2% marrow/cells, and 1.7% residual graft tissue. Tissue engineering for lateral ridge augmentation with combination therapy requires further research to determine predictability and limitations.

Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans

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Takeshi Nishino

2012-11-01

Full Text Available Xanthine oxidoreductase (XOR catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors.

Microdissection of gonadal tissues for gene expression analyses

DEFF Research Database (Denmark)

Jørgensen, Anne; Dalgaard, Marlene Danner; Sonne, Si Brask

2011-01-01

Laser microdissection permits isolation of specific cell types from tissue sections or cell cultures. This may be beneficial when investigating the role of specific cells in a complex tissue or organ. In tissues with easily distinguishable morphology, a simple hematoxylin staining is sufficient...... phosphatase enzyme, such as fetal germ cells, testicular carcinoma in situ cells, and putatively also other early stem cell populations. We have applied these protocols for microdissection of rat Leydig cells, fetal human and zebrafish germ cells, and human testicular germ cell tumors, but the staining...

The metabolism of Tay-Sachs ganglioside: catabolic studies with lysosomal enzymes from normal and Tay-Sachs brain tissue

Science.gov (United States)

Tallman, John F.; Johnson, William G.; Brady, Roscoe O.

1972-01-01

The catabolism of Tay-Sachs ganglioside, N-acetylgalactosaminyl- (N-acetylneuraminosyl) -galactosylglucosylceramide, has been studied in lysosomal preparations from normal human brain and brain obtained at biopsy from Tay-Sachs patients. Utilizing Tay-Sachs ganglioside labeled with 14C in the N-acetylgalactosaminyl portion or 3H in the N-acetylneuraminosyl portion, the catabolism of Tay-Sachs ganglioside may be initiated by either the removal of the molecule of N-acetylgalactosamine or N-acetylneuraminic acid. The activity of the N-acetylgalactosamine-cleaving enzyme (hexosaminidase) is drastically diminished in such preparations from Tay-Sachs brain whereas the activity of the N-acetylneuraminic acid-cleaving enzyme (neuraminidase) is at a normal level. Total hexosaminidase activity as measured with an artificial fluorogenic substrate is increased in tissues obtained from patients with the B variant form of Tay-Sachs disease and it is virtually absent in the O-variant patients. The addition of purified neuraminidase and various purified hexosaminidases exerted only a minimal synergistic effect on the hydrolysis of Tay-Sachs ganglioside in the lysosomal preparations from the control or patient with the O variant of Tay-Sachs disease. Images PMID:4639018

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Science.gov (United States)

Yadav, Manisha C.; de Oliveira, Rodrigo Cardoso; Foster, Brian L.; Fong, Hanson; Cory, Esther; Narisawa, Sonoko; Sah, Robert L.; Somerman, Martha; Whyte, Michael P.; Millán, José Luis

2012-01-01

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl−/−, a.k.a. Akp2−/−) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl−/− mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl−/− mice, histological, μCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP (sALP-FcD10, a.k.a. ENB-0040) at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization, and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP. PMID:22461224

''Normal'' tissues from humans exposed to radium contain an alteration in the c-mos locus

International Nuclear Information System (INIS)

Huberman, E.; Schlenker, R.A.; Hardwick, J.P.

1989-01-01

The structures of a number of human proto-oncogenes from persons with internal systemic exposure to radium were analyzed by restriction enzyme digestion and southern blotting of their DNA. Two extra c-mos Eco R1 restriction-fragment-length bands of 5.0 kb and 5.5 kb were found in tissue DNA from six of seven individuals. The extra c-mos bands were detected in DNA from many, but not all, of the tissues of the individuals exposed to radium. Our results suggest that the c-mos restriction-fragment-length alterations (RFLA) found in individuals exposed to radium were induced rather than inherited, are epigenetic in origin, and most likely result from changes in the methylation of bases surrounding the single exon of the c-mos proto-oncogene. 7 refs., 3 figs., 2 tabs

Effects of the toxic dinoflagellate, Gymnodinium catenatum on hydrolytic and antioxidant enzymes, in tissues of the giant lions-paw scallop Nodipecten subnodosus.

Science.gov (United States)

Estrada, Norma; de Jesús Romero, Maria; Campa-Córdova, Angel; Luna, Antonio; Ascencio, Felipe

2007-11-01

This study documents effects of the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison, on juvenile farmed (5.9+/-0.39 cm) giant lions-paw scallop Nodipecten subnodosus. Scallops were fed bloom concentrations of toxic dinoflagellate G. catenatum for 7 h. The effect of the toxic dinoflagellate in different tissues was determined by analysis of antioxidant enzymes (catalase, superoxide dismutase, gluthathione peroxidase), thiobarbituric acid reactive substances (lipid peroxidation), and hydrolytic enzymes (proteases, glycosidases, phosphatases, lipases, and esterases). Histopathological photos record the effects of the toxic dinoflagellate in various tissues. The results show that juvenile lions-paw scallops produce pseudo-feces, partially close their shell, increase melanization, and aggregate hemocytes. Several enzymes were affected and could serve as biological markers. In general, the adductor muscle was not affected. In the digestive gland, some enzymes could be the result of defensive and digestive processes. Gills and mantle tissue were markedly affected because these sites respond first to toxic dinoflagellates, leading to the idea that proteolytic cascades could be involved.

2010 Great Lakes Human Health Fish Tissue Study Fish Tissue Data Dictionary

Science.gov (United States)

The Office of Science and Technology (OST) is providing the fish tissue results from the 2010 Great Lakes Human Health Fish Tissue Study (GLHHFTS). This document includes the “data dictionary” for Mercury, PFC, PBDE and PCBs.

Human conditions of insulin-like growth factor-I (IGF-I) deficiency

Science.gov (United States)

2012-01-01

Insulin-like growth factor I (IGF-I) is a polypeptide hormone produced mainly by the liver in response to the endocrine GH stimulus, but it is also secreted by multiple tissues for autocrine/paracrine purposes. IGF-I is partly responsible for systemic GH activities although it possesses a wide number of own properties (anabolic, antioxidant, anti-inflammatory and cytoprotective actions). IGF-I is a closely regulated hormone. Consequently, its logical therapeutical applications seems to be limited to restore physiological circulating levels in order to recover the clinical consequences of IGF-I deficiency, conditions where, despite continuous discrepancies, IGF-I treatment has never been related to oncogenesis. Currently the best characterized conditions of IGF-I deficiency are Laron Syndrome, in children; liver cirrhosis, in adults; aging including age-related-cardiovascular and neurological diseases; and more recently, intrauterine growth restriction. The aim of this review is to summarize the increasing list of roles of IGF-I, both in physiological and pathological conditions, underlying that its potential therapeutical options seem to be limited to those proven states of local or systemic IGF-I deficiency as a replacement treatment, rather than increasing its level upper the normal range. PMID:23148873

Human conditions of insulin-like growth factor-I (IGF-I deficiency

Directory of Open Access Journals (Sweden)

Puche Juan E

2012-11-01

Full Text Available Abstract Insulin-like growth factor I (IGF-I is a polypeptide hormone produced mainly by the liver in response to the endocrine GH stimulus, but it is also secreted by multiple tissues for autocrine/paracrine purposes. IGF-I is partly responsible for systemic GH activities although it possesses a wide number of own properties (anabolic, antioxidant, anti-inflammatory and cytoprotective actions. IGF-I is a closely regulated hormone. Consequently, its logical therapeutical applications seems to be limited to restore physiological circulating levels in order to recover the clinical consequences of IGF-I deficiency, conditions where, despite continuous discrepancies, IGF-I treatment has never been related to oncogenesis. Currently the best characterized conditions of IGF-I deficiency are Laron Syndrome, in children; liver cirrhosis, in adults; aging including age-related-cardiovascular and neurological diseases; and more recently, intrauterine growth restriction. The aim of this review is to summarize the increasing list of roles of IGF-I, both in physiological and pathological conditions, underlying that its potential therapeutical options seem to be limited to those proven states of local or systemic IGF-I deficiency as a replacement treatment, rather than increasing its level upper the normal range.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

Directory of Open Access Journals (Sweden)

João A Paredes

Full Text Available Loss of thymidine kinase 2 (TK2 causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA depletion syndrome (MDS in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

Science.gov (United States)

Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Accumulation of free oligosaccharides and tissue damage in cytosolic α-mannosidase (Man2c1)-deficient mice.

Science.gov (United States)

Paciotti, Silvia; Persichetti, Emanuele; Klein, Katharina; Tasegian, Anna; Duvet, Sandrine; Hartmann, Dieter; Gieselmann, Volkmar; Beccari, Tommaso

2014-04-04

Free Man(7-9)GlcNAc2 is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man5GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man(8-9)GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man(8-9)GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman's capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.

Naloxone inhibits superoxide but not enzyme release by human neutrophils

International Nuclear Information System (INIS)

Simpkins, C.; Alailima, S.; Tate, E.

1986-01-01

The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10 -7 to 10 -4 M), naloxone inhibited (p 2 - ) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O 2 - released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of 3 H FMLP to HN. Using 3 H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10 -5 ) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED 50 for naloxone inhibition of O 2 - (1 x 10 -5 M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D 2 or E 2 . Conclusions: (1) naloxone inhibits FMLP-stimulated O 2 but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN

Effects of Copper Oxide Nanoparticles on Antioxidant Enzyme Activities and on Tissue Accumulation of Oreochromis niloticus.

Science.gov (United States)

Tunçsoy, Mustafa; Duran, Servet; Ay, Özcan; Cicik, Bedii; Erdem, Cahit

2017-09-01

Accumulation of copper oxide nanoparticles (CuO NPs) in gill, liver and muscle tissues of Oreochromis niloticus and its effects on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities in gill and liver tissues were studied after exposing the fish to 20 µg/L Cu over 15 days. Copper levels and enzyme activities in tissues were determined using spectrophotometric (ICP-AES and UV) techniques respectively. No mortality was observed during the experiments. Copper levels increased in gill and liver tissues of O. niloticus compared to control when exposed to CuO NPs whereas exposure to metal had no effect on muscle level at the end of the exposure period. Highest accumulation of copper was observed in liver while no accumulation was detected in muscle tissue. SOD, CAT activities decreased and GPx activity increased in gill and liver tissues when exposed to CuO NPs.

Engineering Human Neural Tissue by 3D Bioprinting.

Science.gov (United States)

Gu, Qi; Tomaskovic-Crook, Eva; Wallace, Gordon G; Crook, Jeremy M

2018-01-01

Bioprinting provides an opportunity to produce three-dimensional (3D) tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a method for fabricating human neural tissue by 3D printing human neural stem cells with a bioink, and subsequent gelation of the bioink for cell encapsulation, support, and differentiation to functional neurons and supporting neuroglia. The bioink uniquely comprises the polysaccharides alginate, water-soluble carboxymethyl-chitosan, and agarose. Importantly, the method could be adapted to fabricate neural and nonneural tissues from other cell types, with the potential to be applied for both research and clinical product development.

Ornithine aminotransferase deficiency: Diagnostic difficulties in neonatal presentation

NARCIS (Netherlands)

Cleary, M. A.; Dorland, L.; de Koning, T. J.; Poll-The, B. T.; Duran, M.; Mandell, R.; Shih, V. E.; Berger, R.; Olpin, S. E.; Besley, G. T. N.

2005-01-01

We describe two unrelated cases of ornithine aminotransferase (OAT) deficiency with rare neonatal presentation of hyperammonaemia. The diagnosis in the neonatal presentation of OAT deficiency is hampered as hyperornithinaemia is absent. Enzyme and mutation studies confirmed the diagnosis. OAT

Analyses of Selenotranscriptomes and Selenium Concentrations in Response to Dietary Selenium Deficiency and Age Reveal Common and Distinct Patterns by Tissue and Sex in Telomere-Dysfunctional Mice.

Science.gov (United States)

Cao, Lei; Zhang, Li; Zeng, Huawei; Wu, Ryan Ty; Wu, Tung-Lung; Cheng, Wen-Hsing

2017-10-01

Background: The hierarchies of tissue selenium distribution and selenotranscriptomes are thought to critically affect healthspan and longevity. Objective: We determined selenium status and selenotranscriptomes in response to long-term dietary selenium deficiency and age in tissues of male and female mice. Methods: Weanling telomerase RNA component knockout C57BL/6 mice were fed a selenium-deficient (0.03 mg Se/kg) Torula yeast-based AIN-93G diet or a diet supplemented with sodium selenate (0.15 mg Se/kg) until age 18 or 24 mo. Plasma, hearts, kidneys, livers, and testes were collected to assay for selenotranscriptomes, selected selenoproteins, and tissue selenium concentrations. Data were analyzed with the use of 2-factor ANOVA (diet × age) in both sexes. Results: Dietary selenium deficiency decreased ( P ≤ 0.05) selenium concentrations (65-72%) and glutathione peroxidase (GPX) 3 (82-94%) and selenoprotein P (SELENOP) (17-41%) levels in the plasma of both sexes of mice and mRNA levels (9-68%) of 4, 4, and 12 selenoproteins in the heart, kidney, and liver of males, respectively, and 5, 16, and 14 selenoproteins, respectively, in females. Age increased selenium concentrations and SELENOP levels (27% and 30%, respectively; P ≤ 0.05) in the plasma of males only but decreased (12-46%; P selenium deficiency and age in ≥1 tissue or sex, or both. Dietary selenium deficiency upregulated (40-160%; P ≤ 0.05) iodothyronine deiodinase 2 ( Dio2 ) and selenoprotein N ( Selenon ) in the kidneys of males. Age upregulated (11-44%; P selenium status and selenotranscriptomes because of dietary selenium deficiency and age. © 2017 American Society for Nutrition.

Viscoelastic Properties of Human Tracheal Tissues.

Science.gov (United States)

Safshekan, Farzaneh; Tafazzoli-Shadpour, Mohammad; Abdouss, Majid; Shadmehr, Mohammad B

2017-01-01

The physiological performance of trachea is highly dependent on its mechanical behavior, and therefore, the mechanical properties of its components. Mechanical characterization of trachea is key to succeed in new treatments such as tissue engineering, which requires the utilization of scaffolds which are mechanically compatible with the native human trachea. In this study, after isolating human trachea samples from brain-dead cases and proper storage, we assessed the viscoelastic properties of tracheal cartilage, smooth muscle, and connective tissue based on stress relaxation tests (at 5% and 10% strains for cartilage and 20%, 30%, and 40% for smooth muscle and connective tissue). After investigation of viscoelastic linearity, constitutive models including Prony series for linear viscoelasticity and quasi-linear viscoelastic, modified superposition, and Schapery models for nonlinear viscoelasticity were fitted to the experimental data to find the best model for each tissue. We also investigated the effect of age on the viscoelastic behavior of tracheal tissues. Based on the results, all three tissues exhibited a (nonsignificant) decrease in relaxation rate with increasing the strain, indicating viscoelastic nonlinearity which was most evident for cartilage and with the least effect for connective tissue. The three-term Prony model was selected for describing the linear viscoelasticity. Among different models, the modified superposition model was best able to capture the relaxation behavior of the three tracheal components. We observed a general (but not significant) stiffening of tracheal cartilage and connective tissue with aging. No change in the stress relaxation percentage with aging was observed. The results of this study may be useful in the design and fabrication of tracheal tissue engineering scaffolds.

Predicting Tissue-Specific Enhancers in the Human Genome

Energy Technology Data Exchange (ETDEWEB)

Pennacchio, Len A.; Loots, Gabriela G.; Nobrega, Marcelo A.; Ovcharenko, Ivan

2006-07-01

Determining how transcriptional regulatory signals areencoded in vertebrate genomes is essential for understanding the originsof multi-cellular complexity; yet the genetic code of vertebrate generegulation remains poorly understood. In an attempt to elucidate thiscode, we synergistically combined genome-wide gene expression profiling,vertebrate genome comparisons, and transcription factor binding siteanalysis to define sequence signatures characteristic of candidatetissue-specific enhancers in the human genome. We applied this strategyto microarray-based gene expression profiles from 79 human tissues andidentified 7,187 candidate enhancers that defined their flanking geneexpression, the majority of which were located outside of knownpromoters. We cross-validated this method for its ability to de novopredict tissue-specific gene expression and confirmed its reliability in57 of the 79 available human tissues, with an average precision inenhancer recognition ranging from 32 percent to 63 percent, and asensitivity of 47 percent. We used the sequence signatures identified bythis approach to assign tissue-specific predictions to ~;328,000human-mouse conserved noncoding elements in the human genome. Byoverlapping these genome-wide predictions with a large in vivo dataset ofenhancers validated in transgenic mice, we confirmed our results with a28 percent sensitivity and 50 percent precision. These results indicatethe power of combining complementary genomic datasets as an initialcomputational foray into the global view of tissue-specific generegulation in vertebrates.

Health Deficiencies

Data.gov (United States)

U.S. Department of Health & Human Services — A list of all health deficiencies currently listed on Nursing Home Compare, including the nursing home that received the deficiency, the associated inspection date,...

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress.

Science.gov (United States)

Burrow, Hannah; Kanwar, Rupinder K; Mahidhara, Ganesh; Kanwar, Jagat R

2011-10-01

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H2O2) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione- s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H2O2 exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H2O2 exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be

UK Iatrogenic Creutzfeldt-Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches.

Science.gov (United States)

Ritchie, Diane L; Barria, Marcelo A; Peden, Alexander H; Yull, Helen M; Kirkpatrick, James; Adlard, Peter; Ironside, James W; Head, Mark W

2017-04-01

Creutzfeldt-Jakob disease (CJD) is the prototypic human prion disease that occurs most commonly in sporadic and genetic forms, but it is also transmissible and can be acquired through medical procedures, resulting in iatrogenic CJD (iCJD). The largest numbers of iCJD cases that have occurred worldwide have resulted from contaminated cadaveric pituitary-derived human growth hormone (hGH) and its use to treat primary and secondary growth hormone deficiency. We report a comprehensive, tissue-based and molecular genetic analysis of the largest series of UK hGH-iCJD cases reported to date, including in vitro kinetic molecular modelling of genotypic factors influencing prion transmission. The results show the interplay of prion strain and host genotype in governing the molecular, pathological and temporal characteristics of the UK hGH-iCJD epidemic and provide insights into the adaptive mechanisms involved when prions cross genotypic barriers. We conclude that all of the available evidence is consistent with the hypothesis that the UK hGH-iCJD epidemic resulted from transmission of the V2 human prion strain, which is associated with the second most common form of sporadic CJD.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

Science.gov (United States)

Takahashi, Nozomi; Harada, Miyuki; Hirota, Yasushi; Zhao, Lin; Azhary, Jerilee M K; Yoshino, Osamu; Izumi, Gentaro; Hirata, Tetsuya; Koga, Kaori; Wada-Hiraike, Osamu; Fujii, Tomoyuki; Osuga, Yutaka

2017-01-01

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells. Copyright © 2017 by the Endocrine Society.

Effect of dietary zinc deficiency on the accumulation of cadmium and metallothionein in selected tissues of the rat

Energy Technology Data Exchange (ETDEWEB)

Waalkes, M.P.

1986-01-01

The effect of continuous dietary zinc deficiency on the metabolism of the toxic heavy metal cadmium has not been widely studied. This investigation was designed to assess the effects of subadequate dietary zinc intake on the accumulation of dietary cadmium and on metallothionein (MT) and zinc concentrations in target organs of cadmium toxicity. Adult male Wistar rats (180-200 g) were allowed, ad libitum, diets either adequate (60 ppm) or deficient (7 ppm) in zinc for a total of 9 wk. The zinc-deficient diet resulted in an approximately 40% reduction in plasma zinc (assessed at 3, 6, and 9 wk) in the absence of overt signs of zinc deficiency (i.e., reduced weight gain, alopecia, etc.). Separate groups of rats were also maintained on zinc-defined diets for a total of 9 wk, but cadmium was added to the diet (0, 12.5, 25, 50, 100, and 200 ppm) a the end of wk 3 and maintained at that level throughout the remaining 6 wk of the study, when the rats were killed. The feeding of the zinc-deficient diet markedly enhanced the accumulation of cadmium in the liver, kidney, and testes. Hepatic, renal, and testicular zinc concentrations were not affected by suboptimal zinc intake alone. However, marked reductions in renal and testicular zinc concentrations were caused by zinc deficiency in concert with cadmium exposure. MT levels, when related to tissue cadmium concentrations, were elevated to a significantly lesser extent in the kidneys of zinc-deficient animals. These results indicate that marginal zinc deficiency markedly increases cadmium accumulation in various organs and reduces zinc content and MT induction in some organs.

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

Science.gov (United States)

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Vitamin D for combination photodynamic therapy of skin cancer in individuals with vitamin D deficiency: Insights from a preclinical study in a mouse model of squamous cell carcinoma

Science.gov (United States)

Anand, Sanjay; Thomas, Erik; Hasan, Tayyaba; Maytin, Edward V.

2016-03-01

Combination photodynamic therapy (cPDT) in which vitamin D (VD) is given prior to aminolevulinate, a precursor (pro-drug) for protoporphyrin IX (PpIX), is an approach developed in our laboratory. We previously showed that 1α,25- dihydroxyvitamin D3 (calcitriol), given prior to PDT, enhances accumulation of PpIX and improves cell death post-PDT in a mouse skin cancer model. However, since calcitriol poses a risk for hypercalcemia, we replaced systemic calcitriol with oral cholecalciferol (D3), administered as a high (tenfold, "10K") diet over a ten-day period. Here, we ask whether VD deficiency might alter the response to cPDT. Nude mice were fed a VD-deficient diet for at least 4 weeks ("deficient"); controls were fed a normal 1,000 IU/kg diet ("1K"). Human A431 cells were implanted subcutaneously and mice were switched to the 10K diet or continued on their baseline diets (controls). In other experiments, mice received a human equivalent dose of 50,000 IU D3 by oral gavage, to simulate administration of a single, high-dose VD pill. At various times, tumors were harvested and serum was collected to measure levels of VD metabolic intermediates. A significant increase in PpIX levels and in the expression of differentiation and proliferation markers in tumor tissue was observed after VD supplementation of both the deficient and 1K mice. Further results describing mechanistic details of PpIX enhancement through alteration of heme- and VD-metabolic enzyme levels will be presented. Based on these results, a clinical study using oral vitamin D prior to PDT for human skin cancer should be performed.

Status quo of management of the human tissue banks in Taiwan.

Science.gov (United States)

Chou, Ching-Pang; Chou, Szu-Cheng; Chen, Ying-Hua; Chen, Yu-Hsuan; Lee, Ming-Shin

2017-03-01

As the technologies associated with transplantation and biological tissue engineering continue to advance, human cells and tissues form an integral part to the practice of regenerative medicine. The patient's use of tissues entails the risk of introducing, transmitting and spreading communicable diseases. To prevent such risk and to ensure that the human organs, tissues and cells remain intact and functional after being handled and processed, the transplanted tissues must be subject to good management standards through all stages of collection, screening, processing, storage and distribution as the safety of the users is of the utmost importance. On February 2009, the government of Taiwan promulgated the Regulations for Administration on Human Organ Bank that requires all human tissues banks to adhere to the Good Tissue Practice for Human Organ, Tissue and Cell in terms of establishment and operation in order to cope with the international management trend and the development and management need of the domestic industry. Six years have passed since the law became effective. This article seeks to introduce the current management mechanism and status quo of management of human tissue banks in Taiwan. We also conducted statistical analysis of the data relating to the tissue banks to identify potential risks and the room for improvement. The study concludes that human tissue banks in Taiwan are on the right track with their management practice, leading to a state of steady development and progress.

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

Science.gov (United States)

Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

2017-12-01

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

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Maricilda Palandi de Mello

2011-12-01

Full Text Available The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T to dihydrotestosterone (DHT, and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2 was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency.

Fucosylation Deficiency in Mice Leads to Colitis and Adenocarcinoma

Science.gov (United States)

Wang, Yiwei; Huang, Dan; Chen, Kai-Yuan; Cui, Min; Wang, Weihuan; Huang, Xiaoran; Awadellah, Amad; Li, Qing; Friedman, Ann; Xin, William W.; Di Martino, Luca; Cominelli, Fabio; Miron, Alex; Chan, Ricky; Fox, James; Xu, Yan; Shen, Xiling; Kalady, Mathew F.; Markowitz, Sanford; Maillard, Ivan; Lowe, John B.; Xin, Wei; Zhou, Lan

2016-01-01

Background & Aims De novo synthesis of GDP-fucose, a substrate for fucosylglycans, requires sequential reactions mediated by GDP-mannose 4,6-dehydratase (GMDS) and GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase (FX or TSTA3). GMDS deletions and mutations are found in 6%–13% of colorectal cancers; these mostly affect ascending and transverse colon. We investigated whether lack of fucosylation consequent to loss of GDP-fucose synthesis contributes to colon carcinogenesis. Methods FX deficiency and GMDS deletion produce the same biochemical phenotype of GDP-fucose deficiency. We studied a mouse model of fucosylation deficiency (Fx–/– mice) and mice with the full-length Fx gene (controls). Mice were placed on standard chow or fucose-containing diet (equivalent to a control fucosylglycan phenotype). Colon tissues were collected and analyzed histologically or by ELISAs to measure cytokine levels; T cells were also collected and analyzed. Fecal samples were analyzed by 16s rRNA sequencing. Mucosal barrier function was measured by uptake of fluorescent dextran. We transplanted bone marrow cells from Fx–/– or control mice (Ly5.2) into irradiated 8-week old Fx–/– or control mice (Ly5.1). We performed immunohistochemical analyses for expression of Notch and the hes family bHLH transcription factor (HES1) in colon tissues from mice and a panel of 60 human colorectal cancer specimens (27 left-sided, 33 right-sided). Results Fx–/– mice developed colitis and serrated-like lesions. The intestinal pathology of Fx–/– mice was reversed by addition of fucose to the diet, which restored fucosylation via a salvage pathway. In the absence of fucosylation, dysplasia appeared and progressed to adenocarcinoma in up to 40% of mice, affecting mainly the right colon and cecum. Notch was not activated in Fx–/– mice fed standard chow, leading to decreased expression of its target Hes1. Fucosylation deficiency altered the composition of the fecal microbiota, reduced

Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

Science.gov (United States)

Laganowsky, Arthur; Gómez, Stephen M; Whitelegge, Julian P; Nishio, John N

2009-04-13

The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

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Müller Sylke

2009-05-01

Full Text Available Abstract Background Plasmodium falciparum-parasitized red blood cells (RBCs are equipped with protective antioxidant enzymes and heat shock proteins (HSPs. The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

Science.gov (United States)

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of

Advancing biomaterials of human origin for tissue engineering

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Chen, Fa-Ming; Liu, Xiaohua

2015-01-01

Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for

Variation in alternative splicing across human tissues

OpenAIRE

Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

2004-01-01

Background: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most p...

Study of vitamin D serum level in patients with epilepsy treated with enzyme-inducing and non enzyme-inducing medications

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sima Hashemipour

2014-01-01

Full Text Available Background : Changes of serum minerals and vitamin D have been reported in anticonvulsant drugs user patients. The present study aimed at comparing the changes of serum minerals and vitamin D among two groups of enzyme-inducing and non enzyme-inducing anticonvulsant drug users. Methods: In this study 22 patients treated with enzyme-inducing drugs (carbamazepin, phenytoin, phenobarbital were compared to 25 patients of matched sex, age, and BMI treated with non enzyme-inducing drugs (sodium evaporate, lamotrigine. Serum calcium, phosphate, parathormone, and 25-hydroxy vitamin D were calculated in both groups. Calcium was measured by Calorimetery method. Parathormone and vitamin D were measured using ELISA method. Results: The mean serum vitamin D level was lower in enzyme-inducing than non enzyme-inducing drugs users (15.9±8.3 and 24.2±14.8, P=0.02. Frequency of vitamin D deficiency was higher in enzyme-inducing compared to non enzyme-inducing drugs users, 84% and 48% , respectively (P=0.016. The mean serum calcium level was significantly lower in enzyme-inducing drugs users. (8.7±0.2 vs. 9.0± 0.7, p= 0.05. Four percent in enzyme-inducing group compared to twenty four percent of non enzyme-inducing group had secondary hyperparathyroidism (P=0.016. Conclusion: While vitamin D deficiency is more frequent in enzyme-inducing drug users, secondary hyperparathyroidism is less frequent.

Hematology, plasma biochemistry, and tissue enzyme activities of invasive red lionfish captured off North Carolina, USA.

Science.gov (United States)

Anderson, E T; Stoskopf, M K; Morris, J A; Clarke, E O; Harms, C A

2010-12-01

The red lionfish Pterois volitans is important not only in the aquarium trade but also as an invasive species in the western Atlantic. Introduced to waters off the southeastern coast of the United States, red lionfish have rapidly spread along much of the East Coast and throughout Bermuda, the Bahamas, and much of the Caribbean. Hematology and plasma biochemistry were evaluated in red lionfish captured from the offshore waters of North Carolina to establish baseline parameters for individual and population health assessment. Blood smears were evaluated for total and differential white blood cell counts, and routine clinical biochemical profiles were performed on plasma samples. To improve the interpretive value of routine plasma biochemistry profiles, tissue enzyme activities (alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyl transferase [GGT], lactate dehydrogenase [LD], and creatine kinase [CK]) were analyzed from liver, kidney, skeletal muscle, gastrointestinal tract, and heart tissues from five fish. The hematological and plasma biochemical values were similar to those of other marine teleosts except that the estimated white blood cell counts were much lower than those routinely found in many species. The tissue enzyme activity findings suggest that plasma LD, CK, and AST offer clinical relevance in the assessment of red lionfish.

Deficient UDP-glucuronosyltransferase detoxification enzyme activity in the small intestinal mucosa of patients with coeliac disease.

NARCIS (Netherlands)

Goerres, M.S.; Roelofs, H.M.J.; Jansen, J.B.M.J.; Peters, W.H.M.

2006-01-01

BACKGROUND: Small intestinal malignancies in humans are rare; however, patients with coeliac disease have a relatively high risk for such tumours. Intestinal UDP-glucuronosyltransferases are phase II drug metabolism enzymes also involved in the detoxification of ingested toxins and carcinogens. As

β-Galactosidase Deficiency in Colombia

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Alfredo Uribe PhD

2015-05-01

Full Text Available β-Galactosidase (BGal is the first enzyme involved in the catabolism of sphingolipids. Two pathologies have been directly associated with its deficiency: GM1 gangliosidosis and Morquio B. Morquio B is among the rarest types of mucopolysaccharidosis (MPS. We aim to document the β-galactosidase deficiency in Colombia. We evaluated leukocytes from 1492 healthy Colombian individuals and 923 patients, referred between 2005 and August 2014. Dried blood spot (DBS samples from the same number of patients were evaluated. β-Galactosidase was measured with 4-methylumbelliferyl-β- d -galactoside. As a control enzyme, the total hexosaminidase activity was also evaluated. We identified 14 patients with GM1 gangliosidosis, 5 patients with Morquio B, and 1 patient with I-cell disease. We could establish a reference value for Bgal in Colombian leukocyte samples. GM1 gangliosidosis is the main pathology associated with a direct deficiency of BGal. The high number of patients found with MPS IVB indicates that there are patients who could be misdiagnosed due to an unawareness of the disease.

Effects of sub-lethal dose of γ-irradiation on lysosomal enzymes in tissue of pigeon

International Nuclear Information System (INIS)

Shah, V.C.; Gadhia, P.K.

1979-01-01

Effects of total body γ-irradiation with sub-lethal dose (300 rad) on three lysosomal enzymes namely acid phosphatase, ribonuclease-II and deoxyribonuclease-II have been studied in pigeons. Liver, kidney and spleen were the tissues studied at different intervals like 1-h, 24-h, 48-h, and 72-h of irradiation. The specific activities ('crude' fraction) of acid phosphatase and ribonuclease-II increased significantly in spleen and liver at 48-h of irradiation. The activity of deoxyribonuclease-II in liver and spleen was increased only at 72-h post-irradiation. On the other hand, the total activities of three lysosomal enzymes did not show remarkable change throughout 72-h of irradiation. (author)

Naloxone inhibits superoxide but not enzyme release by human neutrophils

Energy Technology Data Exchange (ETDEWEB)

Simpkins, C.; Alailima, S.; Tate, E.

1986-03-01

The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

[Glucose-6-phosphate dehydrogenase deficiency in children: a case report].

Science.gov (United States)

Verdugo L, Patricia; Calvanese T, Marlene; Rodríguez V, Diego; Cárcamo C, Cassandra

2014-02-01

Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is the most common red blood cell (RBC) enzyme disorder. The decrease as well as the absence of the enzyme increase RBC vulnerability to oxidative stress caused by exposure to certain medications or intake of fava beans. Among the most common clinical manifestations of this condition, acute hemolysis, chronic hemolysis, neonatal hyperbilirubinemia, and an asymptomatic form are observed. To analyze the case of a child who presented hemolytic crisis due to favism. A 2 year and 7 month old boy with a history of hyperbilirubinemia during the newborn period with no apparent cause, no family history of hemolytic anemia or parental consanguinity. He presented a prolonged neonatal jaundice and severe anemia requiring RBC transfusion. An intake of fava beans 48 h prior to onset of symptoms was reported. G6PD qualitative determination was compatible with this enzyme deficiency. G6PD deficiency can be highly variable in its clinical presentation, so it is necessary to keep it in mind during the diagnosis of hemolytic anemia at any age.

Methods for demonstration of enzyme activity in muscle fibres at the muscle/bone interface in demineralized tissue

DEFF Research Database (Denmark)

Kirkeby, S; Vilmann, H

1981-01-01

A method for demonstration of activity for ATPase and various oxidative enzymes (succinic dehydrogenase, alpha-glycerophosphate dehydrogenase, and lactic dehydrogenase) in muscle/bone sections of fixed and demineralized tissue has been developed. It was found that it is possible to preserve...... considerable amounts of the above mentioned enzymes in the muscle fibres at the muscle/bone interfaces. The best results were obtained after 20 min fixation, and 2-3 weeks of storage in MgNa2EDTA containing media. As the same technique previously has been used to describe patterns of resorption and deposition...

Are MAO-A deficiency states in the general population and in putative high-risk populations highly uncommon?

Science.gov (United States)

Murphy, D L; Sims, K; Eisenhofer, G; Greenberg, B D; George, T; Berlin, F; Zametkin, A; Ernst, M; Breakefield, X O

1998-01-01

Lack of monoamine oxidase A (MAO-A) due to either Xp chromosomal deletions or alterations in the coding sequence of the gene for this enzyme are associated with marked changes in monoamine metabolism and appear to be associated with variable cognitive deficits and behavioral changes in humans and in transgenic mice. In mice, some of the most marked behavioral changes are ameliorated by pharmacologically-induced reductions in serotonin synthesis during early development, raising the question of possible therapeutic interventions in humans with MAO deficiency states. At the present time, only one multi-generational family and a few other individuals with marked MAO-A deficiency states have been identified and studied in detail. Although MAO deficiency states associated with Xp chromosomal deletions were identified by distinct symptoms (including blindness in infancy) produced by the contiguous Norrie disease gene, the primarily behavioral phenotype of individuals with the MAO mutation is less obvious. This paper reports a sequential research design and preliminary results from screening several hundred volunteers in the general population and from putative high-risk groups for possible MAO deficiency states. These preliminary results suggest that marked MAO deficiency states are very rare.

Skin wound healing in MMP2-deficient and MMP2 / plasminogen double-deficient mice

DEFF Research Database (Denmark)

Frøssing, Signe; Rønø, Birgitte; Hald, Andreas

2010-01-01

-sensitive MMPs during wound healing. To address whether MMP2 is accountable for the galardin-induced healing deficiency in wildtype and Plg-deficient mice, incisional skin wounds were generated in MMP2 single-deficient mice and in MMP2/Plg double-deficient mice and followed until healed. Alternatively, tissue...... was isolated 7 days post wounding for histological and biochemical analyses. No difference was found in the time from wounding to overt gross restoration of the epidermal surface between MMP2-deficient and wildtype control littermate mice. MMP2/Plg double-deficient mice were viable and fertile, and displayed...... an unchallenged general phenotype resembling that of Plg-deficient mice, including development of rectal prolapses. MMP2/Plg double-deficient mice displayed a slight increase in the wound length throughout the healing period compared with Plg-deficient mice. However, the overall time to complete healing...

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

Science.gov (United States)

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

Science.gov (United States)

Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

Iodine Deficiency

NARCIS (Netherlands)

Zimmermann, M.B.

2009-01-01

Iodine deficiency has multiple adverse effects in humans, termed iodine deficiency disorders, due to inadequate thyroid hormone production. Globally, it is estimated that 2 billion individuals have an insufficient iodine intake, and South Asia and sub-Saharan Africa are particularly affected.

Human natural killer cell development in secondary lymphoid tissues

Science.gov (United States)

Freud, Aharon G.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

For nearly a decade it has been appreciated that critical steps in human natural killer (NK) cell development likely occur outside of the bone marrow and potentially necessitate distinct microenvironments within extramedullary tissues. The latter include the liver and gravid uterus as well as secondary lymphoid tissues such as tonsils and lymph nodes. For as yet unknown reasons these tissues are naturally enriched with NK cell developmental intermediates (NKDI) that span a maturation continuum starting from an oligopotent CD34+CD45RA+ hematopoietic precursor cell to a cytolytic mature NK cell. Indeed despite the detection of NKDI within the aforementioned tissues, relatively little is known about how, why, and when these tissues may be most suited to support NK cell maturation and how this process fits in with other components of the human immune system. With the discovery of other innate lymphoid subsets whose immunophenotypes overlap with those of NKDI, there is also need to revisit and potentially re-characterize the basic immunophenotypes of the stages of the human NK cell developmental pathway in vivo. In this review, we provide an overview of human NK cell development in secondary lymphoid tissues and discuss the many questions that remain to be answered in this exciting field. PMID:24661538

2-Methylbutyryl-coenzyme A dehydrogenase deficiency

DEFF Research Database (Denmark)

Sass, Jörn Oliver; Ensenauer, Regina; Röschinger, Wulf

2008-01-01

2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem...... individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant...

Tissue specific and androgen-regulated expression of human prostate-specific transglutaminase

NARCIS (Netherlands)

H.J. Dubbink (Erik Jan); N.S. Verkaik (Nicole); P.W. Faber; J. Trapman (Jan); F.H. Schröder (Fritz); J.C. Romijn (Johannes)

1996-01-01

textabstractTransglutaminases (TGases) are calcium-dependent enzymes catalysing the post-translational cross-linking of proteins. In the prostate at least two TGases are present, the ubiquitously expressed tissue-type TGase (TGC), and a prostate-restricted TGase (TGP).

Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice

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Margarita Vida

2015-07-01

Full Text Available Interleukin-6 (IL-6 has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD in wild-type (WT and IL-6-deficient (IL-6−/− mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6. Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1 and signal transducer and activator of transcription-3 (STAT3, increased AMP kinase phosphorylation (p-AMPK, and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS and stearoyl-CoA desaturase 1 (SCD1. The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β, FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis.

Adenovirus 36 DNA in human adipose tissue.

Science.gov (United States)

Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

2015-12-01

Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

Geometry Modeling Program Implementation of Human Hip Tissue

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WANG Mo-nan

2017-10-01

Full Text Available Abstract:Aiming to design a simulate software of human tissue modeling and analysis，Visual Studio 2010 is selected as a development tool to develop a 3 D reconstruction software of human tissue with language C++.It can be used alone. It also can be a module of the virtual surgery systems. The system includes medical image segmentation modules and 3 D reconstruction modules，and can realize the model visualization. This software system has been used to reconstruct hip muscles，femur and hip bone accurately. The results show these geometry models can simulate the structure of hip tissues.

Geometry Modeling Program Implementation of Human Hip Tissue

Directory of Open Access Journals (Sweden)

WANG Monan

2017-04-01

Full Text Available Aiming to design a simulate software of human tissue modeling and analysis，Visual Studio 2010 is selected as a development tool to develop a 3 D reconstruction software of human tissue with language C++.It can be used alone. It also can be a module of the virtual surgery systems. The system includes medical image segmentation modules and 3 D reconstruction modules，and can realize the model visualization. This software system has been used to reconstruct hip muscles，femur and hip bone accurately. The results show these geometry models can simulate the structure of hip tissues.

Histone Acetylation Modifications Affect Tissue-Dependent Expression of Poplar Homologs of C4 Photosynthetic Enzyme Genes

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Yuan Li

2017-06-01

Full Text Available Histone modifications play important roles in regulating the expression of C4 photosynthetic genes. Given that all enzymes required for the C4 photosynthesis pathway are present in C3 plants, it has been hypothesized that this expression regulatory mechanism has been conserved. However, the relationship between histone modification and the expression of homologs of C4 photosynthetic enzyme genes has not been well determined in C3 plants. In the present study, we cloned nine hybrid poplar (Populus simonii × Populus nigra homologs of maize (Zea mays C4 photosynthetic enzyme genes, carbonic anhydrase (CA, pyruvate orthophosphate dikinase (PPDK, phosphoenolpyruvate carboxykinase (PCK, and phosphoenolpyruvate carboxylase (PEPC, and investigated the correlation between the expression levels of these genes and the levels of promoter histone acetylation modifications in four vegetative tissues. We found that poplar homologs of C4 homologous genes had tissue-dependent expression patterns that were mostly well-correlated with the level of histone acetylation modification (H3K9ac and H4K5ac determined by chromatin immunoprecipitation assays. Treatment with the histone deacetylase inhibitor trichostatin A further confirmed the role of histone acetylation in the regulation of the nine target genes. Collectively, these results suggest that both H3K9ac and H4K5ac positively regulate the tissue-dependent expression pattern of the PsnCAs, PsnPPDKs, PsnPCKs, and PsnPEPCs genes and that this regulatory mechanism seems to be conserved among the C3 and C4 species. Our findings provide new insight that will aid efforts to modify the expression pattern of these homologs of C4 genes to engineer C4 plants from C3 plants.

In silico prediction of potential chemical reactions mediated by human enzymes.

Science.gov (United States)

Yu, Myeong-Sang; Lee, Hyang-Mi; Park, Aaron; Park, Chungoo; Ceong, Hyithaek; Rhee, Ki-Hyeong; Na, Dokyun

2018-06-13

Administered drugs are often converted into an ineffective or activated form by enzymes in our body. Conventional in silico prediction approaches focused on therapeutically important enzymes such as CYP450. However, there are more than thousands of different cellular enzymes that potentially convert administered drug into other forms. We developed an in silico model to predict which of human enzymes including metabolic enzymes as well as CYP450 family can catalyze a given chemical compound. The prediction is based on the chemical and physical similarity between known enzyme substrates and a query chemical compound. Our in silico model was developed using multiple linear regression and the model showed high performance (AUC = 0.896) despite of the large number of enzymes. When evaluated on a test dataset, it also showed significantly high performance (AUC = 0.746). Interestingly, evaluation with literature data showed that our model can be used to predict not only enzymatic reactions but also drug conversion and enzyme inhibition. Our model was able to predict enzymatic reactions of a query molecule with a high accuracy. This may foster to discover new metabolic routes and to accelerate the computational development of drug candidates by enabling the prediction of the potential conversion of administered drugs into active or inactive forms.

Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

Directory of Open Access Journals (Sweden)

Biljana Musicki

Full Text Available Testosterone deficiency is associated with sickle cell disease (SCD, but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH levels compared with wild type (WT mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR, but not cholesterol side-chain cleavage enzyme (P450scc, in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

Effect of vitamin D deficiency in developed countries.

Science.gov (United States)

Hassan-Smith, Zaki K; Hewison, Martin; Gittoes, Neil J

2017-06-01

Vitamin D deficiency is common worldwide with adverse effects on skeletal health. In recent years, there has been great interest in non-classical actions of vitamin D. Basic research has uncovered actions in a range of non-skeletal tissues, and observational studies have identified inverse relationships with risk of a number of disease states including sarcopenia, obesity, the metabolic syndrome, cardiovascular disease, cancer and autoimmune diseases. PubMed, Medline and Cochrane Systematic Reviews. Current evidence supports the use of vitamin D supplementation in deficiency to improve skeletal outcomes such as falls/fracture risk and bone mineral density. There is debate reflected in guidelines on optimal thresholds for circulating levels of vitamin D. Further studies are required to refine dosing regimens and treatment target levels of vitamin D. A number of studies have investigated the extra-skeletal effects of vitamin D deficiency but causality in humans has yet to be confirmed. Large-scale randomized controlled trials incorporating data on vitamin D status at baseline and follow up, adverse events, and comparison of dosing regimens are required. It is imperative that studies are carried out with a diverse range of participants (age, gender and ethnicity), and settings to allow for a more individualized approach. In addition, we would advocate incorporating cutting-edge research tools into human studies to advance our understanding of the mechanisms of vitamin D action in extra-skeletal disease. This may involve multi-metabolite analysis of vitamin D metabolites, or unbiased approaches to assess regulation of gene/protein expression in tissues of interest. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

X-Linked G6PD Deficiency Protects Hemizygous Males but Not Heterozygous Females against Severe Malaria

OpenAIRE

Guindo, Aldiouma; Fairhurst, Rick M; Doumbo, Ogobara K; Wellems, Thomas E; Diallo, Dapa A

2007-01-01

Editors' Summary Background. “Favism” is a condition that results from a deficiency in an enzyme called glucose-6-phosphate dehydrogenase (G6PD), and this disorder is thought to be the commonest enzyme-deficiency disease worldwide. The disease is named favism after the Italian word for broad beans (fava), which cause a classic reaction when eaten by people with G6PD deficiency. The G6PD enzyme is particularly important in red blood cells, where it protects against damage that can be caused by...

[Ubiquinone: metabolism and functions. Ubiquinone deficiency and its implication in mitochondrial encephalopathies. Treatment with ubiquinone].

Science.gov (United States)

Artuch, R; Colomé, C; Vilaseca, M A; Pineda, M; Campistol, J

Review of ubiquinone-10 metabolism and functions in humans, focusing its implication in the pathogenesis and physiopathology of mitochondrial encephalomyopathies. Ubiquinone-10 is an endogenously synthesized lipid with a wide distribution in tissues. Tyrosine and acetil-CoA are involved in ubiquinone biosynthesis. This molecule has several biological functions in cells: it is a movil electron carrier in the mitochondrial respiratory chain and also acts as antioxidant. Owing to its implication in these functions, ubiquinone deficiency may cause important deletereous effects in tissues. Several authors reported ubiquinone deficient status in some physiological and pathological conditions. Mitochondrial encephalomyopathies may be related to a primary or secondary ubiquinone deficient status, or even to an altered function of ubiquinone in the respiratory chain. Moreover, some relevant aspects about ubiquinone therapy in mitochondrial disorders are reported. According to recent reports about ubiquinone implication in several diseases, its determination in different biological samples seems very useful to elucidate the physiopathological mechanisms involved and even the to start a therapy in cases with ubiquinone deficiency.

[Dinitrosyl iron complexes are endogenous signaling agents in animal and human cells and tissues (a hypothesis)].

Science.gov (United States)

Vanin, A F

2004-01-01

The hypothesis was advanced that dinitrosyl iron complexes generated in animal and human cells and tissues producing nitric oxide can function as endogenous universal regulators of biochemical and physiological processes. This function is realized by the ability of dinitrosyl iron complexes to act as donors of free nitric oxide molecules interacting with the heme groups of proteins, nitrosonium ions, or Fe+(NO+)2 interacting with the thiol groups of proteins. The effect of dinitrosyl iron complexes on the activity of some enzymes and the expression of the genome at the translation and transcription levels was considered.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Science.gov (United States)

Park, Chul Min; Lee, Kyunghoon; Jun, Sun-Hee; Song, Sang Hoon; Song, Junghan

2017-08-15

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R 2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum and tissue angiotensin-converting enzyme in patients with alopecia areata.

Science.gov (United States)

Fahim, Shabnam; Montazer, Fatemeh; Tohidinik, Hamid Reza; Naraghi, Zahra Safaei; Abedini, Robabeh; Nasimi, Maryam; Ghandi, Narges

2018-03-27

Alopecia areata is an immune-dependent disorder characterized by the interaction of T-lymphocytes with follicular antigens. Recent studies have shown the existence of a local renin-angiotensin system in the skin, where angiotensin-converting enzyme (ACE) plays a role in autoimmunity and inflammation. The objective of this study was to evaluate serum and tissue ACE activity in patients with alopecia areata. This case-control study was conducted on patients with alopecia areata and healthy controls. Serum and tissue ACE activity were assessed and compared between the two groups. Twenty-five alopecia areata patients (60% male, mean age 32.1 ± 9.9 years) and 24 controls (50% male, mean age 37.4 ± 8.8 years) were included. Mean serum ACE activity was 52.1 ± 9 U/L in cases and 55.3 ± 14.7 U/L in controls (P = 0.37). Tissue ACE activity was significantly lower in cases in all parts of the skin i.e. epidermis (P = 0.016), follicular epithelium (P = 0.004), and endothelium (P = 0.037). Among cases, serum ACE activity was significantly higher in patients with more severe disease (P = 0.030), nonpatchy alopecia areata (alopecia universalis; ophiasis, patchy and ophiasis, diffuse) (P = 0.029), and with nail involvement (P = 0.027). The sample size was too small to draw definite conclusions. Further, most of the patients had only mild or moderate alopecia areata. Unlike in some other inflammatory diseases, the tissue level of ACE seems to be significantly lower in alopecia areata compared to normal controls. Serum ACE was significantly higher in patients with more severe disease.

Radioimmunoassay of cholecystokinin in human tissue and plasma

International Nuclear Information System (INIS)

Jansen, J.B.M.J.; Lamers, C.B.H.W.

1983-01-01

A highly sensitive radioimmunoassay for cholecystokinin (CCK) without any cross-reactivity with gastrin is described. The antibody was raised in a rabbit by immunisation with 30% CCK and bound to all COOH-terminal CCK-peptides containing at least 14 amino acid residues. The affinity constant of the antibody was 59.4 x 10 10 l/mol. CCK 33 conjugated to [ 125 I]hydroxyphenylpropionic acid-succinimide ester was used as label. The binding between label and antibody was inhibited by 50% (ID 50 ) at a concentration of 2.8 pmol/l cholecystokinin 33. The detection limit of the assay was between 0.5 and 1.0 pmol/l plasma. Concentrations of CCK in aqueous acid extracts of human upper small intestine were 36.5 +- 9.8 pmol/g and of human cerebral cortex 28.2 +- 2.5 pmol/g tissue. Plasma samples were extracted in 96% ethanol prior to assay. No advantage was obtained by adding aprotinin to the tubes. When frozen at -20 0 C plasma CCK was stable for at least 6 months. Basal plasma CCK concentrations in 30 normal subjects were very low, 0.9 +- 0.1 pmol/l, range 0.5 to 3.1 pmol/l. Intraduodenal administration of fat induced significant increases in plasma CCK from 1.1 +- 0.1 to 8.2 +- 1.3 pmol/l (p = 0.01). Infusion of exogenous CCK, resulting in plasma CCK levels slightly lower than those measured during administration of fat, induced pancreatic enzyme secretion and gallbladder contraction. The reliability of this radioimmunoassay for measurements of CCK in human plasma was extensively evaluated. (Auth.)

In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

Science.gov (United States)

Dave, Shruti D; Patel, Chetan N; Vanikar, Aruna V; Trivedi, Hargovind L

2018-01-01

Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 37 0 C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 37 0 C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 10 3/ μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease.

Changes in Lecithin Concentration in the Human Brain Tissue in Some Neurodegenerative Conditions

International Nuclear Information System (INIS)

Ajanovic, A.; Mihaljevic, M.; Hasanbasic, D.; Rukavina, D.; Sofic, E.

2011-01-01

As a consequence of a possible increase in oxidative stress or deterioration of nerve cells during aging, in some states neurodegeneration was demonstrated by multiple biochemical deficiency, especially deficiency of cholesterol and lecithin in brain regions. The aim of this study was to determine the changes in the concentration of lecithin in different regions of brain tissue (MC - motor cortex, NC - nucleus caudates, GT - temporal gyrus) dissected postmortem from people with senile dementia of Alzheimer's type (SDAT), and persons with Parkinson's disease (PD) as compared to people who died without these diseases (C). Spectrophotometric determination of lecithin in 18 postmortem brain tissue regions collected from of 12 persons with SDAT, in 11 postmortem brain tissue regions of 8 persons with PD and in 18 postmortem brain tissue regions of 8 control persons, was performed by enzymatic method. The content of lecithin in MC: 14.4 mg/g fresh tissue (f.t.) and GT: 13.1 mg/g (f.t.) for SDAT was significantly reduced (p < 0.01) by about 30 %, compared to control where there was: 21.6 mg/g (f.t.) in MC and 18.3 mg/g (f.t.) in the GT estimated. In all regions of the brain of PD patients, the content of lecithin was decreased by about 12 % compared to control, but without statistical significance. These results suggest that changes in the content of lecithin in these regions of brain tissue might affect the changes in the membrane potential and cell degeneration. (author)

The human serum metabolome of vitamin B-12 deficiency and repletion, and associations with neurological function

Science.gov (United States)

We characterize the human serum metabolome in sub-clinical vitamin B-12 (B-12) deficiency and repletion. A pre-post treatment study provided one injection of 10 mg B-12 to 27 community-dwelling elderly Chileans with B-12 deficiency evaluated with serum B-12, plasma homocysteine, methylmalonic acid a...

Effects of genetic deficiency of cyclooxygenase-1 or cyclooxygenase-2 on functional and histological outcomes following traumatic brain injury in mice

Directory of Open Access Journals (Sweden)

Scheff Stephen W

2009-08-01

Full Text Available Abstract Background Neuroinflammation contributes to the pathophysiology of acute CNS injury, including traumatic brain injury (TBI. Although prostaglandin lipid mediators of inflammation contribute to a variety of inflammatory responses, their importance in neuroinflammation is not clear. There are conflicting reports as to the efficacy of inhibiting the enzymes required for prostaglandin formation, cyclooxygenase (COX -1 and COX-2, for improving outcomes following TBI. The purpose of the current study was to determine the role of the COX isoforms in contributing to pathological processes resulting from TBI by utilizing mice deficient in COX-1 or COX-2. Results Following a mild controlled cortical impact injury, the amount of cortical tissue loss, the level of microglial activation, and the capacity for functional recovery was compared between COX-1-deficient mice or COX-2-deficient mice, and their matching wild-type controls. The deficiency of COX-2 resulted in a minor (6%, although statistically significant, increase in the sparing of cortical tissue following TBI. The deficiency of COX-1 resulted in no detectable effect on cortical tissue loss following TBI. As determined by 3[H]-PK11195 autoradiography, TBI produced a similar increase in microglial activation in multiple brain regions of both COX-1 wild-type and COX-1-deficient mice. In COX-2 wild-type and COX-2-deficient mice, TBI increased 3[H]-PK11195 binding in all brain regions that were analyzed. Following injury, 3[H]-PK11195 binding in the dentate gyrus and CA1 region of the hippocampus was greater in COX-2-deficient mice, as compared to COX-2 wild-type mice. Cognitive assessment was performed in the wild-type, COX-1-deficient and COX-2-deficient mice following 4 days of recovery from TBI. There was no significant cognitive effect that resulted from the deficiency of either COX-1 or COX-2, as determined by acquisition and spatial memory retention testing in a Morris water maze

Prolactin suppresses malonyl-CoA concentration in human adipose tissue

DEFF Research Database (Denmark)

Nilsson, L. A.; Roepstorff, Carsten; Kiens, Bente

2009-01-01

Prolactin is best known for its involvement in lactation, where it regulates mechanisms that supply nutrients for milk production. In individuals with pathological hyperprolactinemia, glucose and fat homeostasis have been reported to be negatively influenced. It is not previously known, however......, whether prolactin regulates lipogenesis in human adipose tissue. The aim of this study was to investigate the effect of prolactin on lipogenesis in human adipose tissue in vitro. Prolactin decreased the concentration of malonyl-CoA, the product of the first committed step in lipogenesis, to 77......+/-6% compared to control 100+/-5% (p=0.022) in cultured human adipose tissue. In addition, prolactin was found to decrease glucose transporter 4 ( GLUT4) mRNA expression, which may cause decreased glucose uptake. In conclusion, we propose that prolactin decreases lipogenesis in human adipose tissue...

Widespread episodic thiamine deficiency in Northern Hemisphere wildlife

Science.gov (United States)

Balk, Lennart; Hägerroth, Per-Åke; Gustavsson, Hanna; Sigg, Lisa; Åkerman, Gun; Ruiz Muñoz, Yolanda; Honeyfield, Dale C.; Tjärnlund, Ulla; Oliveira, Kenneth; Ström, Karin; McCormick, Stephen D.; Karlsson, Simon; Ström, Marika; van Manen, Mathijs; Berg, Anna-Lena; Halldórsson, Halldór P.; Strömquist, Jennie; Collier, Tracy K.; Börjeson, Hans; Mörner, Torsten; Hansson, Tomas

2016-12-01

Many wildlife populations are declining at rates higher than can be explained by known threats to biodiversity. Recently, thiamine (vitamin B1) deficiency has emerged as a possible contributing cause. Here, thiamine status was systematically investigated in three animal classes: bivalves, ray-finned fishes, and birds. Thiamine diphosphate is required as a cofactor in at least five life-sustaining enzymes that are required for basic cellular metabolism. Analysis of different phosphorylated forms of thiamine, as well as of activities and amount of holoenzyme and apoenzyme forms of thiamine-dependent enzymes, revealed episodically occurring thiamine deficiency in all three animal classes. These biochemical effects were also linked to secondary effects on growth, condition, liver size, blood chemistry and composition, histopathology, swimming behaviour and endurance, parasite infestation, and reproduction. It is unlikely that the thiamine deficiency is caused by impaired phosphorylation within the cells. Rather, the results point towards insufficient amounts of thiamine in the food. By investigating a large geographic area, by extending the focus from lethal to sublethal thiamine deficiency, and by linking biochemical alterations to secondary effects, we demonstrate that the problem of thiamine deficiency is considerably more widespread and severe than previously reported.

Widespread episodic thiamine deficiency in Northern Hemisphere wildlife

Science.gov (United States)

Balk, Lennart; Hägerroth, Per-Åke; Gustavsson, Hanna; Sigg, Lisa; Akerman, Gun; Ruiz Muñoz, Yolanda; Honeyfield, Dale C.; Tjarnlund, Ulla; Oliveira, Kenneth; Strom, Karin; McCormick, Stephen D.; Karlsson, Simon; Strom, Marika; van Manen, Mathijs; Berg, Anna-Lena; Halldórsson, Halldór P.; Stromquist, Jennie; Collier, Tracy K.; Borjeson, Hans; Morner, Torsten; Hansson, Tomas

2016-01-01

Many wildlife populations are declining at rates higher than can be explained by known threats to biodiversity. Recently, thiamine (vitamin B1) deficiency has emerged as a possible contributing cause. Here, thiamine status was systematically investigated in three animal classes: bivalves, ray-finned fishes, and birds. Thiamine diphosphate is required as a cofactor in at least five life-sustaining enzymes that are required for basic cellular metabolism. Analysis of different phosphorylated forms of thiamine, as well as of activities and amount of holoenzyme and apoenzyme forms of thiamine-dependent enzymes, revealed episodically occurring thiamine deficiency in all three animal classes. These biochemical effects were also linked to secondary effects on growth, condition, liver size, blood chemistry and composition, histopathology, swimming behaviour and endurance, parasite infestation, and reproduction. It is unlikely that the thiamine deficiency is caused by impaired phosphorylation within the cells. Rather, the results point towards insufficient amounts of thiamine in the food. By investigating a large geographic area, by extending the focus from lethal to sublethal thiamine deficiency, and by linking biochemical alterations to secondary effects, we demonstrate that the problem of thiamine deficiency is considerably more widespread and severe than previously reported.

Purification of PON1 from human serum and assessment of enzyme kinetics against metal toxicity.

Science.gov (United States)

Ekinci, Deniz; Beydemir, Sükrü

2010-06-01

Paraoxonase-1 (PON1) is an organophosphate hydrolyser enzyme which has also antioxidant properties in metabolism. Due to its crucial functions, inhibition of the enzyme is undesirable and very dangerous. PON1 enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Pb(+2), Cr(+2), Fe(+2), and Zn(+2), on the activity of human serum PON1 (hPON1; EC 3.1.8.1.). For this purpose, we purified the enzyme from human serum and analyzed the alterations in the enzyme activity in the presence of metal ions. The results show that metal ions exhibit inhibitory effects on hPON1 at low concentrations with IC (50) values ranging from 0.838 to 7.410 mM. Metal ions showed different inhibition mechanisms: lead and iron were competitive, chrome was noncompetitive, and zinc was uncompetitive. Lead was determined to be the most effective inhibitor.

Structure of the periodontium in cathepsin C-deficient mice

NARCIS (Netherlands)

de Haar, Susanne F.; Tigchelaar-Gutter, Wikky; Everts, Vincent; Beertsen, Wouter

2006-01-01

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency

Fire Safety Deficiencies

Data.gov (United States)

U.S. Department of Health & Human Services — A list of all fire safety deficiencies currently listed on Nursing Home Compare, including the nursing home that received the deficiency, the associated inspection...

Trace element deficiency and its diagnosis by biochemical criteria

International Nuclear Information System (INIS)

Kirchgessner, M.; Grassmann, E.; Roth, H.P.; Spoerl, R.; Schnegg, A.

1976-01-01

The effect of trace element deficiency on growth of rats and dairy cows is demonstrated using zinc and nickel. The effect of copper deficiency on reproductive performance is shown to be associated with increased death rates of pregnant animals and their foetuses. For the diagnosis of suboptimum states of trace element supply, biochemical criteria are needed. The mere analysis of the trace element content of various body tissues may lead to falase diagnoses because of the often slow response to varying intake and because of interactions with other dietary ingredients affecting absorption and metabolic efficiency of utilization. Thus copper deficiency is associated with a decrease in the serum level of both copper and iron, despite adequate iron intake, and simultaneously with an accumulation of iron in the liver of the animal. Enzymes and hormones containing the essential trace element as an integral constituent may serve as biochemical criteria. A sensitive response to zinc intake is exhibited by the activity of the alkaline phosphatase of serum or bones, and by the activity of the pancreatic carboxypeptidase A, all of which show a significant reaction to deficient intake within two to four days, and perhaps by the biopotency of insulin. Ceruloplasmin responds to the supply of copper. Its biosynthesis in the liver is possible only from copper available for this purpose. Thus, the determination of ceruloplasmin may take account of at least part of the copper available to the body for metabolic functions. Among various criteria, the catalase activity in blood may provide additional information on the state of iron supply. Malate dehydrogenase and glucose-6-phosphate dehydrogenase respond to nickel-deficient intake. Nickel deficiency also involves anaemia due to disorders in iron absorption

Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels.

Science.gov (United States)

Herman, Mark A; She, Pengxiang; Peroni, Odile D; Lynch, Christopher J; Kahn, Barbara B

2010-04-09

Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

Uptakes of trace elements in Zn-deficient mice

International Nuclear Information System (INIS)

Ohyama, T.; Yanaga, M.; Yoshida, T.; Maetsu, H.; Suganuma, H.; Omori, T.

2002-01-01

A multitracer technique was used to obtain uptake rates of essential trace elements in various organs and tissues in Zn-deficient mice. A multitracer solution, containing more than 20 radioisotopes, was injected intraperitoneally into Zn-deficient state mice and control ones. Uptake rates of the radioisotopes were compared with concentrations of trace elements determined by instrumental neutron activation analysis (INAA) in order to study a specific metabolism of Zn and other essential trace elements, such as Mn, Co, Se, Rb, and Sr. The result suggests that Zn is supplied from bone to other organs and tissues and an increase in Co concentration in all organs and tissues depends on its chemical form, under the Z-deficient state. (author)

Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia.

Science.gov (United States)

McKee, M D; Nakano, Y; Masica, D L; Gray, J J; Lemire, I; Heft, R; Whyte, M P; Crine, P; Millán, J L

2011-04-01

Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2(-/-)) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B(6)-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2(-/-) mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD(10)). sALP-FcD(10) prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin--a marker of acellular cementum--was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2(-/-) mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life--where the majority of tooth root development occurs, including acellular cementum formation--could prevent the accelerated tooth loss seen in individuals with HPP.

A deficiency in chromatin repair, genetic instability, and predisposition to cancer

International Nuclear Information System (INIS)

Sanford, K.K.; Parshad, R.; Gantt, R.R.; Tarone, R.E.

1989-01-01

This review traces steps leading to malignant neoplastic transformation of rodent and human cells in culture and in vivo. Emphasis is placed on an abnormal response characterized by persistent chromatid damage following irradiation of cells in culture with X-rays or fluorescent light during G2 phase of the cell cycle. Evidence is presented that deficient or unbalanced DNA repair during G2 accounts for the abnormal response. This G2 repair deficiency can be inherited or acquired by normal tissue cells during the process of or following attainment of infinite lifespan. It appears as an early, possibly initiating step in neoplastic transformation. It characterizes all human tumor cells examined irrespective of histopathology or tissue of origin. It has a genetic basis. In an animal model, the BALB/c mouse, this phenotype is associated with genes on chromosomes 1 and 4. It characterizes skin fibroblasts and blood lymphocytes from individuals with genetic or familial conditions predisposing to cancer and can be used to identify clinically normal family members carrying a gene(s) for any one of the three cancer-prone genetic disorders studied to date. Furthermore, it can provide the basis of a test for carriers of genes predisposing to a high risk of cancer. We conclude that the G2 repair deficiency, whether inherited or acquired, is a prerequisite for cancer development and that it accounts for the genetic instability of the cancer cell. 167 refs

The Human Tissue Act 2004 and the child donor.

Science.gov (United States)

Baston, Jenny

2009-05-01

In 2001, the inquiry panel appointed to investigate the removal, retention and disposal of human organs and tissues at the Royal Liverpool Children's Hospital published its report. The panel's recommendations led to a new approach to consent for organ removal and storage under the new Human Tissue Act 2004. For child bone marrow donors, the new consent process requires all donor children or their parent to undergo a separate assessment before the bone marrow donation. They must be assessed by an accredited assessor who will submit a recommendation to the Human Tissue Authority for consideration. The unfortunate circumstances highlighted in the inquiry have led to changes to law, practice and culture that are benefiting other children and families.

Polychlorinated naphthalenes in human adipose tissue from New York, USA

International Nuclear Information System (INIS)

Kunisue, Tatsuya; Johnson-Restrepo, Boris; Hilker, David R.; Aldous, Kenneth M.; Kannan, Kurunthachalam

2009-01-01

Polychlorinated naphthalenes (PCNs) are persistent, bioaccumulative, and toxic contaminants. Prior to this study, the occurrence of PCNs in human adipose tissues from the USA has not been analyzed. Here, we have measured concentrations of PCNs in human adipose tissue samples collected in New York City during 2003-2005. Concentrations of PCNs were in the range of 61-2500 pg/g lipid wt. in males and 21-910 pg/g lipid wt. in females. PCN congeners 52/60 (1,2,3,5,7/1,2,4,6,7) and 66/67 (1,2,3,4,6,7/1,2,3,5,6,7) were predominant, collectively accounting for 66% of the total PCN concentrations. Concentrations of PCNs in human adipose tissues were 2-3 orders of magnitude lower than the previously reported concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). Concentrations of PCNs were not correlated with PCB concentrations. The contribution of PCNs to dioxin-like toxic equivalents (TEQs) in human adipose tissues was estimated to be <1% of the polychlorinated dibenzo-p-dioxin/dibenzofuran (PCDD/F)-TEQs. - Polychlorinated naphthalenes have been measured in human adipose tissues from the USA for the first time

NCI’s Cooperative Human Tissue Network

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Quality biospecimens are a foundational resource for cancer research. One of NCI’s longest running biospecimen programs is the Cooperative Human Tissue Network, a resource mainly for basic discovery and early translational research.

Lack of oxygen effect in glutathione-deficient human cells in culture

International Nuclear Information System (INIS)

Edgren, M.; Larsson, A.; Nilsson, K.; Revesz, L.; Scott, O.C.A.

1980-01-01

The frequency of X-ray-induced DNA breaks was determined in human cell lines which are deficient in glutathione synthetase and have a greatly reduced glutathione content. Hydroxyapatite chromatography was used for the estimation of the DNA breaks in cell cultures, which were derived either from lymphoblasts transformed by infection with EB virus or from fibroblasts. The dose-effect relationship for the induction of breaks when radiation exposure was made in argon, was similar to that found when exposure was made in air. In control cultures with normal glutathione content, the induction of breaks was enhanced when irradiation was made under aerobic, instead of anaerobic, conditions. Treatment of the glutathione-deficient cells with the hypoxic radiosensitizer misonidazole did not enhance the induction of breaks by radiation delivered either in air or in argon. In control cultures, radiation induction of breaks was enhanced by misonidazole under anaerobic but not under aerobic conditions. When the glutathione-deficient cells were pretreated with cysteamine however, irradiation in the absence of oxygen resulted in a decreased frequency of DNA breaks. (author)

In a model of Batten disease, palmitoyl protein thioesterase-1 deficiency is associated with brown adipose tissue and thermoregulation abnormalities.

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Alfia Khaibullina

Full Text Available Infantile neuronal ceroid lipofuscinosis (INCL is a fatal neurodegenerative disorder caused by a deficiency of palmitoyl-protein thioesterase-1 (PPT1. We have previously shown that children with INCL have increased risk of hypothermia during anesthesia and that PPT1-deficiency in mice is associated with disruption of adaptive energy metabolism, downregulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, and mitochondrial dysfunction. Here we hypothesized that Ppt1-knockout mice, a well-studied model of INCL that shows many of the neurologic manifestations of the disease, would recapitulate the thermoregulation impairment observed in children with INCL. We also hypothesized that when exposed to cold, Ppt1-knockout mice would be unable to maintain body temperature as in mice thermogenesis requires upregulation of Pgc-1α and uncoupling protein 1 (Ucp-1 in brown adipose tissue. We found that the Ppt1-KO mice had lower basal body temperature as they aged and developed hypothermia during cold exposure. Surprisingly, this inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with an adequate upregulation of Pgc-1α and Ucp-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. In addition, during baseline conditions, brown adipose tissue of Ppt1-KO mice had less vacuolization (lipid droplets compared to wild-type animals. After cold stress, wild-type animals had significant decreases whereas Ppt1-KO had insignificant changes in lipid droplets compared with baseline measurements, thus suggesting that Ppt1-KO had less lipolysis in response to cold stress. These results uncover a previously unknown phenotype associated with PPT1 deficiency, that of altered thermoregulation, which is associated with impaired lipolysis and neurotransmitter release to brown adipose tissue during cold exposure. These findings suggest that INCL should be added to the list of

Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

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Landry Losi

2010-08-01

Full Text Available Abstract Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration.

Browning of Subcutaneous White Adipose Tissue in Humans

OpenAIRE

Sidossis, Labros S.; Porter, Craig; Saraf, Manish K.; Børsheim, Elisabet; Radhakrishnan, Ravi S.; Chao, Tony; Ali, Arham; Chondronikola, Maria; Mlcak, Ronald; Finnerty, Celeste C.; Hawkins, Hal K.; Toliver-Kinsky, Tracy; Herndon, David N.

2015-01-01

Since the presence of brown adipose tissue (BAT) was confirmed in adult humans, BAT has become a therapeutic target for obesity and insulin resistance. We examined whether human subcutaneous white adipose tissue (sWAT) can adopt a BAT-like phenotype using a clinical model of prolonged and severe adrenergic stress. sWAT samples were collected from severely burned and healthy individuals. A subset of burn victims were prospectively followed during their acute hospitalization. Browning of sWAT w...

Characterization of muscarinic receptor subtypes in human tissues

International Nuclear Information System (INIS)

Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

1988-01-01

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [ 3 H]Pirenzepine and [ 3 H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M 1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M 1 , the cardiac M 2 and the glandular M 3

THE MAIN DEFICIENCIES IN THE IMPLEMENTATION OF THE SECTORAL OPERATIONAL PROGRAMME HUMAN RESOURCES DEVELOPMENT

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Pautu Sorina

2013-07-01

Full Text Available The absorption of EU funds for Romania is a necessity in the nowadays context. The slow pace of absorption of these structural funds earmarked for Romania as EU member state is a deficiency with negative effects on the economic and social development of our country. Their low absorption shows deficiencies in their coordination and implementation at central level and also at the level of beneficiaries. Their coordinative authorities, in particular the Managing Authority of Structural Instruments, together with its subordinated institutions presents deficiencies in their coordination and implementation as having negative effects on their absorption. The main weaknesses identified on national level mainly consist in the lack of specialized personnel, in excessive bureaucracy and a mismatch of national legislation with the European one. The lack of transparency and change is specific to these structural funds, representing deficiencies that lead to beneficiaries’ discouragement to implement projects financed from structural funds. In the Sectoral Operational Programme, the Human Resources Development Program is a leader in the rate of absorption but it also has the largest number of problems and deficiencies in implementation. Due to the deficiencies identified by the auditing European Commission of the Sectoral Operational Programme Human Resources Development, payments were suspended for a period of four months. Following this situation, it was necessary to implement the necessary corrective measures at the level of POSDRU, leading to its release. Taking action and removing the deficiencies at the POSDRU level, and also at the level of other operational programs, it is a necessity and a priority to increase the absorption of these funds. The main measures that need to be taken mainly consist of training the personnel involved in the management of these funds, reimbursements release funds to the final beneficiaries, creating a more transparent

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

Science.gov (United States)

Kato, Kosuke; Zemskova, Marina A; Hanss, Alec D; Kim, Marianne M; Summer, Ross; Kim, Kwang Chul

2017-11-25

MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation

A family of hyperelastic models for human brain tissue

Science.gov (United States)

Mihai, L. Angela; Budday, Silvia; Holzapfel, Gerhard A.; Kuhl, Ellen; Goriely, Alain

2017-09-01

Experiments on brain samples under multiaxial loading have shown that human brain tissue is both extremely soft when compared to other biological tissues and characterized by a peculiar elastic response under combined shear and compression/tension: there is a significant increase in shear stress with increasing axial compression compared to a moderate increase with increasing axial tension. Recent studies have revealed that many widely used constitutive models for soft biological tissues fail to capture this characteristic response. Here, guided by experiments of human brain tissue, we develop a family of modeling approaches that capture the elasticity of brain tissue under varying simple shear superposed on varying axial stretch by exploiting key observations about the behavior of the nonlinear shear modulus, which can be obtained directly from the experimental data.

MAP3K8 (TPL2/COT affects obesity-induced adipose tissue inflammation without systemic effects in humans and in mice.

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Dov B Ballak

Full Text Available Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8. Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1β, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1β, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1β and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

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Cereser, Biancastella; Jansen, Marnix; Austin, Emily; Elia, George; McFarlane, Taneisha; van Deurzen, Carolien Hm; Sieuwerts, Anieta M; Daidone, Maria G; Tadrous, Paul J; Wright, Nicholas A; Jones, Louise; McDonald, Stuart Ac

2018-01-01

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Delayed O-methylation of l-DOPA in MB-COMT-deficient mice after oral administration of l-DOPA and carbidopa.

Science.gov (United States)

Tammimäki, Anne; Aonurm-Helm, Anu; Männistö, Pekka T

2018-04-01

1. Catechol-O-methyltransferase (COMT) is involved in the O-methylation of l-DOPA, dopamine, and other catechols. The enzyme is expressed in two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-COMT), which is anchored to intracellular membranes. 2. To obtain specific information on the functions of COMT isoforms, we studied how a complete MB-COMT deficiency affects the total COMT activity in the body, peripheral l-DOPA levels, and metabolism after l-DOPA (10 mg kg -1 ) plus carbidopa (30 mg kg -1 ) administration by gastric tube in wild-type (WT) and MB-COMT-deficient mice. l-DOPA and 3-O-methyl-l-DOPA (3-OMD) levels were assayed in plasma, duodenum, and liver. 3. We showed that the selective lack of MB-COMT did not alter the total COMT activity, COMT enzyme kinetics, l-DOPA levels, or the total O-methylation of l-DOPA but delayed production of 3-OMD in plasma and peripheral tissues.

Calpain 1 inhibitor BDA-410 ameliorates α-klotho-deficiency phenotypes resembling human aging-related syndromes.

Science.gov (United States)

Nabeshima, Yoko; Washida, Miwa; Tamura, Masaru; Maeno, Akiteru; Ohnishi, Mutsuko; Shiroishi, Toshihiko; Imura, Akihiro; Razzaque, M Shawkat; Nabeshima, Yo-ichi

2014-08-01

Taking good care of elderly is a major challenge of our society, and thus identification of potential drug targets to reduce age-associated disease burden is desirable. α-klotho(-/-) (α-kl) is a short-lived mouse model that displays multiple phenotypes resembling human aging-related syndromes. Such ageing phenotype of α-kl(-/-) mice is associated with activation of a proteolytic enzyme, Calpain-1. We hypothesized that uncontrolled activation of calpain-1 might be causing age-related phenotypes in α-kl-deficient mice. We found that daily administration of BDA-410, a calpain-1 inhibitor, strikingly ameliorated multiple aging-related phenotypes. Treated mice showed recovery of reproductive ability, increased body weight, reduced organ atrophy, and suppression of ectopic calcifications, bone mineral density reduction, pulmonary emphysema and senile atrophy of skin. We also observed ectopic expression of FGF23 in calcified arteries of α-kl(-/-) mice, which might account for the clinically observed association of increased FGF23 level with increased risk of cardiovascular mortality. These findings allow us to propose that modulation of calpain-1 activity is a potential therapeutic option for delaying age-associated organ pathology, particularly caused by the dysregulation of mineral ion homeostasis.

Microwave non-contact imaging of subcutaneous human body tissues.

Science.gov (United States)

Kletsov, Andrey; Chernokalov, Alexander; Khripkov, Alexander; Cho, Jaegeol; Druchinin, Sergey

2015-10-01

A small-size microwave sensor is developed for non-contact imaging of a human body structure in 2D, enabling fitness and health monitoring using mobile devices. A method for human body tissue structure imaging is developed and experimentally validated. Subcutaneous fat tissue reconstruction depth of up to 70 mm and maximum fat thickness measurement error below 2 mm are demonstrated by measurements with a human body phantom and human subjects. Electrically small antennas are developed for integration of the microwave sensor into a mobile device. Usability of the developed microwave sensor for fitness applications, healthcare, and body weight management is demonstrated.

Evidence for the ectopic synthesis of melanin in human adipose tissue.

Science.gov (United States)

Randhawa, Manpreet; Huff, Tom; Valencia, Julio C; Younossi, Zobair; Chandhoke, Vikas; Hearing, Vincent J; Baranova, Ancha

2009-03-01

Melanin is a common pigment in animals. In humans, melanin is produced in melanocytes, in retinal pigment epithelium (RPE) cells, in the inner ear, and in the central nervous system. Previously, we noted that human adipose tissue expresses several melanogenesis-related genes. In the current study, we confirmed the expression of melanogenesis-related mRNAs and proteins in human adipose tissue using real-time polymerase chain reaction and immunohistochemical staining. TYR mRNA signals were also detected by in situ hybridization in visceral adipocytes. The presence of melanin in human adipose tissue was revealed both by Fontana-Masson staining and by permanganate degradation of melanin coupled with liquid chromatography/ultraviolet/mass spectrometry determination of the pyrrole-2,3,5-tricarboxylic acid (PTCA) derivative of melanin. We also compared melanogenic activities in adipose tissues and in other human tissues using the L-[U-(14)C] tyrosine assay. A marked heterogeneity in the melanogenic activities of individual adipose tissue extracts was noted. We hypothesize that the ectopic synthesis of melanin in obese adipose may serve as a compensatory mechanism that uses its anti-inflammatory and its oxidative damage-absorbing properties. In conclusion, our study demonstrates for the first time that the melanin biosynthesis pathway is functional in adipose tissue.

Prenatal diagnosis in adenylosuccinate lyase deficiency

NARCIS (Netherlands)

Marie, S.; Flipsen, J. W.; Duran, M.; Poll-The, B. T.; Beemer, F. A.; Bosschaart, A. N.; Vincent, M. F.; van den Berghe, G.

2000-01-01

Adenylosuccinate lyase deficiency, an autosomal recessive inborn error of purine synthesis, provokes accumulation in body fluids of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Most patients display severe

Comparative in silico profiling of epigenetic modifiers in human tissues.

Science.gov (United States)

Son, Mi-Young; Jung, Cho-Rok; Kim, Dae-Soo; Cho, Hyun-Soo

2018-04-06

The technology of tissue differentiation from human pluripotent stem cells has attracted attention as a useful resource for regenerative medicine, disease modeling and drug development. Recent studies have suggested various key factors and specific culture methods to improve the successful tissue differentiation and efficient generation of human induced pluripotent stem cells. Among these methods, epigenetic regulation and epigenetic signatures are regarded as an important hurdle to overcome during reprogramming and differentiation. Thus, in this study, we developed an in silico epigenetic panel and performed a comparative analysis of epigenetic modifiers in the RNA-seq results of 32 human tissues. We demonstrated that an in silico epigenetic panel can identify epigenetic modifiers in order to overcome epigenetic barriers to tissue-specific differentiation.

Translational neuropharmacology: the use of human isolated gastrointestinal tissues.

Science.gov (United States)

Sanger, G J; Broad, J; Kung, V; Knowles, C H

2013-01-01

Translational sciences increasingly emphasize the measurement of functions in native human tissues. However, such studies must confront variations in patient age, gender, genetic background and disease. Here, these are discussed with reference to neuromuscular and neurosecretory functions of the human gastrointestinal (GI) tract. Tissues are obtained after informed consent, in collaboration with surgeons (surgical techniques help minimize variables) and pathologists. Given the difficulties of directly recording from human myenteric neurones (embedded between muscle layers), enteric motor nerve functions are studied by measuring muscle contractions/relaxations evoked by electrical stimulation of intrinsic nerves; responses are regionally dependent, often involving cholinergic and nitrergic phenotypes. Enteric sensory functions can be studied by evoking the peristaltic reflex, involving enteric sensory and motor nerves, but this has rarely been achieved. As submucosal neurones are more accessible (after removing the mucosa), direct neuronal recordings are possible. Neurosecretory functions are studied by measuring changes in short-circuit current across the mucosa. For all experiments, basic questions must be addressed. Because tissues are from patients, what are the controls and the influence of disease? How long does it take before function fully recovers? What is the impact of age- and gender-related differences? What is the optimal sample size? Addressing these and other questions minimizes variability and raises the scientific credibility of human tissue research. Such studies also reduce animal use. Further, the many differences between animal and human GI functions also means that human tissue research must question the ethical validity of using strains of animals with unproved translational significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency.

Science.gov (United States)

Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C; Wallace, John C; Polyak, Steven W

2008-01-01

The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.

Biochemical characteristics of glucose-6-phosphate dehydrogenase variants among the Malays of Singapore with report of a new non-deficient (GdSingapore) and three deficient variants.

Science.gov (United States)

Saha, N; Hong, S H; Wong, H A; Jeyaseelan, K; Tay, J S

1991-12-01

Biochemical characteristics of one non-deficient fast G6PD variant (GdSingapore) and six different deficient variants (three new, two Mahidol, one each of Indonesian and Mediterranean) were studied among the Malays of Singapore. The GdSingapore variant had normal enzyme activity (82%) and fast electrophoretic mobilities (140% in TEB buffer, 160% in phosphate and 140% in Tris-HCl buffer systems respectively). This variant is further characterized by normal Km for G6P; utilization of analogues (Gal6P, 2dG6P; dAmNADP), heat stability and pH optimum. The other six deficient G6PD variants had normal electrophoretic mobility in TEB buffer with enzyme activities ranging from 1 to 12% of GdB+. The biochemical characteristics identity them to be 2 Mahidol, 1 Indonesian and 1 Mediterranean variants and three new deficient variants.

Transfer RNA and human disease

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Jamie A Abbott

2014-06-01

Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

Transfer RNA and human disease.

Science.gov (United States)

Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

2014-01-01

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

Familial LCAT deficiency: from renal replacement to enzyme replacement

NARCIS (Netherlands)

Stoekenbroek, R. M.; van den Bergh Weerman, M. A.; Hovingh, G. K.; Potter van Loon, B. J.; Siegert, C. E. H.; Holleboom, A. G.

2013-01-01

Familial LCAT deficiency (FLD) is a recessive lipid disorder ultimately leading to end-stage renal disease (ESRD). We present two brothers with considerable variation in the age at which they developed ESRD. Kidney biopsies revealed both tubular and glomerular pathology. To date, no causal therapy

Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

Science.gov (United States)

Nakajima, Masahiro; Mizumoto, Shuji; Miyake, Noriko; Kogawa, Ryo; Iida, Aritoshi; Ito, Hironori; Kitoh, Hiroshi; Hirayama, Aya; Mitsubuchi, Hiroshi; Miyazaki, Osamu; Kosaki, Rika; Horikawa, Reiko; Lai, Angeline; Mendoza-Londono, Roberto; Dupuis, Lucie; Chitayat, David; Howard, Andrew; Leal, Gabriela F; Cavalcanti, Denise; Tsurusaki, Yoshinori; Saitsu, Hirotomo; Watanabe, Shigehiko; Lausch, Ekkehart; Unger, Sheila; Bonafé, Luisa; Ohashi, Hirofumi; Superti-Furga, Andrea; Matsumoto, Naomichi; Sugahara, Kazuyuki; Nishimura, Gen; Ikegawa, Shiro

2013-06-06

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

NARCIS (Netherlands)

Miners, J.S.; Verbeek, M.M.; Olde Rikkert, M.G.M.; Kehoe, P.G.; Love, S.

2008-01-01

Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and

Effects of Nrf2 deficiency on arsenic metabolism in mice.

Science.gov (United States)

Wang, Huihui; Zhu, Jiayu; Li, Lu; Li, Yongfang; Lv, Hang; Xu, Yuanyuan; Sun, Guifan; Pi, Jingbo

2017-12-15

Inorganic arsenic (iAs) is a known toxicant and carcinogen. Worldwide arsenic exposure has become a threat to human health. The severity of arsenic toxicity is strongly correlated with the speed of arsenic metabolism (methylation) and clearance. Furthermore, oxidative stress is recognized as a major mechanism for arsenic-induced toxicity. Nuclear factor-E2-related factor 2 (Nrf2), a key regulator in cellular adaptive antioxidant response, is clearly involved in alleviation of arsenic-induced oxidative damage. Multiple studies demonstrate that Nrf2 deficiency mice are more vulnerable to arsenic-induced intoxication. However, what effect Nrf2 deficiency might have on arsenic metabolism in mice is still unknown. In the present study, we measured the key enzymes involved in arsenic metabolism in Nrf2-WT and Nrf2-KO mice. Our results showed that basal transcript levels of glutathione S-transferase omega 2 (Gsto2) were significantly higher and GST mu 1 (Gstm1) lower in Nrf2-KO mice compared to Nrf2-WT control. Arsenic speciation and methylation rate in liver and urine was then studied in mice treated with 5mg/kg sodium arsenite for 12h. Although there were some alterations in arsenic metabolism enzymes between Nrf2-WT and Nrf2-KO mice, the Nrf2 deficiency had no significant effect on arsenic methylation. These results suggest that the Nrf2-KO mice are more sensitive to arsenic than Nrf2-WT mainly because of differences in adaptive antioxidant detoxification capacity rather than arsenic methylation capacity. Copyright © 2017. Published by Elsevier Inc.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Hepatic NAD(+) deficiency as a therapeutic target for non-alcoholic fatty liver disease in ageing.

Science.gov (United States)

Zhou, Can-Can; Yang, Xi; Hua, Xia; Liu, Jian; Fan, Mao-Bing; Li, Guo-Qiang; Song, Jie; Xu, Tian-Ying; Li, Zhi-Yong; Guan, Yun-Feng; Wang, Pei; Miao, Chao-Yu

2016-08-01

Ageing is an important risk factor of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD(+) ), a ubiquitous coenzyme, links ageing with NAFLD. Hepatic concentrations of NAD(+) , protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD(+) biosynthesis, were compared in middle-aged and aged mice or patients. The influences of NAD(+) decline on the steatosis and steatohepatitis were evaluated in wild-type and H247A dominant-negative, enzymically-inactive NAMPT transgenic mice (DN-NAMPT) given normal or high-fat diet (HFD). Hepatic NAD(+) level decreased in aged mice and humans. NAMPT-controlled NAD(+) salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and humans. Given normal chow, middle-age DN-NAMPT mice displayed systemic NAD(+) reduction and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially release of pro-inflammatory factors, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway and hepatic fibrosis (Masson's staining and α-SMA staining), deteriorated further under HFD challenge. Oral administration of nicotinamide riboside, a natural NAD(+) precursor, completely corrected these NAFLD phenotypes induced by NAD(+) deficiency alone or HFD, whereas adenovirus-mediated SIRT1 overexpression only partially rescued these phenotypes. These results provide the first evidence that ageing-associated NAD(+) deficiency is a critical risk factor for NAFLD, and suggest that supplementation with NAD(+) substrates may be a promising therapeutic strategy to prevent and treat NAFLD. © 2016 The British Pharmacological Society.

Caffeine and Cannabis Effects on Vital Neurotransmitters and Enzymes in the Brain Tissue of Juvenile Experimental Rats.

Science.gov (United States)

Owolabi, J O; Olatunji, S Y; Olanrewaju, A J

2017-05-01

Caffeine and cannabis are globally consumed and abused psychoactive substances. While caffeine is legally used in various forms, including in tea and coffee as beverages, it is also consumed in soda and energy drinks as additives. Cannabis, on the other hand, is considered illegal in most countries; albeit, it is being consumed globally particularly by adolescents. The adolescent stage marks a critical stage of brain development and maturation. Influences of agents on the brain at this stage may affect neuronal structural and functional attributes. To this end, the current experiment considered the effects of cannabis and caffeine on selected key neurotransmitters and enzymes in the brain tissues after regimented caffeine and cannabis treatment for 21 days. A total of 72 juvenile Wistar rats that were approximately 40 days old were divided into 6 groups A-F. The group A served as the control. Other groups were administered various dosages of caffeine or cannabis in distilled water, using oral gavages as follows: group B animals received 100 mg/kg body weight of caffeine, group C animals received 50 mg/kg body weight of caffeine, group D animals received 500 mg/kg body weight of cannabis, group E animals received 200 mg/kg body weight of cannabis, and group F received a low dose of cannabis (200 mg/kg body weight) plus a low dose of caffeine (50 mg/kg body weight). The animals were killed by cervical dislocation 24 h after the last administration. The brain tissues were excised and homogenized. The enzymes cytochrome C oxidase and glucose-6-phosphate dehydrogenase were assayed to observe tissue energy metabolism while the neurotransmitters gamma-amino butyric acid (GABA), glutamate, and dopamine were assayed to observe the effects of the psychoactive substances on their activities relative to mental activities. GABA, glutamate, and dopamine were generally higher in the treated groups of animals. The levels of G-6-PDH were higher in all treated animals' brains

Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

Science.gov (United States)

Drozdzik, M; Oswald, S

2016-01-01

Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

Angiotensin I-Converting Enzyme (ACE Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

Directory of Open Access Journals (Sweden)

Małgorzata Darewicz

2014-08-01

Full Text Available The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes and ex vivo digestion (with human gastrointestinal enzymes. Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50% of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

International Nuclear Information System (INIS)

Richardson, Terrilyn A.; Klaassen, Curtis D.

2010-01-01

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T 4 ), thus reducing serum T 4 , and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T 4 glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T 4 glucuronidation, decreased serum T 4 , and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T 4 , triiodothyronine (T 3 ), and TSH concentrations, hepatic T 4 /T 3 glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T 4 , whereas serum T 3 was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T 4 glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T 3 glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T 3 glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T 4 glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T 4 , increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T 4 glucuronidation, and cannot be attributed to Ugt1a enzymes.

Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

International Nuclear Information System (INIS)

Rappuoli, R.; Leoncini, P.; Tarli, P.; Neri, P.

1981-01-01

Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzyme-labeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated

Aminotransferaza asparaginianowa – kluczowy enzym w metabolizmie ogólnoustrojowym człowieka

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Dagmara Otto-Ślusarczyk

2016-03-01

Full Text Available Aspartate aminotransferase is an organ - nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms - cytoplasmic (AST1 and mitochondrial (AST2, that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys – 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs.

Engineered human broncho-epithelial tissue-like assemblies

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

2012-01-01

Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

Preliminary Examination of X-ray Scattering from Human Tissues

International Nuclear Information System (INIS)

Desouky, O.S.; Wilkinson, S.; Hall, C.; Rogers, K.; Round, A.

2008-01-01

Small Angle x-ray scattering (SAXS) and wide angle x-ray scattering (WAXS) patterns have been recorded from different human soft tissues using x-ray synchrotron radiation.Pathological breast, normal kidney and lung tissues show SAXS peaks at q-values equal to 0.291 nm -1 and 0.481 nm -1 (d 21.6 nm and d =13. nm) which are the 3 r d and 5 t h order of the well known axial D-spacing of collagen fibrils. The diffraction is particularly intense in the meridional direction indicating some febrile alignment. In contrast, the normal tissue of brain, liver and heart shows diffuse scatter.The wide-angle coherent scattering from normal human tissues of brain, liver, heart, lung, and kidney is typical of that for amorphous materials. The scatter of the healthy adipose breast tissue shows a sharp peak at momentum transfer 1.24 nm -1 (d= 0.417 nm). The data of the other tissues appears to consist of a broad scattering peak. The two scattering regimes succeed in differentiating between the two major components of breast tissue, collagen and adipose tissue. The results of this study suggest that the soft tissues may have scattering patterns that are characteristics for the particular tissue types and tissue disease state. These results indicate that it may be possible use the coherent scattering as a diagnostic tool

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

Science.gov (United States)

Herrera Sanchez, Maria Beatriz; Previdi, Sara; Bruno, Stefania; Fonsato, Valentina; Deregibus, Maria Chiara; Kholia, Sharad; Petrillo, Sara; Tolosano, Emanuela; Critelli, Rossana; Spada, Marco; Romagnoli, Renato; Salizzoni, Mauro; Tetta, Ciro; Camussi, Giovanni

2017-07-27

Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme

HepG2 cells develop signs of riboflavin deficiency within four days of culture in riboflavin-deficient medium*

OpenAIRE

Werner, Ricarda; Manthey, Karoline C.; Griffin, Jacob B.; Zempleni, Janos

2005-01-01

Flavin mononucleotide and flavin adenine dinucleotide are essential coenzymes in redox reactions. For example, flavin adenine dinucleotide is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/L) medium for up to six days; controls ...

Research Applications of Proteolytic Enzymes in Molecular Biology

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József Tőzsér

2013-11-01

Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

International Nuclear Information System (INIS)

Chen, Qi-Liang; Luo, Zhi; Pan, Ya-Xiong; Zheng, Jia-Lang; Zhu, Qing-Ling; Sun, Lin-Dan; Zhuo, Mei-Qin; Hu, Wei

2013-01-01

Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

Energy Technology Data Exchange (ETDEWEB)

Chen, Qi-Liang; Luo, Zhi, E-mail: luozhi99@yahoo.com.cn; Pan, Ya-Xiong; Zheng, Jia-Lang; Zhu, Qing-Ling; Sun, Lin-Dan; Zhuo, Mei-Qin; Hu, Wei

2013-07-15

Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

Science.gov (United States)

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Carnitine palmityl transferase I deficiency

NARCIS (Netherlands)

Al-Aqeel, A. I.; Rashed, M. S.; Ruiter, J. P.; Al-Husseini, H. F.; Al-Amoudi, M. S.; Wanders, R. J.

2001-01-01

Carnitine palmityl transferase I is the key enzyme in the carnitine dependent transport of long chain fatty acids across the mitochondrial inner membrane and its deficiency results in a decrease rate of fatty acids beta-oxidation with decreased energy production. We reported a family of 3 affected

ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage

Science.gov (United States)

Jordan, Jennifer J; Chhim, Sophea; Margulies, Carrie M; Allocca, Mariacarmela; Bronson, Roderick T; Klungland, Arne; Samson, Leona D; Fu, Dragony

2017-01-01

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents. PMID:28726787

Cross-species and tissue variations in cyanide detoxification rates in rodents and non-human primates on protein-restricted diet.

Science.gov (United States)

Kimani, S; Moterroso, V; Morales, P; Wagner, J; Kipruto, S; Bukachi, F; Maitai, C; Tshala-Katumbay, D

2014-04-01

We sought to elucidate the impact of diet, cyanide or cyanate exposure on mammalian cyanide detoxification capabilities (CDC). Male rats (~8 weeks old) (N=52) on 75% sulfur amino acid (SAA)-deficient diet were treated with NaCN (2.5mg/kg bw) or NaOCN (50mg/kg bw) for 6 weeks. Macaca fascicularis monkeys (~12 years old) (N=12) were exclusively fed cassava for 5 weeks. CDC was assessed in plasma, or spinal cord, or brain. In rats, NaCN induced seizures under SAA-restricted diet whereas NaOCN induced motor deficits. No deficits were observed in non-human primates. Under normal diet, the CDC were up to ~80× faster in the nervous system (14 ms to produce one μmol of thiocyanate from the detoxification of cyanide) relative to plasma. Spinal cord CDC was impaired by NaCN, NaOCN, or SAA deficiency. In M. fascicularis, plasma CDC changed proportionally to total proteins (r=0.43; pcyanide may result from a "multiple hit" by the toxicity of cyanide or its cyanate metabolite, the influences of dietary deficiencies, and the tissue variations in CDC. Chronic dietary reliance on cassava may cause metabolic derangement including poor CDC. Copyright © 2014 Elsevier Ltd. All rights reserved.

Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

International Nuclear Information System (INIS)

Sakai, T.; Kisiel, W.

1990-01-01

Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which 125 I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

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Lushchak V.I.

1998-01-01

Full Text Available The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

The effect of ZnO Nps of 20 nm on changes of enzyme and liver tissues of pregnant NMRI mice

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bager Seyed Alipour

2015-01-01

Full Text Available Background :Nowadays, nanotechnology has been developing rapidly and may have considerable effects on industry, society and the environment. In this research the toxicity properties of zinc oxide nanoparticles with a size of 20 nm on enzyme and liver tissue of NMRI mice were studied. Materials and Methods: This experimental study was performed in standard conditions on 25 NMRI mice with an average weight of 30 ± 3 g so that they received different doses of zinc oxide nanoparticles, an a days, for 15 days intraperitoneally. Then, blood samples were taken on day 17 of NMRI mice. The collected tissues were washed with saline and fixed in Boin΄s fluied buffer and stained with hematoxylin and eosin for histopathology evaluation. After data collection, statistical analysis was done using SAS software. Results: The results showed that activity of ALT enzyme at concentrations 50, 100, 150 and 200 mg/kg ZnO Nps at a significant level (p<0.05 increased in comparison with the control group. Histopathological investigation showed that zinc oxide nanoparticles caused severe damage in liver. Damaged liver cells develop leaky membranes and escape of intracellular enzymes into the bloodstream. Conclusion: Our findings showed that using different concentration of zinc oxide nanoparticles could be caused undesirable effects on liver with damage to hepatocyte and level elevation of liver enzymes.

Partial IGF-1 deficiency induces brain oxidative damage and edema, which are ameliorated by replacement therapy.

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Puche, Juan E; Muñoz, Úrsula; García-Magariño, Mariano; Sádaba, María C; Castilla-Cortázar, Inma

2016-01-01

Insulin-like growth factor 1 (IGF-1) induces multiple cytoprotective effects on every tissue, including the brain. Since the mechanisms by which IGF-1 produces neuroprotection are not fully understood, the aim of this work was to delve into the underlying mechanisms. IGF-1 deficient mice (Hz) were compared with wild type (WT) and Hz mice treated with low doses of IGF-1 (2 µg/100 g body weight/day) for 10 days (Hz + IGF). Gene expression, quantitative PCR, histology, and magnetic resonance imaging were performed in the three groups. IGF-1 deficiency induced increased oxidative damage determined by markers of lipid peroxidation and hypoxia, as well as gene expression of heat shock proteins, antioxidant enzymes, and molecules involved in inflammation, apoptosis, and mitochondrial protection. These changes correlated with edema and learning impairment in Hz mice. IGF-1 therapy improved all these alterations. In conclusion, IGF-1 deficiency is responsible for increased brain oxidative damage, edema, and impaired learning and memory capabilities which are rescued by IGF-1 replacement therapy. © 2016 International Union of Biochemistry and Molecular Biology.

Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

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Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

2015-11-02

Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

Advancing biomaterials of human origin for tissue engineering

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Chen, Fa-Ming; Liu, Xiaohua

2015-01-01

Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking in...

Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

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Khan, Sheeba; Priyamvada, Shubha; Khan, Sara A; Khan, Wasim; Farooq, Neelam; Khan, Farah; Yusufi, A N K

2009-07-01

Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

Genetic effects on gene expression across human tissues

NARCIS (Netherlands)

Battle, Alexis; Brown, Christopher D.; Engelhardt, Barbara E.; Montgomery, Stephen B.; Aguet, François; Ardlie, Kristin G.; Cummings, Beryl B.; Gelfand, Ellen T.; Getz, Gad; Hadley, Kane; Handsaker, Robert E.; Huang, Katherine H.; Kashin, Seva; Karczewski, Konrad J.; Lek, Monkol; Li, Xiao; MacArthur, Daniel G.; Nedzel, Jared L.; Nguyen, Duyen T.; Noble, Michael S.; Segrè, Ayellet V.; Trowbridge, Casandra A.; Tukiainen, Taru; Abell, Nathan S.; Balliu, Brunilda; Barshir, Ruth; Basha, Omer; Bogu, Gireesh K.; Brown, Andrew; Castel, Stephane E.; Chen, Lin S.; Chiang, Colby; Conrad, Donald F.; Cox, Nancy J.; Damani, Farhan N.; Davis, Joe R.; Delaneau, Olivier; Dermitzakis, Emmanouil T.; Eskin, Eleazar; Ferreira, Pedro G.; Frésard, Laure; Gamazon, Eric R.; Garrido-Martín, Diego; Gewirtz, Ariel D. H.; Gliner, Genna; Gloudemans, Michael J.; Guigo, Roderic; Hall, Ira M.; Han, Buhm; He, Yuan

2017-01-01

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression

Normal bone density in male pseudohermaphroditism due to 5a- reductase 2 deficiency

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Costa Elaine Maria Frade

2001-01-01

Full Text Available Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4 and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

Astrocyte calcium signal and gliotransmission in human brain tissue.

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Navarrete, Marta; Perea, Gertrudis; Maglio, Laura; Pastor, Jesús; García de Sola, Rafael; Araque, Alfonso

2013-05-01

Brain function is recognized to rely on neuronal activity and signaling processes between neurons, whereas astrocytes are generally considered to play supportive roles for proper neuronal function. However, accumulating evidence indicates that astrocytes sense and control neuronal and synaptic activity, indicating that neuron and astrocytes reciprocally communicate. While this evidence has been obtained in experimental animal models, whether this bidirectional signaling between astrocytes and neurons occurs in human brain remains unknown. We have investigated the existence of astrocyte-neuron communication in human brain tissue, using electrophysiological and Ca(2+) imaging techniques in slices of the cortex and hippocampus obtained from biopsies from epileptic patients. Cortical and hippocampal human astrocytes displayed spontaneous Ca(2+) elevations that were independent of neuronal activity. Local application of transmitter receptor agonists or nerve electrical stimulation transiently elevated Ca(2+) in astrocytes, indicating that human astrocytes detect synaptic activity and respond to synaptically released neurotransmitters, suggesting the existence of neuron-to-astrocyte communication in human brain tissue. Electrophysiological recordings in neurons revealed the presence of slow inward currents (SICs) mediated by NMDA receptor activation. The frequency of SICs increased after local application of ATP that elevated astrocyte Ca(2+). Therefore, human astrocytes are able to release the gliotransmitter glutamate, which affect neuronal excitability through activation of NMDA receptors in neurons. These results reveal the existence of reciprocal signaling between neurons and astrocytes in human brain tissue, indicating that astrocytes are relevant in human neurophysiology and are involved in human brain function.

The case for applying tissue engineering methodologies to instruct human organoid morphogenesis.

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Marti-Figueroa, Carlos R; Ashton, Randolph S

2017-05-01

Three-dimensional organoids derived from human pluripotent stem cell (hPSC) derivatives have become widely used in vitro models for studying development and disease. Their ability to recapitulate facets of normal human development during in vitro morphogenesis produces tissue structures with unprecedented biomimicry. Current organoid derivation protocols primarily rely on spontaneous morphogenesis processes to occur within 3-D spherical cell aggregates with minimal to no exogenous control. This yields organoids containing microscale regions of biomimetic tissues, but at the macroscale (i.e. 100's of microns to millimeters), the organoids' morphology, cytoarchitecture, and cellular composition are non-biomimetic and variable. The current lack of control over in vitro organoid morphogenesis at the microscale induces aberrations at the macroscale, which impedes realization of the technology's potential to reproducibly form anatomically correct human tissue units that could serve as optimal human in vitro models and even transplants. Here, we review tissue engineering methodologies that could be used to develop powerful approaches for instructing multiscale, 3-D human organoid morphogenesis. Such technological mergers are critically needed to harness organoid morphogenesis as a tool for engineering functional human tissues with biomimetic anatomy and physiology. Human PSC-derived 3-D organoids are revolutionizing the biomedical sciences. They enable the study of development and disease within patient-specific genetic backgrounds and unprecedented biomimetic tissue microenvironments. However, their uncontrolled, spontaneous morphogenesis at the microscale yields inconsistences in macroscale organoid morphology, cytoarchitecture, and cellular composition that limits their standardization and application. Integration of tissue engineering methods with organoid derivation protocols could allow us to harness their potential by instructing standardized in vitro morphogenesis

The expression of Egfl7 in human normal tissues and epithelial tumors.

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Fan, Chun; Yang, Lian-Yue; Wu, Fan; Tao, Yi-Ming; Liu, Lin-Sen; Zhang, Jin-Fan; He, Ya-Ning; Tang, Li-Li; Chen, Guo-Dong; Guo, Lei

2013-04-23

To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors.
 RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results.
 Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. 
 Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.

Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

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Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

2015-01-01

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

Human umbilical cord mesenchymal stem cells: osteogenesis in vivo as seed cells for bone tissue engineering.

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Diao, Yinze; Ma, Qingjun; Cui, Fuzhai; Zhong, Yanfeng

2009-10-01

Mesenchymal stem cells (MSCs) are ideal seed cells for bone tissue engineering. However, intrinsic deficiencies exist for the autologous transplantation strategy of constructing artificial bone with MSCs derived from bone marrow of patients. In this study, MSCs-like cells were isolated from human umbilical cords and were expanded in vitro. Flow cytometric analysis revealed that cells from the fourth passage were positive for CD29, CD44, CD71, CD73, CD90, and CD105 whereas they were negative for CD14, CD34, CD45, and CD117. Furthermore, these cells expressed HLA-A, B, C (MHC-I), but not HLA-DP, DQ, DR (MHC-II), or costimulatory molecules such as CD80 and CD86. Following incubation in specific inductive media for 3 weeks, cultured cells were shown to possess potential to differentiate into adipogenic, osteogenic or chondrogenic lineages in vitro. The umbilical cord-derived MSCs (UC-MSCs) were loaded with a biomimetic artificial bone scaffold material before being implanted subcutaneously in the back of Balb/c nude mice for four to twelve weeks. Our results revealed that UC-MSCs loaded with the scaffold displayed capacity of osteogenic differentiation leading to osteogenesis with human origin in vivo. As a readily available source of seed cells for bone tissue engineering, UC-MSCs should have broad application prospects.

Severe Vitamin D Deficiency in Human Immunodeficiency Virus-Infected Pregnant Women is Associated with Preterm Birth.

Science.gov (United States)

Jao, Jennifer; Freimanis, Laura; Mussi-Pinhata, Marisa M; Cohen, Rachel A; Monteiro, Jacqueline Pontes; Cruz, Maria Leticia; Branch, Andrea; Sperling, Rhoda S; Siberry, George K

2017-04-01

Background  Low maternal vitamin D has been associated with preterm birth (PTB). Human immunodeficiency virus (HIV)-infected pregnant women are at risk for PTB, but data on maternal vitamin D and PTB in this population are scarce. Methods  In a cohort of Latin American HIV-infected pregnant women from the National Institute of Child Health and Human Development International Site Development Initiative protocol, we examined the association between maternal vitamin D status and PTB. Vitamin D status was defined as the following 25-hydroxyvitamin D levels: severe deficiency (PTBs = 36 weeks [interquartile range: 34-36]). In multivariate analysis, severe vitamin D deficiency was associated with PTB (odds ratio = 4.7, 95% confidence interval: 1.3-16.8]). Conclusion  Severe maternal vitamin D deficiency is associated with PTB in HIV-infected Latin American pregnant women. Further studies are warranted to determine if vitamin D supplementation in HIV-infected women may impact PTB. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Identification of human tissue cross-presenting dendritic cells

OpenAIRE

Haniffa, Muzlifah; Collin, Matthew; Ginhoux, Florent

2013-01-01

Dendritic cells (DCs) are a heterogeneous group of functionally specialized antigen-presenting cells. We recently characterized the human tissue cross-presenting DCs and aligned the human and mouse DC subsets. Our findings will facilitate the translation of murine DC studies to the human setting and aid the design of DC-based vaccine strategies for infection and cancer immunotherapy.

Up-Regulation of the Lymphatic Marker Podoplanin, a Mucin-Type Transmembrane Glycoprotein, in Human Squamous Cell Carcinomas and Germ Cell Tumors

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Schacht, Vivien; Dadras, Soheil S.; Johnson, Louise A.; Jackson, David G.; Hong, Young-Kwon; Detmar, Michael

2005-01-01

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers. PMID:15743802

Effects of potassium or potassium/magnesium supplementation on potassium content of body tissues and fluids in furosemide-treated rats on magnesium-deficient or magnesium-sufficient diet

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Coram, W.M.; Kapeghian, J.C.; Plocinski, A.F.; Toledo, L.M.; Douglas, F.L.; Weiss, G.B. (Univ. of New Jersey, Newmark (USA))

1990-01-01

Persistent Mg{sup 2+} deficiency may interfere with restoration of normal tissue K{sup +} levels. This study examined: (a) the effects of chronic furosemide treatment of K{sup +} of sartorius, aorta and ventricle of rats fed Mg{sup 2+}-deficient or Mg{sup 2+} sufficient diet and deionized water; (b) whether normal tissue K{sup +} is restored by oral K{sup +} or K{sup +}/Mg{sup 2+} supplementation with continued furosemide therapy. Levels of Mg{sup 2+} were also measured. Furosemide decreased K{sup +} in sartorius, aorta and ventricle by 5.5, 4.3 and 19.9 {mu}Eq/gm, respectively, in rats fed 100 ppm Mg{sup 2+} diet. Furosemide did not alter K{sup +} levels in rats fed 400 ppm Mg{sup 2+} diet. K{sup +} supplementation restored K{sup +} to normal in sartorius but the addition of Mg{sup 2+} supplementation was necessary to restore K+ levels to normal in ventricle and aorta. These data indicate that furosemide can decrease tissue K{sup +} in rats on a Mg{sup 2+}- deficient diet. This decrease can be reversed during diuretic administration by K{sup +} supplementation in sartorius, or K{sup +} plus Mg{sup 2+} supplementation in ventricle and aorta.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

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Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

2014-01-01

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

In vitro generation of functional insulin-producing cells from lipoaspirated human adipose tissue-derived stem cells.

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Mohamad Buang, Mohamad Lizan; Seng, Heng Kien; Chung, Lee Han; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

2012-01-01

Tissue engineering strategy has been considered as an alternative treatment for diabetes mellitus due to lack of permanent pharmaceutical treatment and islet donors for transplantation. Various cell lines have been used to generate functional insulin-producing cells (IPCs) including progenitor pancreatic cell lines, embryonic stem cells (ESCs), umbilical cord blood stem cells (UCB-SCs), adult bone marrow stem cells (BMSCs), and adipose tissue-derived stem cells (ADSCs). Human ADSCs from lipoaspirated abdominal fat tissue was differentiated into IPCs following a two-step induction protocol based on a combination of alternating high and low glucose, nicotinamide, activin A and glucagon-like peptide 1 (GLP-1) for a duration of 3 weeks. During differentiation, histomorphological changes of the stem cells towards pancreatic β-islet characteristics were observed via light microscope and transmission electron microscope (TEM). Dithizone (DTZ) staining, which is selective towards IPCs, was used to stain the new islet-like cells. Production of insulin hormone by the cells was analyzed via enzyme-linked immunosorbent assay (ELISA), whereas its hormonal regulation was tested via a glucose challenge test. Histomorphological changes of the differentiated cells were noted to resemble pancreatic β-cells, whereas DTZ staining positively stained the cells. The differentiated cells significantly produced human insulin as compared to the undifferentiated ADSCs, and its production was increased with an increase of glucose concentration in the culture medium. These initial data indicate that human lipoaspirated ADSCs have the potential to differentiate into functional IPCs, and could be used as a therapy to treat diabetes mellitus in the future. Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.

Induction of hyperresponsiveness in human airway tissue by neutrophils--mechanism of action.

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Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

1996-05-01

The two main features of asthma are bronchial hyperresponsiveness and inflammation. The inflammatory response in asthma consists of infiltration and activation of a variety of inflammatory cells including neutrophils. Our previous studies have shown that stimulated neutrophil supernatants cause hyperresponsiveness of human bronchial tissue in vitro. To investigate the effect of the sensitization status of the tissue and the albumin concentration used to prepare supernatants on the response of human bronchial tissue to stimulated neutrophil supernatants. Neutrophil supernatants were prepared from human isolated blood in the presence of varying concentrations of albumin (0%, 0.1% and 4%). Neutrophil supernatants were added to sensitized and non-sensitized human isolated bronchial tissue which was stimulated with electrical field stimulation (EFS) (20 s every 4 min). Receptor antagonists specific for the prostaglandin and thromboxane (10(-7) M GR32191), platelet activating factor (10(-6) M WEB 2086), leukotriene D4 (10(-6) M MK-679) and neurokinin A (10(-7) M SR48968) receptors were used to identify neutrophil products responsible for the effects observed in the bronchial tissue. In non-sensitized human bronchial tissue, stimulated neutrophil supernatants induced a direct contraction in the presence of 0% and 0.1% but not 4% albumin. This contraction was due to leukotriene D4 as MK-679 completely inhibited the contraction. In contrast, stimulated neutrophil supernatants increased responsiveness of sensitized human bronchial tissue to EFS. The increased responsiveness was observed only in the presence of 0.1% albumin, with the site of modulation likely to be prejunctional on the parasympathetic nerve. The increased responsiveness was not inhibited by any of the antagonists tested. Sensitization status of the tissue and albumin concentration effect the responsiveness of human bronchial tissue to stimulated neutrophil supernatant. Our results suggest a possible role for

The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

Science.gov (United States)

Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

2013-01-01

BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

Adipose tissue macrophages impair preadipocyte differentiation in humans.

Directory of Open Access Journals (Sweden)

Li Fen Liu

Full Text Available The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation.Abdominal subcutaneous(SAT and visceral(VAT adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified.Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001. With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance.The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

Immunolocalization of transforming growth factor alpha in normal human tissues

DEFF Research Database (Denmark)

Christensen, M E; Poulsen, Steen Seier

1996-01-01

anchorage-independent growth of normal cells and was, therefore, considered as an "oncogenic" growth factor. Later, its immunohistochemical presence in normal human cells as well as its biological effects in normal human tissues have been demonstrated. The aim of the present investigation was to elucidate...... the distribution of the growth factor in a broad spectrum of normal human tissues. Indirect immunoenzymatic staining methods were used. The polypeptide was detected with a polyclonal as well as a monoclonal antibody. The polyclonal and monoclonal antibodies demonstrated almost identical immunoreactivity. TGF......-alpha was found to be widely distributed in cells of normal human tissues derived from all three germ layers, most often in differentiated cells. In epithelial cells, three different kinds of staining patterns were observed, either diffuse cytoplasmic, cytoplasmic in the basal parts of the cells, or distinctly...

Biological Roles of Aberrantly Expressed Glycosphingolipids and Related Enzymes in Human Cancer Development and Progression

Directory of Open Access Journals (Sweden)

Dinghao Zhuo

2018-05-01

Full Text Available Glycosphingolipids (GSLs, which consist of a hydrophobic ceramide backbone and a hydrophilic carbohydrate residue, are an important type of glycolipid expressed in surface membranes of all animal cells. GSLs play essential roles in maintenance of plasma membrane stability, in regulation of numerous cellular processes (including adhesion, proliferation, apoptosis, and recognition, and in modulation of signal transduction pathways. GSLs have traditionally been classified as ganglio-series, lacto-series, or globo-series on the basis of their diverse types of oligosaccharide chains. Structures and functions of specific GSLs are also determined by their oligosaccharide chains. Different cells and tissues show differential expression of GSLs, and changes in structures of GSL glycan moieties occur during development of numerous types of human cancer. Association of GSLs and/or related enzymes with initiation and progression of cancer has been documented in 100s of studies, and many such GSLs are useful markers or targets for cancer diagnosis or therapy. In this review, we summarize (i recent studies on aberrant expression and distribution of GSLs in common human cancers (breast, lung, colorectal, melanoma, prostate, ovarian, leukemia, renal, bladder, gastric; (ii biological functions of specific GSLs in these cancers.

Long-term culture of human liver tissue with advanced hepatic functions.

Science.gov (United States)

Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

2017-06-02

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

A regenerative biology view on artificial tissue construction and 3D bioprinting: what may we learn from natural regenerative phenomena?

DEFF Research Database (Denmark)

Lauridsen, Henrik

2017-01-01

The implications of the low tissue regenerative potential in humans are severe and widespread. Several of our major diseases are direct results of this deficiency that leaves us vulnerable to events of tissue damage. This is opposed to some animal groups, such as the urodele amphibians (salamanders...

Vibrational Micro-Spectroscopy of Human Tissues Analysis: Review.

Science.gov (United States)

Bunaciu, Andrei A; Hoang, Vu Dang; Aboul-Enein, Hassan Y

2017-05-04

Vibrational spectroscopy (Infrared (IR) and Raman) and, in particular, micro-spectroscopy and micro-spectroscopic imaging have been used to characterize developmental changes in tissues, to monitor these changes in cell cultures and to detect disease and drug-induced modifications. The conventional methods for biochemical and histophatological tissue characterization necessitate complex and "time-consuming" sample manipulations and the results are rarely quantifiable. The spectroscopy of molecular vibrations using mid-IR or Raman techniques has been applied to samples of human tissue. This article reviews the application of these vibrational spectroscopic techniques for analysis of biological tissue published between 2005 and 2015.

Immunolocalisation of oestrogen receptor beta in human tissues.

Science.gov (United States)

Taylor, A H; Al-Azzawi, F

2000-02-01

Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.

Deficiency of UV-induced excision repair in human thymocytes

International Nuclear Information System (INIS)

Gensler, H.L.; Lindberg, R.E.; Pinnas, J.L.; Jones, J.F.

1985-01-01

The capacity of human thymocytes and of differentiated lymphocytes circulating in peripheral blood to perform unscheduled DNA synthesis (a measure of nucleotide excision repair) after UV irradiation was measured by radioautographic analysis. Only 4% of immature T lymphocytes, but 68% of circulating lymphocytes exhibited unscheduled DNA synthesis. When UV sensitivity of peripheral blood lymphocytes and thymocytes from the same donor were compared, the thymocytes, in each case, were significantly more UV sensitive than were the circulating lymphocytes. Peripheral blood lymphocytes from subjects undergoing halothane and morphine anesthesia during surgery showed 56% less excision repair capacity than those from unanesthetized donors. The difference occurred in the number of cells capable of repair rather than in the extent of repair synthesis per cell. Ultraviolet-induced unscheduled DNA synthesis occurred in only 3% of the thymocytes removed from rats killed by cervical dislocation. Therefore, the deficiency of excision repair was observed in rat thymocytes which had not been affected by anesthesia or surgical trauma. The results indicate that immature T-cells are deficient in nucleotide excision repair whereas the majority of mature peripheral blood lymphocytes exhibit such repair. (author)

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

DEFF Research Database (Denmark)

Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

DEFF Research Database (Denmark)

Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

Increased variability of bone tissue mineral density resulting from estrogen deficiency influences creep behavior in a rat vertebral body.

Science.gov (United States)

Kim, Do-Gyoon; Navalgund, Anand R; Tee, Boon Ching; Noble, Garrett J; Hart, Richard T; Lee, Hye Ri

2012-11-01

Progressive vertebral deformation increases the fracture risk of a vertebral body in the postmenopausal patient. Many studies have observed that bone can demonstrate creep behavior, defined as continued time-dependent deformation even when mechanical loading is held constant. Creep is a characteristic of viscoelastic behavior, which is common in biological materials. We hypothesized that estrogen deficiency-dependent alteration of the mineral distribution of bone at the tissue level could influence the progressive postmenopausal vertebral deformity that is observed as the creep response at the organ level. The objective of this study was thus to examine whether the creep behavior of vertebral bone is changed by estrogen deficiency, and to determine which bone property parameters are responsible for the creep response of vertebral bone at physiological loading levels using an ovariectomized (OVX) rat model. Correlations of creep parameters with bone mineral density (BMD), tissue mineral density (TMD) and architectural parameters of both OVX and sham surgery vertebral bone were tested. As the vertebral creep was not fully recovered during the post-creep unloading period, there was substantial residual displacement for both the sham and OVX groups. A strong positive correlation between loading creep and residual displacement was found (r=0.868, pcreep behavior of the OVX group (pcreep caused progressive, permanent reduction in vertebral height for both the sham and OVX groups. In addition, estrogen deficiency-induced active bone remodeling increased variability of trabecular TMD in the OVX group. Taken together, these results suggest that increased variability of trabecular TMD resulting from high bone turnover influences creep behavior of the OVX vertebrae. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of single and binary combinations of plant-derived molluscicides on different enzyme activities in the nervous tissue of Achatina fulica.

Science.gov (United States)

Rao, I G; Singh, Amrita; Singh, V K; Singh, D K

2003-01-01

Effect of single and binary treatments of plant-derived molluscicides on different enzymes--acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and acid/alkaline phosphatase (ACP/ALP)--in the nervous tissue of the harmful terrestrial snail Achatina fulica were studied. Sublethal in vivo 24-h exposure to 40% and 80% LC(50) of Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, Nerium indicum bark powder and binary combinations of A. sativum (AS) + C. deodara (CD) and CD + A. indica (AI) oils significantly altered the activity of these enzymes in the nervous tissue of Achatina fulica. The binary treatment of AS + CD was more effective against AChE, LDH, and ALP than the single ones. However, binary treatment of AI + CD was more effective against ALP. Copyright 2003 John Wiley & Sons, Ltd.

Combined spectroscopic imaging and chemometric approach for automatically partitioning tissue types in human prostate tissue biopsies

Science.gov (United States)

Haka, Abigail S.; Kidder, Linda H.; Lewis, E. Neil

2001-07-01

We have applied Fourier transform infrared (FTIR) spectroscopic imaging, coupling a mercury cadmium telluride (MCT) focal plane array detector (FPA) and a Michelson step scan interferometer, to the investigation of various states of malignant human prostate tissue. The MCT FPA used consists of 64x64 pixels, each 61 micrometers 2, and has a spectral range of 2-10.5 microns. Each imaging data set was collected at 16-1 resolution, resulting in 512 image planes and a total of 4096 interferograms. In this article we describe a method for separating different tissue types contained within FTIR spectroscopic imaging data sets of human prostate tissue biopsies. We present images, generated by the Fuzzy C-Means clustering algorithm, which demonstrate the successful partitioning of distinct tissue type domains. Additionally, analysis of differences in the centroid spectra corresponding to different tissue types provides an insight into their biochemical composition. Lastly, we demonstrate the ability to partition tissue type regions in a different data set using centroid spectra calculated from the original data set. This has implications for the use of the Fuzzy C-Means algorithm as an automated technique for the separation and examination of tissue domains in biopsy samples.

Cystathionine-gamma-lyase deficient mice are protected against the development of multiorgan failure and exhibit reduced inflammatory response during burn.

Science.gov (United States)

Ahmad, Akbar; Druzhyna, Nadiya; Szabo, Csaba

2017-08-01

Considering the role of H 2 S in critical illness, the aim of this study was to compare the outcome of burn in wild-type mice and in mice deficient in CSE, one of the principal mammalian H 2 S-generating enzymes. Animals were subjected to scald burn. Outcome variables included indices of organ injury, clinical chemistry parameters and plasma levels of inflammatory mediators. Plasma levels of H 2 S significantly increased in response to burn in wild-type mice, but remained unchanged in CSE -/- mice. Expression of the three H 2 S-producing enzymes (CSE, CBS and 3-MST) in the lung and liver, and the capacity of tissue homogenates to produce H 2 S, however, was not affected by burn. In CSE deficient mice there was a significant amelioration of burn-induced accumulation of myeloperoxidase levels in heart, lung, liver and kidney and significantly lower degree of malon dialdehyde accumulation in the heart, lung and kidney than in wild-type mice. CSE deficient mice, compared to wild-type mice, showed a significant attenuation of the burn-induced elevation in circulating alkaline aminotransferase and blood urea nitrogen and creatinine levels, indicative of protective effects of CSE deficiency against burn-induced hepatic, and renal functional impairment. Multiple burn-induced inflammatory mediators (TNF-α, IL-1β, IL-4, IL-6, IL-10 and IL-12) were significantly lower in the plasma of CSE -/- animals after burn than in the plasma of wild-type controls subjected to burns. In conclusion, CSE deficiency improves organ function and attenuates the inflammatory response in a murine model of burn. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

FXR-deficiency confers increased susceptibility to torpor

NARCIS (Netherlands)

Cariou, Bertrand; Bouchaert, Emmanuel; Abdelkarim, Mouaadh; Dumont, Julie; Caron, Sandrine; Fruchart, Jean-Charles; Burcelin, Remy; Kuipers, Folkert; Staels, Bart

2007-01-01

The role of the nuclear receptor FXR in adaptive thermogenesis was investigated using FXR-deficient mice. Despite elevated serum bile acid concentrations and increased mRNA expression profiles of thermogenic genes in brown adipose tissue, FXR-deficiency did not alter energy expenditure under basal

High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

Directory of Open Access Journals (Sweden)

Ehud Goldin

Full Text Available Gaucher disease (GD, the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase. Previously, wildtype GCase was used for high throughput screening (HTS of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

1994-12-31

Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

Implications of human tissue studies

International Nuclear Information System (INIS)

Kathren, R.L.

1986-10-01

Through radiochemical analysis of voluntary tissue donations, the United States Transuranium and Uranium Registries are gaining improved understanding of the distribution and biokinetics of actinide elements in occupationally exposed persons. Evaluation of the first two whole body contributions to the Transuranium Registry revealed an inverse proportionality between actinide concentration and bone ash fraction. The analysis of a whole body with a documented 241 Am deposition indicated a significantly shorter half-time in liver and a greater fraction resident in the skeleton than predicted by existing models. Other studies of the Registries are designed to evaluate in vivo estimates of actinide deposition with those derived from postmortem tissue analysis, compare results of animal experiments with human data, and reviw histopathologic slides for tissue toxicity that might be attributable to exposure to uranium and the transuranic elements. The implications of these recent findings and other work of the Registries are discussed from the standpoint of their potential impact on biokinetic modeling, internal dose assessment, safety standards, and operational health physics practices

Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

Energy Technology Data Exchange (ETDEWEB)

Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (USA)); Birkenmeier, E.H.; Gwynn, B. (Jackson Laboratory, Bar Harbor, ME (USA))

1990-05-01

The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

Iron deficiency anemia from diagnosis to treatment in children

OpenAIRE

Özdemir, Nihal

2015-01-01

Iron deficiency is the most common nutritional deficiency worldwide and an important public health problem especially in developing countries. Since the most important indicator of iron deficieny is anemia, the terms “iron deficiency” and “iron deficiency anemia” are often used interchangeably. However, iron deficiency may develop in the absence of anemia and the tissues may be affected from this condition. The most common causes of iron deficiency in children include insufficient intake toge...

Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

Science.gov (United States)

Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

2014-01-01

There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to

Construction of retroviral recombinant containing human tissue ...

African Journals Online (AJOL)

USER

2010-03-29

Mar 29, 2010 ... Recombinant retroviral vector containing human tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene was ..... heavy metal ions, the protein could be express in an .... involves adhesion, degradation and movement. To.

Lymphoid Tissue Grafts in Man

Energy Technology Data Exchange (ETDEWEB)

Kay, H. E.M. [Royal Marsden Hospital, Institute of Cancer Research, London (United Kingdom)

1969-07-15

Grafts of lymphoid tissue or of lymphoid stem cells may be appropriate in the treatment of some congenital immune deficiency disorders. The reasons for preferring tissues of foetal origin are discussed and the evidence for foetal immunocompetence is briefly summarized. Methods of storing foetal liver cells and cells or fragments of thymus are mentioned, and the organization of the Foetal Tissue Bank of the Royal Marsden Hospital is described. Clinical data from transplantation of lymphoid cells in various immune deficiency disorders are briefly presented. (author)

Injury Response of Resected Human Brain Tissue In Vitro

NARCIS (Netherlands)

Verwer, Ronald W. H.; Sluiter, Arja A.; Balesar, Rawien A.; Baaijen, Johannes C.; de Witt Hamer, Philip C.; Speijer, Dave; Li, Yichen; Swaab, Dick F.

2015-01-01

Brain injury affects a significant number of people each year. Organotypic cultures from resected normal neocortical tissue provide unique opportunities to study the cellular and neuropathological consequences of severe injury of adult human brain tissue in vitro. The in vitro injuries caused by

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

Energy Technology Data Exchange (ETDEWEB)

Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

1988-03-25

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human x-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, the authors introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

Directory of Open Access Journals (Sweden)

Lushchak V.I.

2001-01-01

Full Text Available The effects of short-term burst (5 min at 1.8 m/s swimming and long-term cruiser (60 min at 1.2 m/s swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK, pyruvate kinase (PK, fructose-1,6-bisphosphatase (FBPase, and phosphoglucomutase (PGM all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

Science.gov (United States)

Jackson, Matilda; Derrick Roberts, Ainslie; Martin, Ellenore; Rout-Pitt, Nathan; Gronthos, Stan; Byers, Sharon

2015-04-01

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage. Copyright © 2015 Elsevier Inc. All rights reserved.

Water hardness and cardiovascular disease. Elements in water and human tissues

Energy Technology Data Exchange (ETDEWEB)

Sharrett, A R

1977-05-01

The hypothesis that the hardness of drinking water has a causal role in the development of cardiovascular disease will be strengthened if it can be demonstrated that elements in drinking water find their way into human tissues in significant amounts. For biologically important metals, the evidence is reviewed for a relationship of tissue levels to levels in drinking water. Hard water can contribute significantly to daily magnesium intake. Residents of hard-water areas may have raised levels of magnesium in coronary arteries, bone, and myocardial tissue. Lead levels in bone and in blood have been shown to be elevated in individuals living in homes with lead plumbing and soft water. Cadmium intake from water is probably small compared to that from other sources, and there is no convincing evidence of alteration in human tissue levels via drinking water cadmium. Human zinc and copper tissue levels are of interest but have not been adequately studied in relation to drinking water levels.

ERC/mesothelin is expressed in human gastric cancer tissues and cell lines.

Science.gov (United States)

Ito, Tomoaki; Kajino, Kazunori; Abe, Masaaki; Sato, Koichi; Maekawa, Hiroshi; Sakurada, Mutsumi; Orita, Hajime; Wada, Ryo; Kajiyama, Yoshiaki; Hino, Okio

2014-01-01

ERC/mesothelin is expressed in mesothelioma and other malignancies. The ERC/mesothelin gene (MSLN) encodes a 71-kDa precursor protein, which is cleaved to yield 31-kDa N-terminal (N-ERC/mesothelin) and 40-kDa C-terminal (C-ERC/mesothelin) proteins. N-ERC/mesothelin is a soluble protein and has been reported to be a diagnostic serum marker of mesothelioma and ovarian cancer. Gastric cancer tissue also expresses C-ERC/mesothelin, but the significance of serum N-ERC levels for diagnosing gastric cancer has not yet been studied. We examined the latter issue in the present study as well as C-ERC/mesothelin expression in human gastric cancer tissues and cell lines. We immunohistochemically examined C-ERC/mesothelin expression in tissue samples from 50 cases of gastric cancer, and we also assessed the C-ERC/mesothelin expression in 6 gastric cancer cell lines (MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1) using reverse transcription-polymerase chain reaction, flow cytometry, immunohistochemistry and immunoblotting. We also examined the N-ERC/mesothelin concentrations in the supernatants of cultured cells and in the sera of gastric cancer patients using an enzyme-linked immunosorbent assay (ELISA). N-ERC/mesothelin was detected in the supernatants of 3 gastric cancer cell lines (MKN-1, NUGC-4 and TMK-1) by ELISA, but its concentration in the sera of gastric cancer patients was almost same as that observed in the sera of the normal controls. In the gastric cancer tissues, C-ERC/mesothelin expression was associated with lymphatic invasion. N-ERC/mesothelin was secreted into the supernatants of gastric cancer cell lines, but does not appear to be a useful serum marker of gastric cancer.

Reparative Osteogenesis in Normal Conditions and in Micronutrient Iodine and Selenium Deficiency

Directory of Open Access Journals (Sweden)

P.Ye. Kovalchuk

2015-04-01

Full Text Available Today, a number of unresolved issues remains without researchers’ attention and should be explored, among them: the impact of selenium and iodine deficiencies on bone tissue, healing of bone defects and morphological peculiarities of the process under micronutrient iodine and selenium deficiency. This paper presents the results of experimental study of physiological features and reparative osteogenesis in posttraumatic defects of femoral metadiaphysis under selenium and iodine deficiency. The data that we have shown testify the negative impact of micronutrient deficiency on reparative osteogenesis that is manifested by inhibition of this process and is accompanied by the formation of bone regenerate, deterioration of structural and functional state of bone tissue, development of degenerative and necrotic changes in bone tissue and epiphyseal cartilage.

Radioisotope-enzymes and cancer study

International Nuclear Information System (INIS)

Luyen, T. van

2008-01-01

Cancer is a pathological sign, when the abnormal cells appear in certain human tissues or organs. These cells can reproduce beyond the control of normal biological protection mechanism. Because they reproduce very fast, the metabolic process is accelerated, which causes the extreme need for more energy, substrate and catalyzing enzymes. Based on these needs, we can control the metabolic process by: Stopping supplying the energy. Stopping supplying the substrate and the materials to build up the cell's structure. Stopping operating catalysis by breaking out the enzyme's structure. Destroying the tumor cell by extra agents such as radiations and chemicals. All of these methods have been studied for a long time, which costs too much money, time and labor. Although we succeeded in some ways, the results are still not satisfactory. There are many reasons for this situation but the main one is the lack of information to understand all the processes taking place in the cell and our body. However, as far as we studied, we would like to propose the method to break the structure of the enzyme by nuclear decay process. (author)

Serum cholinesterases are differentially regulated in normal and dystrophin-deficient mutant mice

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Andrea R. Durrant

2012-06-01

Full Text Available The cholinesterases, acetylcholinesterase and butyrylcholinesterase (pseudocholinesterase, are abundant in the nervous system and in other tissues. The role of acetylcholinesterase in terminating transmitter action in the peripheral and central nervous system is well understood. However, both knowledge of the function(s of the cholinesterases in serum, and of their metabolic and endocrine regulation under normal and pathological conditions, is limited. This study investigates acetylcholinesterase and butyrylcholinesterase in sera of dystrophin-deficient mdx mutant mice, an animal model for the human Duchenne muscular dystrophy and in control healthy mice. The data show systematic and differential variations in the concentrations of both enzymes in the sera, and specific changes dictated by alteration of hormonal balance in both healthy and dystrophic mice. While acetylcholinesterase in mdx-sera is elevated, butyrylcholinesterase is markedly diminished, resulting in an overall cholinesterase decrease compared to sera of healthy controls. The androgen testosterone (T is a negative modulator of butyrylcholinesterase, but not of acetylcholinesterase, in male mouse sera. T-removal elevated both butyrylcholinesterase activity and the butyrylcholinesterase/acetylcholinesterase ratio in mdx male sera to values resembling those in healthy control male mice. Mechanisms of regulation of the circulating cholinesterases and their impairment in the dystrophic mice are suggested, and clinical implications for diagnosis and treatment are considered.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

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Kytidou, Kassiani; Beekwilder, Jules; Artola, Marta; van Meel, Eline; Wilbers, Ruud H P; Moolenaar, Geri F; Goosen, Nora; Ferraz, Maria J; Katzy, Rebecca; Voskamp, Patrick; Florea, Bogdan I; Hokke, Cornelis H; Overkleeft, Herman S; Schots, Arjen; Bosch, Dirk; Pannu, Navraj; Aerts, Johannes M F G

2018-04-19

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lysoGb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lysoGb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Enzyme immunoassay of oestrogen receptors in needle biopsies from human liver

DEFF Research Database (Denmark)

Becker, U; Andersen, J; Poulsen, H S

1991-01-01

For quantitative assessments of sex hormone receptors in liver tissue, ligand binding assays are inconvenient, as they require large biopsies (0.5-1.0 g). The present study shows that it is possible to measure oestrogen receptors (ER) quantitatively in needle biopsy specimens as small as 10 mg...... by modifications of a commercial enzyme immunoassay employing monoclonal antibodies. Sucrose gradient centrifugation and the dextran charcoal method served as reference methods. A consecutive series of needle biopsies from patients suspected of liver disease were investigated. The biopsies (n = 37) had a median...

Novel targets for ATM-deficient malignancies

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Winkler, Johannes; Hofmann, Kay; Chen, Shuhua

2014-01-01

Conventional chemo- and radiotherapies for the treatment of cancer target rapidly dividing cells in both tumor and non-tumor tissues and can exhibit severe cytotoxicity in normal tissue and impair the patient's immune system. Novel targeted strategies aim for higher efficacy and tumor specificity. The role of ATM protein in the DNA damage response is well known and ATM deficiency frequently plays a role in tumorigenesis and development of malignancy. In addition to contributing to disease development, ATM deficiency also renders malignant cells heavily dependent on other pathways that cooperate with the ATM-mediated DNA damage response to ensure tumor cell survival. Disturbing those cooperative pathways by inhibiting critical protein components allows specific targeting of tumors while sparing healthy cells with normal ATM status. We review druggable candidate targets for the treatment of ATM-deficient malignancies and the mechanisms underlying such targeted therapies. PMID:27308314

JCL Roundtable: enzyme replacement therapy for lipid storage disorders.

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Brown, W Virgil; Desnick, Robert J; Grabowski, Gregory A

2014-01-01

There are several inherited disorders that involve abnormal storage of lipids in tissues leading to severe compromise of organs. Sadly, these are often accompanied by lifelong morbidity and early mortality. Disorders such as Gaucher, Fabry, and lysosomal acid lipase deficiencies (Wolman and cholesteryl ester storage diseases) have been known for many years, and provide a difficult and frustrating set of problems for patients, their families, and their physicians. With recombinant methods of protein synthesis, it is now possible to literally replace the defective enzymes that underlie the basic pathophysiology of many such disorders. The delivery of these enzymes into the affected cells is possible because of their location in the lysosomes where the natural degradation of their lipid substrates occurs. I have asked 2 well-known investigators to join us for this Roundtable. These are professors who have been involved with the research that has made this type of therapy possible and who have participated in the clinical trials that demonstrated the value of enzyme replacement therapy. They are Dr. Robert Desnick, dean of Genetic and Genomic Medicine and professor and chairman emeritus of the Department of Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai in New York City, and Dr. Gregory Grabowski, professor of Microbiology, Biochemistry, and Pediatrics, at the University of Cincinnati College of Medicine. Dr. Grabowski recently retired from that school to become the chief science officer of Synageva, a company involved in producing enzymes for this type of therapy. Copyright © 2014 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Association of the Plasma and Tissue Riboflavin Levels with C20orf54 Expression in Cervical Lesions and Its Relationship to HPV16 Infection

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Kelimu, Alimujiang; Guo, Xia; Mamtimin, Batur; Abudula, Abuliz; Upur, Halmurat

2013-01-01

Riboflavin deficiency can cause a variety of metabolic problems that lead to skin and mucosal disorders. Limited evidence suggests that high intake of riboflavin may reduce overall risks of cancer. However, association of this deficiency with cervical cancer and precancerous lesions are still not definitively known. In this study, we characterized the relationship between plasma and tissue riboflavin levels and C20orf54 protein expression in patients with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) as well as the relationship of these levels with human papillomavirus virus 16, 18 (HPV16/18) infections. High-performance liquid chromatography (HPLC) was used to measure blood riboflavin levels in patients with CIN and CSCC, and an enzyme-linked immunosorbent assay (ELISA) was used to determine tissue riboflavin levels in patients with CSCC and matched normal mucous epithelia. The expression of C20orf54 in fresh CSCC and matched tissues were detected by qRT-PCR and western blot, respectively. And it was further confirmed by immunohistochemistry (IHC) with formalin-fixed, paraffin-embedded CIN and CSCC. An HPV genotyping chip was used to analyze HPV infection and typing. The results showed that patients with CIN and CSCC had decreased plasma riboflavin levels as compared with normal controls. There was also significantly decreased riboflavin in tissues from CSCC patients, when compared with normal cervical epithelia. C20orf54 expression were significantly up-regulated in CSCC compared to matched control on both mRNA and protein level. Tissue riboflavin levels were significantly lower in HPV16/18 positive tissue compared with HPV16/18-negative tissue, and an inverse association was found between tissue riboflavin levels and C20orf54 mRNA and protein expression in CSCC. Additionally, C20orf54 was significantly correlated with tumor stages. In conclusion, C20orf54 tend to play a protective role in Uyghur cervical carcinogenesis of

[Human brown adipose tissue].

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Virtanen, Kirsi A; Nuutila, Pirjo

2015-01-01

Adult humans have heat-producing and energy-consuming brown adipose tissue in the clavicular region of the neck. There are two types of brown adipose cells, the so-called classic and beige adipose cells. Brown adipose cells produce heat by means of uncoupler protein 1 (UCP1) from fatty acids and sugar. By applying positron emission tomography (PET) measuring the utilization of sugar, the metabolism of brown fat has been shown to multiply in the cold, presumably influencing energy consumption. Active brown fat is most likely present in young adults, persons of normal weight and women, least likely in obese persons.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

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Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

Directory of Open Access Journals (Sweden)

Zvonimir Marelja

2018-02-01

Full Text Available Iron sulfur (Fe-S clusters and the molybdenum cofactor (Moco are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii increased iron transiently displaces manganese on superoxide