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Carbamoyl phosphate synthetase 1 (CPS1) plays a paramount role in liver ureagenesis since it catalyzes the first and rate-limiting step of the urea cycle, the major pathway for nitrogen disposal in humans. CPS1 deficiency (CPS1D) is an autosomal recessive inborn error which leads to hyperammonemia due to mutations in the CPS1 gene, or is caused secondarily by lack of its allosteric activator NAG. Proteolytic, immunological and structural data indicate that human CPS1 resembles Escherichia coli CPS in structure, and a 3D model of CPS1 has been presented for elucidating the pathogenic role of missense mutations. Recent availability of CPS1 expression systems also can provide valuable tools for structure-function analysis and pathogenicity-testing of mutations in CPS1. In this paper, we provide a comprehensive compilation of clinical CPS1 mutations, and discuss how structural knowledge of CPS enzymes in combination with in vitro analyses can be a useful tool for diagnosis of CPS1D. Show less

CPS1 is a mitochondrial matrix enzyme that catalyzes the first committed step of the urea cycle, the primary system for removing nitrogen produced by protein metabolism using N-acetylglutamate. Patients with CPS1 deficiency have severe hyperammonemia that results in serious neurologic sequelae and sometimes death. LT has been indicated for neonatal-onset CPS1 deficiency. This study retrospectively reviewed five children with a diagnosis of CPS1 deficiency who underwent LDLT from heterozygous donors. Between November 2005 and May 2010, 124 children underwent LDLT with an overall patient and graft survival of 91.0%. Five patients were indicated for LDLT because of CPS1 deficiency. All recipients achieved resolution of their metabolic derangement, without donor complication, with a normal feeding regimen without medication for their original metabolic liver disease. LDLT, even from heterozygous donors, appears to be a feasible option, associated with a better quality of life for treating patients with CPS1 deficiency. Long-term observation may therefore be necessary to collect sufficient data to confirm the efficacy of this treatment modality. Show less

The ESCRT complexes are required for multivesicular body biogenesis, macroautophagy, cytokinesis, and the budding of HIV-1. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA+ ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-III substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the betadomains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the two-fold axis of the Vta1 VSL domain is parallel to within approximately 20 degrees of the six-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly. Show less

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an autosomal recessive disorder of the urea cycle which causes hyperammonemia. Two forms of CPS1D are recognized: a lethal neonatal type and a less severe, delayed onset type. Neonatal CPS1D cases often present their symptoms within the first days of life. Delayed onset type were adolescents or adults, and infantile cases were rare. We report a case of CPS1D in a boy who developed symptoms at one month of age. He showed excellent response to treatments including continuous hemodialysis, drugs and a low-protein diet. His development and weight gain were good at the last follow-up at 1 year and three months of age. Molecular assay of the CPS1 gene demonstrated that the patient was heterozygous for c.2407C>G (R803G: maternal) in exon 20 and c.3784C>T (R1262X: paternal) in exon 32. Our clinical experience suggests that CPS1D could be one of the causes of hyperammonemia in early infantile cases. Show less

Sirtuins are NAD-dependent protein deacetylases that connect metabolism and aging. In mammals, there are seven sirtuins (SIRT1-7), three of which are associated with mitochondria. Here, we show that SIRT5 localizes in the mitochondrial matrix and interacts with carbamoyl phosphate synthetase 1 (CPS1), an enzyme, catalyzing the initial step of the urea cycle for ammonia detoxification and disposal. SIRT5 deacetylates CPS1 and upregulates its activity. During fasting, NAD in liver mitochondria increases, thereby triggering SIRT5 deacetylation of CPS1 and adaptation to the increase in amino acid catabolism. Indeed, SIRT5 KO mice fail to upregulate CPS1 activity and show elevated blood ammonia during fasting. Similar effects occur during long-term calorie restriction or a high protein diet. These findings demonstrate SIRT5 plays a pivotal role in ammonia detoxification and disposal by activating CPS1. Show less

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps. Show less

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes. Show less

The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases. Show less

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice, regardless of the route of challenge infection. Remarkably, cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts, cps1-1 strain-elicited CD8(+) immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition, we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively, these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain. Show less

Deficiency of the urea cycle enzyme carbamylphosphate synthetase 1 (CPS1) causes hyperammonemia with a vast range of clinical severity from neonatal onset with early lethality to onset after age 40 with rare episodes of hyperammonemic confusion. The cause for this variability is not understood. We report two patients from one family with highly divergent clinical course, one presenting neonatally with a fatal form and the other at age 45 with benign diet-responsive disease. The patients are compound heterozygous for two mutations of the CPS1 gene, c.3558 + 1G > C and c.4101 + 2T > C. The haplotypes containing each mutation are identical between the two patients, as are the sequences of CPS1 exons and flanking introns. Transcriptional experiments show that the abnormal CPS1 transcripts generated by both mutations are identical in these two patients. We characterize promoter and enhancer sequences of the CPS1 gene and find also in these regions no sequence differences between patients. Finally, we perform cloning experiments and find that in the neonatal-onset case, clones of messenger RNA (mRNA) expressed from the allele carrying the c.4101 + 2T > C mutation are threefold more than clones of mRNA from the allele with the c.3558 + 1G > C mutation, whereas in the adult-onset case the two types of clones are equal, indicating skewed expression towards the c.4101 + 2T > C allele in the neonatal case. Although we are yet to understand the mechanism of this differential expression, our work suggests that allelic imbalance may explain clinical variability in CPS1 deficiency in some families. Show less

Carbamoyl phosphate synthetase-I (CPS1) is a key enzyme in the urea cycle and patients with defects in the function or expression of CPS1 suffer from hyperammonemia. CPS1 is expressed in the liver at neonatal and adult stages in a CCAAT enhancer-binding protein-alpha (C/EBPalpha)-dependent manner. Despite expression of C/EBPalpha, CPS1 is not expressed in fetal liver, indicating an additional factor is involved in the regulation of CPS1 expression. The aim of this study was to elucidate the mechanism of CPS1 expression. Microarray was performed to find Y-box binding protein-1 (YB-1) that was expressed in mouse fetal liver. The role of YB-1 in CPS1 expression was investigated by overexpression of YB-1 in mouse fetal liver culture and luciferase reporter assays using the CPS1 promoter. Chromatin immunoprecipitation assay was used to examine recruitment of YB-1 to the CPS1 promoter in vivo. Expression of YB-1 and CPS1 was inversely correlated in vivo, and YB-1 inhibited CPS1 expression and ammonia clearance in fetal liver culture. Although YB-1 was not expressed in adult liver, acute liver injury up-regulated YB-1 and down-regulated CPS1, accompanying an increase of the serum ammonia level. YB-1 inhibited C/EBPalpha-induced transcription from the CPS1 promoter via the Y-box near the C/EBPalpha-binding site. Chromatin immunoprecipitation assays demonstrated that YB-1 was recruited to the CPS1 promoter in fetal and injured adult liver, but not in normal adult liver. YB-1 is a key regulator of ammonia detoxification by negatively regulating CPS1 expression via suppression of C/EBPalpha function. Show less

In Schizosaccharomyces pombe, Bgs1/Cps1p is a beta(1,3)-glucan synthase required for linear beta(1,3)-glucan synthesis and primary septum formation. Here, we have studied the regulation of Bgs1p by Cfh3/Chr4p, a member of a family of conserved adaptor proteins, which resembles the chitin synthase regulator Chs4p from Saccharomyces cerevisiae and Candida albicans. cfh3Delta cells showed a genetic interaction with cps1-191, and Cfh3p co-immunoprecipitated with Bgs1/Cps1p. In the absence of cfh3(+), cells were more sensitive to digestion by glucanases, and both Calcofluor staining and glucan synthesis were reduced. We found that in a wild-type strain, beta(1,3)-glucan synthesis was reduced under stress conditions. In the cfh3Delta, cps1-191, and cfh3Delta cps1-191 strains, beta(1,3)-glucan synthesis was further reduced, and growth was impaired under stress conditions, suggesting that Cfh3p and Bgs1p might play a role in ensuring growth in unfavorable environments. In a cfh3Delta mutant, Bgs1p was delocalized when the cells were distressed, but a blockade in endocytosis prevented this delocalization. Finally, we found that the SEL1 repeats are required for Cfh3p function. These results show that Cfh3p is a regulatory protein for Bgs1p and that its function is particularly necessary when the cells are undergoing stress. Show less

A single inoculation of mice with the live, attenuated Toxoplasma gondii uracil auxotroph strain cps1-1 induces long-lasting immunity against lethal challenge with hypervirulent strain RH. The mechanism for this robust immunity in the absence of parasite replication has not been addressed. The mechanism of long-lasting immunity, the importance of route of immunization, cellular recruitment to the site of infection, and local and systemic inflammation were evaluated. Our results show that infection with cps1-1 elicits long-lasting CD8+ T cell- mediated immunity. We show that immunization with cps1-1-infected dendritic cells elicits long-lasting immunity. Intraperitoneal infection with cps1-1 induced a rapid influx of GR1+ neutrophils and two stages of GR1+CD68+ inflammatory monocyte infiltration into the site of inoculation. CD19+ B cells and CD3+ T cells steadily increase for 8 days after infection. CD8+ T cells were rapidly recruited to the site of infection and increased faster than CD4+ T cells. Surprisingly, cps1-1 infection induced high systemic levels of bioactive IL-12p70 and a very low level and transient systemic IFN-gamma. Furthermore, we show significant levels of these inflammatory cytokines were locally produced at the site of cps1-1 inoculation. These findings offer new insight into immunological mechanisms and local host responses to a non-replicating type I parasite infection associated with development of long-lasting immunity to Toxoplasma gondii. Show less

Mammalian sirtuins have diverse roles in aging, metabolism and disease. Recently we reported a new function for SIRT5 in urea cycle regulation. Our study uncovered that SIRT5 localized to mitochondria matrix and deacetylates carbamoyl phosphate synthetase 1 (CPS1), an enzyme which is the first and rate-limiting step of urea cycle. Deacetylation of CPS1 by SIRT5 resulted in activation of CPS1 enzymatic activity. Indeed, SIRT5-deficient mice failed to up-regulate CPS1 activity and showed hyper ammonemia during fasting. Similar effects are also observed on high protein diet or calorie restriction. These data indicate SIRT5 also has an emerging role in the metabolic adaptation to fasting, high protein diet and calorie restriction. Show less

Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon, including the non-dysplastic mucosa; we hypothesized that the same field defect will include abnormally expressed proteins. Here we applied proteomics to study the protein expression of UC neoplastic progression. The protein profiles of colonic epithelium were compared from 1) UC patients without dysplasia (non-progressors); 2) none-dysplastic colonic tissue from UC patient with high-grade dysplasia or cancer (progressors); 3) high-grade dysplastic tissue from UC progressors and 4) normal colon. We identified protein differential expression associated with UC neoplastic progression. Proteins relating to mitochondria, oxidative activity, calcium-binding proteins were some of interesting classes of these proteins. Network analysis discovered that Sp1 and c-myc proteins may play roles in UC early and late stages of neoplastic progression, respectively. Two over-expressed proteins in the non-dysplastic tissue of UC progressors, CPS1 and S100P, were further confirmed by IHC analysis. Our study provides insight into the molecular events associated with UC neoplastic progression, which could be exploited for the development of protein biomarkers in fields of non-dysplastic mucosa that identify a patient's risk for UC dysplasia. Show less

To find out the mechanisms of HBx gene inducing oval cell malignant transformation into hepatoma carcinoma cell. The changes of morphology, cell cycle, differentiated markers, c-myc and TGF-alpha in pEGFP-HBx oval cells strain, which stably expressed HBx gene, were studied by inversion phase contrast microscope and transmission electron microscopy, flow cytometry, periodic acid-schiff (PAS) staining, soft agar growth assay, real-time PCR, immunocytochemistry. pEGFP-oval cells and LE/6 oval cells were used as control groups. (1) The pEGFP-HBx oval cells showed bigger in size with malformed nucleus as compared with control groups. (2) Flow cytometry showed that, in contrast with the control groups, the proportion of pEGFP-HBx oval cells arrested in G0/G1 phase decreased but in S or G2/M phase rose. Moreover, the population of aneuploid cells increased obviously. (3) PAS staining showed that there were many glycogen granules in the cytoplasm of pEGFP-HBx oval cell. (4) The pEGFP-HBx oval cell formed colonies in the soft agar. (5) Compared with the control groups, the expression of HNF-4 alpha, AFP, c-myc and TGF-alpha rose obviously, whereas the expression of CK-7 and CK-19 decreased. And the expression of cps1 mRNA was not in the extent of detection. The HBx gene can provoke abnormal differentiation of oval cell and induce oval cell malignant transformation. Show less

Homocysteine is a sulfur amino acid whose plasma concentration has been associated with the risk of cardiovascular diseases, neural tube defects, and loss of cognitive function in epidemiological studies. Although genetic variants of MTHFR and CBS are known to influence homocysteine concentration, common genetic determinants of homocysteine remain largely unknown. To address this issue comprehensively, we performed a genome-wide association analysis, testing 336 469 single-nucleotide polymorphisms in 13 974 healthy white women. Although we confirm association with MTHFR (1p36.22; rs1801133; P=8.1 x 10(-35)) and CBS (21q22.3; rs6586282; P=3.2 x 10(-10)), we found novel associations with CPS1 (2q34; rs7422339; P=1.9 x 10(-11)), MUT (6p12.3; rs4267943; P=2.0 x 10(-9)), NOX4 (11q14.3; rs11018628; P=9.6 x 10(-12)), and DPEP1 (16q24.3; rs1126464; P=1.2 x 10(-12)). The associations at MTHFR, DPEP1, and CBS were replicated in an independent sample from the PROCARDIS study, whereas the association at CPS1 was only replicated among the women. These associations offer new insight into the biochemical pathways involved in homocysteine metabolism and provide opportunities to better delineate the role of homocysteine in health and disease. Show less

Carbamoyl phosphate synthetase I (CPS1), normally found in hepatocytes and small-intestine (SI) enterocytes, is the antigen of Hep Par 1 antibody. Expression of CPS1 in invasive SI adenocarcinoma seems to be lost. We retrospectively collected 36 total specimens, which included 31 SI adenomas and 21 adenocarcinomas. We used 34 cases of duodenitis as a control group. Immunohistochemical and Western blot analyses were performed to determine CPS1 expression. The normal SI mucosa, all 34 cases of duodenitis, and all 29 adenomas with low-grade dysplasia demonstrated diffuse Hep Par 1 expression. Of the 21 invasive adenocarcinomas, 15 lost antigen expression (71%). These data are statistically significant (P < .05). Western blot analysis confirmed the immunohistochemical findings, with strong CPS1 expression within the normal mucosa and adenoma and complete loss in the invasive tumor. The differential expression of Hep Par 1 in dysplastic vs malignant tumors of the SI may be diagnostically useful in difficult cases. Show less

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni-specific bacteriophages (CPS1-6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni, but not other Gram-negative bacteria, including C. coli, Escherichia coli, Salmonella spp., and Gram-positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni. Show less

A water-soluble polysaccharide named CPS1 had been isolated from C. sinensis mycelium by hot water extraction, ethanol precipitation, anion-exchange, and gel-permeation chromatography. UV spectra, FTIR spectra, partial acid hydrolysis, PMP precolumn derivation, periodate oxidation and Smith degradation studies were conducted to elucidate its structure. The results indicated that CPS1 was a glucomannogalactan with the monosaccharide composition of glucose: mannose: galactose = 2.8: 2.9: 1. The total carbohydrate content of CPS1 was 99.0%. The weight-average molecular weight was 8.1 x 10(3) Da. The results predicted (1-->2) and (1-->4)-linkage of mannose, (1-->3)-linkage of galactose, (1--> ) and (1-->3, 6)-linkage of glucose composed the backbone of CPS1. CPS1 was also evaluated for its antioxidant activity in vitro, including scavenging effects on the hydroxyl radicals, the reducing power, Fe(2+)-chelating activity, scavenging effect on superoxide radicals, as well as the inhibition of hydrogen peroxide induced haemolysis. CPS1 showed a high antioxidant effect, especially scavenging effect of hydroxyl radicals, the reducing power and Fe(2+)-chelating activity. The results provide scientific support for the antioxidant activity and indicated a connection between antioxidant activity and reparation of renal failure. Show less

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs. Show less

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise approximately 16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42-86 bp) genotyping on the LightScanner. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067 degrees C before calibration to 0.022 degrees C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086 degrees C to 0.032 degrees C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical. Show less

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC. Show less

To prepare and characterize the monoclonal antibody (mAb) against human carbamyl phosphate synthetase I (CPSI) and make a study of its application. Normal human liver tissues were homogenized, and their mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAb using the routine hybridoma technique. The mAb was detected by ELISA, Western blot immunohistochemistry and immunofluorecent staining. The specificity of mAb was identified by mass spectrometry (MS) and immunoprecipitation (IP) and then confirmed by Uni-ZAP expression library screening. The antibody was used to isolate potential enzymatic complexes by immunocapturing. Three hybridoma cell lines BEH045, ACB271 and BFG021 secreting specific mAb against CPS1 were obtained. The Ig subclass of the mAb was IgG(1), which was used in ELISA, Western blot immunohistochemistry, immunoprecipitation, immunofluorecent staining and the isolation of potential enzymatic complexes. A hybridoma cell line which can secre specific mAb against CPSI stably has been established. The specific mAb against CPSI is of value to the research into the functions and distribution of CPSI. Show less

Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis. Show less

Carbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan. Show less

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT. Show less

A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been correlated with low plasma concentrations of L-arginine in neonates. As plasma L-arginine concentrations are decreased in premature infants with necrotizing enterocolitis (NEC), we hypothesized that the CPS1 T1405N polymorphism would correlate with the presence of NEC. We analyzed the CPS1 genotypes for the T1405N polymorphism in 17 preterm infants (Show less

Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Delta strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast. Show less