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Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein. Show less

Carbamoylphosphate synthetase 1 (CPS1) is the first enzyme of the urea cycle. CPS1 deficiency is a rare autosomal-recessively inherited disorder that can lead to life-threatening hyperammonemia. Since there is no reliable biochemical marker for this disease, diagnosis relies on molecular means which is often done by RNA-based mutation analysis. Skin fibroblasts have been frequently used as a source of RNA while peripheral blood cells do not yield sufficient amounts of specific RNA. To avoid the costly and laborious use of cultured fibroblasts, we tried to use stimulated lymphocytes as an alternative. This was effectively achieved by short-term culture of full heparin blood in the presence of phytohemagglutinin. Hereby, subsequent reverse transcriptase-PCR of the CPS1 transcript became feasible and allowed to detect 16 different mutations (10 missense, 3 deletions, 2 nonsense, 1 duplication; 7 novel mutations) in 14 consecutive patients with CPS1 deficiency. When compared to retrospective data on cultured fibroblasts, the adapted method allowed substantial shortening of the median time to diagnosis (24 days versus 122 days, respectively). Besides disease causing mutations, we detected CPS1 transcript variants including one cryptic exon in RNA from lymphocytes with higher frequency than in RNA from fibroblasts. This underlines that all mutations found in RNA need to be confirmed by DNA sequencing. In conclusion, the presented approach improves the diagnostics of CPS1 deficiency. Besides the shortened time to diagnosis, the method is of particular importance for confirmation of findings of next generation sequencing and gene chips. Show less

The exchange of genetically engineered mouse strains between research facilities requires transporting fresh mouse sperm under refrigerated temperatures. Although sperm generally maintains fertility for 48 h at cold temperatures, in vitro fertilization rates of C57BL/6 mouse sperm are low after 48-h cold storage. Furthermore, 48 h is often not sufficient for the specimens to reach their destinations. To increase the availability of this technology, we aimed to extend the cold storage period while maintaining sperm fertility. In this study, we determined the optimal medium for sperm preservation and evaluated the effect of reduced glutathione in the fertilization medium on sperm fertility after cold storage. We found that higher fertility levels were maintained after 72-h cold storage in the preservation medium Lifor compared with storage in paraffin oil, M2 medium, or CPS-1 medium. In addition, 1.0 mM glutathione enhanced sperm fertility. After transporting sperm from Asahikawa Medical University to our laboratory, embryos were efficiently produced from the cold-stored sperm. After transfer, these embryos developed normally into live pups. Finally, we tested the transport system using genetically engineered mouse strains and obtained similar high fertilization rates with all specimens. In summary, we demonstrated that cold storage of sperm in Lifor maintains fertility, and glutathione supplementation increased the in vitro fertilization rates of sperm after up to 96 h of cold storage. This improved protocol provides a simple alternative to transporting live animals or cryopreserved samples for the exchange of genetically engineered mouse strains among research facilities. Show less

Nonalcoholic fatty liver disease (NAFLD) is being increasingly recognized as a major cause of liver-related morbidity and mortality. Given the increasing prevalence of obesity in western countries, NAFLD has become an important public health problem. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls. Changes in protein expression of liver samples from controls, nonalcoholic steatosis, and nonalcoholic steatohepatitis (NASH) subjects were analyzed by two-dimensional differential in-gel electrophoresis (DIGE). With this proteomic technique, hundreds of proteins can be analyzed simultaneously and their relative abundance can be calculated. Proteins showing significant changes (ratio ≥ 1.5, p < 0.05) were identified by MALDI TOF/TOF mass spectrometry. Western blot of tissue homogenates was then used as a complementary method to validate protein expression changes observed by DIGE. With the aim to have a noninvasive approach to detect changes produced in NAFLD-affected liver, validated proteins were further tested in serum samples of different cohorts of patients. Following this approach, we identified two candidate markers CPS1 and GRP78 that were differentially expressed between control, steatosis, and NASH. This proteomics approach demonstrates that DIGE combined with MALDI TOF/TOF and Western blot analysis of tissue and serum samples is a useful approach to identify candidate markers associated with NAFLD. Show less

We had previously identified the mutant allele of apm1(+) that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4(+)/sip1(+), which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl(-) and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking. Show less

In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure. Show less

Toxoplasma gondii is a protozoan of worldwide distribution. This unit describes murine models of acute T. gondii infection, toxoplasmic encephalitis, and Toxoplasma retinochoroiditis. T. gondii infection in SCID mice allows the study of T cell-independent mechanisms of defense. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii allow studies of immunization and adoptive transfer. In vivo study of parasite host-interaction is possible with the use of parasites that express fluorescent proteins and model antigens, plus the use of transgenic mice that express the appropriate T cell receptor and fluorescently labeled leukocytes. Parasites that express bioluminescent markers make it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. Show less

CPSI deficiency usually results in severe hyperammonemia presenting in the first days of life warranting prompt diagnosis. Most CPS1 defects are non-recurrent, private mutations, including point mutation, small insertions and deletions. In this study, we report the detection of large deletions varying from 1.4 kb to >130 kb in the CPS1 gene of 4 unrelated patients by targeted array CGH. These results underscore the importance of analysis of large deletions when only one mutation or no mutations are identified in cases where CPSI deficiency is strongly indicated. Show less

High doses of T3 are mitogenic in liver, causing hyperplasia that has numerous differences from the compensatory regeneration induced by partial hepatectomy (PH). T3 binds to the thyroid hormone receptor (TR), which directly regulates transcription, while PH acts indirectly through signal transduction pathways. We therefore carried out a proteomic analysis to compare early effects of the two treatments. Transcriptome analysis by DNA microarray also confirmed the observed proteomic changes, demonstrating that they were caused by transcriptional regulation. Among the differentially expressed proteins, many are directly or indirectly involved in energy metabolism and response to oxidative stress. Several enzymes of lipid metabolism (e.g., Acaa2, Acads, Hadh, and Echs1) were differentially regulated by T3. In addition, altered expression levels of several mitochondrial proteins (e.g., Hspa9, Atp5b, Cps1, Glud1, Aldh2, Ak2, Acads) demonstrated the known increase of mitochondrial biogenesis mediated by T3. The present results provide insights in changes in metabolic balance occurring following T3-stimulation and define a basis for dissecting the molecular pathways of hepatocyte hyperplasia. Show less

To evaluate the histopathologic biocompatibility of two new calcium phosphate-based sealers (CPS-1 & CPS-2) with a commercially available calcium hydroxide-based sealer (Acroseal). Polyethylene tubes were filled with freshly mixed sealers and implanted subcutaneously in the dorsal connective tissue of rats. Empty tubes were used as controls. Histopathological examinations were conducted at 7, 15, 30, 60 and 90 days after the implantation procedure. The presence of inflammation and predominant cell types were analysed statistically with Mann-Whitney U and Kruskal-Wallis non-parametric tests. Fibrous connective tissue thickness adjacent to each sample was recorded. Differences were tested for significance using anova and 'Duncan's' multiple comparison test (P < 0.05). CPS-1 sealer was associated with severe inflammation and remained an irritation throughout the 90-day implantation period; the tissue reaction pattern was stromal fibrosis. The control, CPS-2 and Acroseal sealers had similar patterns of irritation, which were more severe initially and diminished with time creating a thin fibrous capsule around the implant with a complete absence of inflammatory cells. There was no difference in tissue reaction between the control, CPS-1, CPS-2 and Acroseal groups amongst the first two observation periods (P > 0.05). However, there was a highly significant difference between the same groups at the last two observation periods (P < 0.01). Also, there were highly significant differences between the observation periods within all four groups at 7, 15, 30, 60 and 90 days (P < 0.01). CPS-1 sealer was not biocompatible. CPS-2 sealer and Acroseal had a favourable biocompatibility level based on the histological findings. Show less

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states. Show less

The ESCRT-0 and ESCRT-I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85-Å crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centred on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP-binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is non-essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms. Show less

Hepatocyte spheroids have been proposed for drug metabolism studies and in bioartificial liver devices. However, the optimal conditions required to meet the aerobic demands of mitochondria-rich hepatocyte spheroids is not well studied. We hypothesized that an optimal concentration of oxygen could be identified and that the health of hepatocyte spheroids might be further improved by antioxidant therapy. Rat hepatocyte spheroids were maintained in suspension culture for 7 days under a mixture of 5% CO(2) plus O(2):N(2) to achieve fractional oxygen contents of 6%(C1), 21%(C2), 58%(C3), and 95%(C4). Spheroid health was assessed under each condition by vital staining, TEM, oxygen consumption, and mitochondrial counts. Hepatocyte differentiation was assessed by expression of 10 liver-related genes (HNF4a, HNF6, Cyp1A1, albumin, Nags, Cps1, Otc, Ass, Asl, Arg1). Functional markers (albumin and urea) were measured. The influence of oxygen tension and antioxidant treatment on the production of reactive oxygen species (ROS) was assessed by confocal microscopy. We observed that the hepatocyte spheroids were healthiest under normal atmospheric (C2) conditions with antioxidants ascorbic acid and L-carnitine. Cell death and reduced functionality of hepatocyte spheroids correlated with the formation of ROS. Normal atmospheric conditions provided the optimal oxygen tension for suspension culture of hepatocyte spheroids. The formation and deleterious effects of ROS were further reduced by adding antioxidants to the culture medium. These findings have direct application to development of the spheroid reservoir bioartificial liver and the use of hepatocyte spheroids in drug metabolism studies. Show less

Carbamoyl phosphate synthetase 1 (CPS1) is a liver-specific, intramitochondrial, rate-limiting enzyme in the urea cycle. A previous study showed that CPS1 is the antigen for hepatocyte paraffin 1 antibody, a commonly used antibody in surgical pathology practice; and CPS1 expression appears to be down-regulated in liver cancer tissue and cell lines. The aim of this study is to understand how the CPS1 gene is regulated in liver carcinogenesis. In this report, we show that human hepatocellular carcinoma (HCC) cells do not express CPS1, whereas cultured human primary hepatocytes express abundant levels. In addition, CPS1 was silenced or down-regulated in liver tumor tissues compared with the matched noncancerous tissues. The expression of CPS1 in HCC cells was restored with a demethylation agent, 5-azacytidine. We show that two CpG dinucleotides, located near the transcription start site, and a CpG-rich region in the first intron were hypermethylated in HCC cells. The hypermethylation of the two CpG dinucleotides was also detected in HCC tumor tissues compared with noncancerous tissues. Further molecular analysis with mutagenesis indicated that the two CpG dinucleotides play a role in promoter activity of the CPS1 gene. In conclusion, our study demonstrates that DNA methylation is a key mechanism of silencing CPS1 expression in human HCC cells, and CPS1 gene hypermethylation of the two CpG dinucleotides is a potential biomarker for HCC. Show less

Bacteriophages are ubiquitous in various environments. Our previous study revealed the diversity of the cyanophage community in paddy floodwater. In this study, the phylogeny and genetic diversity of cyanophage communities in paddy field soils were reported. The viral capsid assembly protein gene (g20) of cyanophage was amplified with the primers CPS1 and CPS8 from soil DNA extracted during two different sampling times at three sampling sites in Japan. The sequencing results indicated that about 93% of the clones were g20 genes. In total, 70 clones of g20 genes were obtained in this study, of which 69 clones were of cyanophage origin. As evaluated by g20 sequence assemblages in paddy field soils, the unifrac analyses results indicated that cyanophage communities changed among the sampling sites and times and differed from those communities detected in paddy floodwater. The phylogenetic analysis showed that the g20 sequences in paddy field soils were very diverse and distributed into Clusters α, β and ɛ, as well as four newly formed clusters. Within Clusters β and ɛ, four unique subclusters were formed from the g20 clones that were only observed in this study. These findings suggested that the cyanophage communities in paddy field soils are different from those found in freshwater, marine water and paddy floodwater. Show less

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. Show less

Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ∼10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein. Show less

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples. We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample. Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples. The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations. Show less

Several common genomic loci, involving various immunity- and metabolism-related genes, have been associated with plasma fibrinogen in European Americans (EAs). The genetic determinants of fibrinogen in African Americans (AAs) are poorly characterized. Using a vascular gene-centric array in 23,634 EA and 6657 AA participants from 6 studies comprising the Candidate Gene Association Resource project, we examined the association of 47,539 common and lower frequency variants with fibrinogen concentration. We identified a rare Pro265Leu variant in FGB (rs6054) associated with lower fibrinogen. Common fibrinogen gene single nucleotide polymorphisms (FGB rs1800787 and FGG rs2066861) significantly associated with fibrinogen in EAs were prevalent in AAs and showed consistent associations. Several fibrinogen locus single nucleotide polymorphism associated with lower fibrinogen were exclusive to AAs; these include a newly reported association with FGA rs10050257. For IL6R, IL1RN, and NLRP3 inflammatory gene loci, associations with fibrinogen were concordant between EAs and AAs, but not at other loci (CPS1, PCCB, and SCL22A5-IRF1). The association of FGG rs2066861 with fibrinogen differed according to assay type used to measure fibrinogen. Further characterization of common and lower-frequency genetic variants that contribute to interpopulation differences in fibrinogen phenotype may help refine our understanding of the contribution of hemostasis and inflammation to atherothrombotic risk. Show less

Patients with extensive ulcerative colitis (UC) have an increased risk of colorectal cancer. Although UC patients generally undergo lifelong colonoscopic surveillance to detect dysplasia or cancer in the colon, detection of cancer in this manner is expensive and invasive. An objective biomarker of dysplasia would vastly improve the clinical management of cancer risk in UC patients. In the current study, accurate mass and time methods with ion intensity-based label-free proteomics are applied to profile individual rectal and colon samples from UC patients with dysplasia or cancer (UC progressors) compared to rectal samples from patients that are dysplasia/cancer free (UC nonprogressors) to identify a set of proteins in the rectum mucosa that differentiate the two groups. In addition to the identification of proteins in UC dysplastic colon tissue, we for the first time identified differentially expressed proteins in nondysplastic rectal tissue from UC progressors. This provides a candidate pool of biomarkers for dysplasia/cancer that could be detected in a random nondysplastic rectal biopsy. Mitochondrial proteins, cytoskeletal proteins, RAS superfamily, proteins relating to apoptosis and metabolism were important protein clusters differentially expressed in the nondysplastic and dysplastic tissues of UC progressors, suggesting their importance in the early stages of UC neoplastic progression. Among the differentially expressed proteins, immunohistochemistry analysis confirmed that TRAP1 displayed increased IHC staining in UC progressors, in both dysplastic and nondysplastic tissue, and CPS1 showed a statistically significant difference in IHC staining between the nonprogressor and progressor groups. Furthermore, rectal CPS1 staining could be used to predict dysplasia or cancer in the colon with 87% sensitivity and 45% specificity, demonstrating the feasibility of using surrogate biomarkers in rectal biopsies to predict dysplasia and/or cancer in the colon. Show less

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8×10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8×10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation. Show less

Cirrhosis is a complex process that involves a dynamic modification of liver cell phenotype associated to gene expression changes. This study investigates the reversing capacity of an adenosine derivative compound (IFC305) on a rat model of liver cirrhosis and gene expression changes associated with it. Rats were treated with IFC305 or saline for 5 or 10 weeks after cirrhosis induction (CCl(4) treatment for 10 weeks). Fibrosis score, collagenase activity and amount of hepatic stellate cells (HSC, activated and with a lipid-storing phenotype) were measured in livers. In addition, gene expression analysis was performed using 5K DNA microarrays and quantitative RT-PCR. Treatment of cirrhotic rats with IFC305 for 5 or 10 weeks compared to saline control, induced: (1) reduction of fibrosis (50-70%) and of collagen, of alpha-SMA and desmin proteins, as well as of activated HSCs in liver, (2) increased collagenase activity and cell number of lipid-storing HSC, (3) improved serum parameters of liver function, such as reduced activity of aminotransferases and bilirubin. Expression of 413 differential genes, deregulated in cirrhotic samples, tended to be normalized by IFC305 treatment. Some genes modulated at transcript level by IFC305 were Tgfb1, Fn1, Col1a1, C9, Apoa1, Ass1, Cps1, and Pparg. The present study shows that IFC305 reverses liver fibrosis through modulation of adipogenic and fibrosis-related genes and by ameliorating hepatic function. Thus, understanding of the anti-cirrhotic effect of IFC305 might have therapeutical potential in patients with cirrhosis. Show less

Oxidative stress is involved in the progression of non-alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2-nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. Proteins extracted from phosphate buffered saline- and hydrogen peroxide-loaded human primary hepatocyte were labeled with the [(12)C]- and [(13)C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [(13)C]-NBS labeling was more intense than the [(12)C]-NBS labeling were detected by MALDI-TOF/MS and identified by MS/MS ion searching. Four pairs of peaks, m/z 1705-1711, m/z 1783-1789, m/z 1902-1908 and m/z 2790-2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl-phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH. Show less

Plasma homocysteine (Hcy) level is associated with cardiovascular disease and may play an etiologic role in vascular damage, a precursor for atherosclerosis. We performed a genome-wide association study for Hcy in 1786 unrelated Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). The most strongly associated single-nucleotide polymorphism (SNP) (rs7422339, P = 4.7 x 10(-13)) encodes Thr1405Asn in the gene CPS1 and explained 3.0% of variation in the Hcy level. The widely studied MTHFR C677T SNP (rs1801133) was also highly significant (P = 8.7 x 10(-10)) and explained 1.6% of the trait variation. We also genotyped these two SNPs in 1679 CLHNS young adult offspring. The MTHFR C677T SNP was strongly associated with Hcy (P = 1.9 x 10(-26)) and explained approximately 5.1% of the variation in the offspring. In contrast, the CPS1 variant was significant only in females (P = 0.11 in all; P = 0.0087 in females). Combined analysis of all samples confirmed that the MTHFR variant was more strongly associated with Hcy in the offspring (interaction P = 1.2 x 10(-5)). Furthermore, although there was evidence for a positive synergistic effect between the CPS1 and MTHFR SNPs in the offspring (interaction P = 0.0046), there was no significant evidence for an interaction in the mothers (P = 0.55). These data confirm a recent finding that CPS1 is a locus influencing Hcy levels in women and suggest that genetic effects on Hcy may differ across developmental stages. Show less

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 A resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole. Show less

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1. Show less

Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 individuals of European ancestry from 20 predominantly population-based studies in order to identify new susceptibility loci for reduced renal function as estimated by serum creatinine (eGFRcrea), serum cystatin c (eGFRcys) and CKD (eGFRcrea < 60 ml/min/1.73 m(2); n = 5,807 individuals with CKD (cases)). Follow-up of the 23 new genome-wide-significant loci (P < 5 x 10(-8)) in 22,982 replication samples identified 13 new loci affecting renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2 and SLC7A9) and 7 loci suspected to affect creatinine production and secretion (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72 and BCAS3). These results further our understanding of the biologic mechanisms of kidney function by identifying loci that potentially influence nephrogenesis, podocyte function, angiogenesis, solute transport and metabolic functions of the kidney. Show less

Perfluorooctanoic acid (PFOA), with an array of industrial uses, is one of the most common perfluoroalkyl acids. Resistance to biological degradation and a global distribution are characteristics that have caused PFOA to become a frequent subject of toxicological studies. PFOA treatment in rodents causes peroxisome proliferation, mitochondrial biogenesis, and transactivation of PPARs. Prior work has shown urea cycle gene expression to be reduced in mice by another PPARalpha ligand, WY14643. In light of these findings, the aim of our investigation was to determine if PFOA treatment in rats alters expression of genes responsible for ureogenesis. 30 mg/kg of PFOA was administered to adult male Sprague-Dawley rats via oral gavage for 28 days and their livers were harvested. Gene transcription was measured using real time PCR and protein expression was determined through western blotting. We observed a decrease in mRNA for the coordinately expressed urea cycle genes Cps1, Ass1, and Asl; mRNA of the ammonia generating Gls2 was also reduced. Protein amounts for CPS1, ASS1, and OTC were all decreased in the PFOA treated rats, and interestingly there was an increase in the amount of S133 phosphorylated CREB, which is a regulator of urea cycle gene transcription. We conclude that the transactivation of PPARalpha by PFOA leads to a metabolic shift that favors the catabolism of lipids over proteins, thereby suppressing urea cycle gene expression. Our findings provide further evidence of the effect of PFOA on intermediary metabolism in rodents and add valuable information in assessing the potential risks of PFOA exposure. Show less

A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been associated with changes in plasma concentrations of L-arginine in term and near term infants but not in adults. In preterm infants homozygosity for the CPS1 Thr1405 variant (CC genotype) was associated with an increased risk of having necrotizing enterocolitis (NEC). Plasma L-arginine concentrations are decreased in preterm infants with NEC. To examine the putative association between the CPS1 T1405N polymorphism and plasma arginine concentrations in preterm infants. Prospective multicenter cohort study. Plasma and DNA samples were collected from 128 preterm infants (<30 weeks) between 6 and 12 hours after birth. Plasma amino acid and CPS1 T1405N polymorphism analysis were performed. Distribution of genotypes did not differ between the preterm (CC:CA:AA = 55.5%:33.6%:10.9%, n = 128) and term infants (CC:CA:AA = 54.2%:35.4%:10.4%, n = 96). There was no association between the CPS1 genotype and plasma L-arginine or L-citrulline concentration, or the ornithine to citrulline ratio, which varies inversely with CPS1 activity. Also the levels of asymmetric dimethylarginine, and symmetric dimethylarginine were not significantly different among the three genotypes. The present study in preterm infants did not confirm the earlier reported association between CPS1 genotype and L-arginine levels in term infants. Show less

Carbamoyl-phosphate synthetase I (CPS1) deficiency (CPS1D), a recessively inherited urea cycle error due to CPS1 gene mutations, causes life-threatening hyperammonemia. The disease-causing potential of missense mutations in CPS1 deficiency can be ascertained with the recombinant CPS1 expression and purification system reported here, which uses baculovirus and insect cells. We study with this system the effects of nine clinical mutations and one polymorphism on CPS1 solubility, stability, activity, and kinetic parameters for NAG. Five of the mutations (p.T471N, p.Q678P, p.P774L, p.R1453Q, and p.R1453W) are first reported here, in three severe CPS1D patients. p.P774L, p.R1453Q, and p.R1453W inactivate CPS1, p.T471N and p.Y1491H greatly decrease the apparent affinity for NAG, p.Q678P hampers correct enzyme folding, and p.S123F, p.H337R, and p.P1411L modestly decrease activity. p.G1376S is confirmed a trivial polymorphism. The effects of the C-terminal domain mutations are rationalized in the light of this domain crystal structure, including the NAG site structure [Pekkala et al. Biochem J 424:211-220]. The agreement of clinical observations and in vitro findings, and the possibility to identify CPS1D patients who might benefit from specific treatment with NAG analogues because they exhibit reduced affinity for NAG highlight the value of this novel CPS1 expression/purification system. Show less