A phase II clinical study on the efficacy and predictive biomarker of pegylated recombinant arginase on hepatocellular carcinoma.

BACKGROUND: Pegylated recombinant human arginase (PEG-BCT-100) is an arginine depleting drug. Preclinical studies showed that HCC is reliant on exogenous arginine for growth due to the under-expression of the arginine regenerating enzymes argininosuccinate synthetase (ASS) and ornithine transcarbamylase (OTC). METHODS: This is a single arm open-label Phase II trial to assess the potential clinical efficacy of PEG-BCT-100 in chemo naïve sorafenib-failure HCC patients. Pre-treatment tumour biopsy was mandated for ASS and OTC expression by immunohistochemistry (IHC). Weekly intravenous PEG-BCT-100 at 2.7 mg/kg was given. Primary endpoint was time to progression (TTP); secondary endpoints included radiological response as per RECIST1.1, progression free survival (PFS) and overall survival (OS). Treatment outcomes were correlated with tumour immunohistochemical expressions of ASS and OTC. RESULTS: In total 27 patients were recruited. The median TTP and PFS were both 6 weeks (95% CI, 5.9-6.0 weeks). The disease control rate (DCR) was 21.7% (5 stable disease). The drug was well tolerated. Post hoc analysis showed that duration of arginine depletion correlated with OS. For patients with available IHC results, 10 patients with ASS-negative tumour had OS of 35 weeks (95% CI: 8.3-78.0 weeks) vs. 15.14 weeks (95% CI: 13.4-15.1 weeks) in 3 with ASS-positive tumour; expression of OTC did not correlate with treatment outcomes. CONCLUSIONS: PEG-BCT-100 in chemo naïve post-sorafenib HCC is well tolerated with moderate DCR. ASS-negative confers OS advantage over ASS-positive HCC. ASS-negativity is a potential biomarker for OS in HCC and possibly for other ASS-negative arginine auxotrophic cancers. TRIAL REGISTRATION NUMBER: NCT01092091. Date of registration: March 23, 2010.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.

Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification.

HepG2 is an immortalized human hepatoma cell line that has been used for research into bioartificial liver systems. However, a low level of ammonia detoxification is its biggest drawback. In this work, a recombinant HepG2 cell line with stable overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC), HepG2/(hArgI + hOTC)4, was developed using a eukaryotic dual gene expression vector pBudCE4.1. (1) The hArgI and hOTC enzymatic activity in HepG2/(hArgI + hOTC)4 cells were higher than in the control cells. (2) The ammonia tolerance capacity of HepG2/(hArgI + hOTC)4 cells was three times that of HepG2 cells and 37.5% of that of primary human hepatocytes in cultivation. In the experiment of ammonia detoxification, HepG2/(hArgI + hOTC)4 cells produced 3.1 times more urea (at 180 mM NH(4) Cl) and 3.1 times more glutamine (at 120 mM NH(4) Cl and 15 mM glutamate) than HepG2 cells, reaching 63.1% and 36.0% that of primary human hepatocytes, respectively. (3) The hArgI and hOTC overexpression did not influence the growth of HepG2 cells and also promoted the expression of other ammonia detoxification associated proteins including glutamine synthetase (GS), arginase II (ArgII), arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) in HepG2 cells. This work illustrates that the modification reported here made significant progress in the improvement of HepG2 cell function and the HepG2/(hArgI + hOTC)4 cells will provide a better selection for the application of bioartificial liver system.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves.

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii.

The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8% (w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 A. Conversely, crystals grown in 20% (v/v) 2-methyl-2,4-pentanediol, 7.5% (w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 A, beta = 100.25 degrees. Diffraction data were collected in-house to 3.00 and 2.91 A resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 A3 Da(-1), respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code 1dxh) as the search model.

Kinetic analysis of the L-ornithine transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola that is resistant to the transition state analogue (R)-N delta-(N'-sulfodiaminophosphinyl)-L-ornithine.

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

A suspected case of ornithine transcarbamylase deficiency in a cat.

An 18 month-old, intact female American Shorthair cat was presented for evaluation of stunted growth and postprandial depression. Fasting serum ammonia and serum bile acid concentrations were above reference ranges at 396 microg/dl and 6.5 micromol/ l and their postprandial concentrations were 785 microg/dl and 9.5 micromol/l, respectively. The initial tentative diagnosis of a portosystemic shunt was excluded by mesenteric portography and histopathology of the liver. The cat was then suspected of a urea cycle enzyme deficiency and its urine was analyzed by gas chromatography-mass spectrometry. A presumptive diagnosis of ornithine transcarbamylase deficiency was made on the basis of the detection of orotic acid and uracil.

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes.

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.

The arcABC gene cluster encoding the arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene.

Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.

Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors.

Mouse models of ornithine transcarbamylase (OTC) deficiency are being used to test the efficacy of viral vectors as possible vehicles for gene therapy. However, it has been demonstrated that virus containing the human OTC cDNA failed to express functional OTC enzyme in the recipient animals. Because functional OTC is assembled as a homotrimer in the mitochondria, there are at least two possible explanations for these results. Either endogenous mutant protein coassembles with the human OTC and has a "dominant-negative effect," or the human version of the protein is not appropriately imported or processed in the mouse mitochondria. To test the importance of processing, which in rodents is thought to depend on the leader peptide, adenoviral vectors containing chimeric OTC cDNAs were prepared. These vectors were evaluated in the OTC-deficient sparse fur mouse models. Although comparable levels of transgene expression were observed in all groups of mice, the only mice that had high levels of OTC activity and mitochondrial OTC immunoreactivity were those mice injected with the vectors containing the mouse leader peptide (mouse OTC and a mouse-human chimera of OTC). To address possible dominant-negative effects, adenoviruses containing mutant human or mouse OTC cDNAs were prepared and evaluated in cell lines or normal C3H mice, respectively. No inhibition of normal OTC activity was observed in either model system. Together, these studies provide no evidence of a dominant-negative effect and suggest that the human and rodent enzymes responsible for transporting of OTC and possibly other mitochondrial proteins have different specificity.

Expression of wild-type and mutant human ornithine transcarbamylase genes in Chinese hamster ovary cells and lack of dominant negative effect of R141Q and R40H mutants.

Chinese hamster ovary cultured cells were transformed to continuously express wild-type and two mutant ornithine transcarbamylase genes, R141Q and R40H. In addition, these cells were transfected to transiently express the same genes. The R141Q mutation abolishes the enzymatic activity, and the amount of "mature" protein present in transfected cells is equivalent to the wild type. The R40H mutation causes a reduction of enzymatic activity to approximately 26 to 35% of wild type concomitant with a significant reduction in the amount of protein present. Transfection with wild-type and mutant genes together in various proportions did not reveal dominant negative effects of the two mutations studied. This expression system can be used to examine the deleterious effect of private mutations or lack thereof in families with ornithine transcarbamylase deficiency as well as evaluate the potential dominant negative effects of gene delivery for treatment of ornithine transcarbamylase deficiency.

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media.

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was approximately 25-33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was approximately 15-30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

Effects of growth hormone on steroid-induced increase in ability of urea synthesis and urea enzyme mRNA levels.

Growth hormone (GH) reduces the catabolic side effects of steroid treatment due to its effects on tissue protein synthesis/degradation. Little attention is focused on hepatic amino acid degradation and urea synthesis. Five groups of rats were given 1) placebo, 2) prednisolone, 3) placebo, pair fed to the steroid group, 4) GH, and 5) prednisolone and GH. After 7 days, the in vivo capacity of urea N synthesis (CUNS) was determined by saturating alanine infusion, in parallel with measurements of liver mRNA levels of urea cycle enzymes, N contents of organs, N balance, and hormones. Prednisolone increased CUNS (micromol . min-1 . 100 g-1, mean +/- SE) from 9.1 +/- 1.0 (pair-fed controls) to 13.2 +/- 0.8 (P < 0.05), decreased basal blood alpha-amino N concentration from 4.2 +/- 0.5 to 3.1 +/- 0.3 mmol/l (P < 0.05), increased mRNA levels of the rate- and flux-limiting urea cycle enzymes by 20 and 65%, respectively (P < 0. 05), and decreased muscle N contents and N balance. In contrast, GH decreased CUNS from 6.1 +/- 0.9 (free-fed controls) to 4.2 +/- 0.5 (P < 0.05), decreased basal blood alpha-amino N concentration from 3. 8 +/- 0.3 to 3.2 +/- 0.2, decreased mRNA levels of the rate- and flux-limiting urea cycle enzymes to 60 and 40%, respectively (P < 0. 05), and increased organ N contents and N balance. Coadministration of GH abolished all steroid effects. We found that prednisolone increases the ability of amino N conversion into urea N and urea cycle gene expression. GH had the opposite effects and counteracted the N-wasting side effects of prednisolone.

Expression analysis of two mutant human ornithine transcarbamylases in COS-7 cells.

Ornithine transcarbamylase (OTC) is located in the mitochondrial matrix of the liver and small intestine and catalyzes the second step of the urea cycle. OTC deficiency (OTCD) is an X-linked inborn error of metabolism and causes hyperammonemia. We reported in 1992 the A152V and G195R mutations in patients with OTCD. These mutant OTC cDNAs were prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The wild-type and mutant cDNAs were transiently expressed in COS-7 cells. The wild-type cDNA gave an OTC activity of 1180 +/- 47 nmol/min per mg protein. The OTC activities of the A152V and the G195R mutants were 3.7% and 2.5% of that of wild-type, respectively. Immunoblot analysis showed that the quantities of OTC proteins in the A152V and G195R mutants were 29% and 12% of that of wild-type, respectively. In pulse-labeling and pulse-chase experiments, the precursor form of OTC was synthesized and processed to the mature form. The A152V mutant OTC was processed to the mature form as rapidly as the wild-type precursor. However, the processed, mature form of the mutant OTC was rapidly degraded, presumably in the mitochondrial matrix. These results indicate that OTCD with the A152V mutation is due both to rapid degradation of the processed, mature form, and to a lower specific activity of the remaining protein.

Tissue-selective expression of enzymes of arginine synthesis.

Arginine is a non-essential amino acid in mammals as judged from nitrogen balance study. Citrulline is synthesized from glutamate in the small intestine, whilst kidneys and some other tissues convert citrulline to arginine. Ornithine transcarbamylase and carbamylphosphate synthetase are expressed in liver and small intestine. Tissue-selective expression depends on the regulatory elements in the promoter, or far 5', region of these genes to which tissue-selective transcription factors bind and activate transcription. Argininosuccinate synthetase and argininosuccinate lyase do not appear to have such elements, therefore their expression is more or less ubiquitous. The selective expression of pyrroline-5-carboxylate synthase activity in the intestine remains to be clarified.

Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed.

A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation. The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster. Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented. Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found. Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase. A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L. plantarum and one with ArgF of B. subtilis, which are paralogous. This divergence was not observed in vivo because an L. plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L. plantarum and B. subtilis. No OTCase activity was detectable when L. plantarum was grown in the presence of saturating amounts of arginine or citrulline. Arginine may repress the citrulline biosynthetic genes in L. plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn. The carA and argCJBDF genes are divergently transcribed. Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation.

Coordinate control and variation in X-linked gene expression among female mice.

In normal female mammals, one of the two X Chromosome (Chr) homologs per cell is silenced coordinately during early embryogenesis. The genes located on the inactivated X homolog are predicted to be influenced by the same underlying repression mechanism. To test the uniformity of cis-acting gene repression, 32 genetically identical F1 female mice were analyzed for differential expression of homologous alleles at three X-linked genes-Otc, Atp7a (= Mottled), and Hprt. Gene expression was assayed by the single-nucleotide primer extension (SNuPE) method, thereby allowing the three genes to be quantitated from the same RNA sample. Although variable between individual animals, the relative expression of the two alleles (allelic expression ratio) of the genes is significantly correlated within each steady-state RNA pool. When examined by animal age (3 months to 12 months), no statistically significant differences were observed in the mean or variance of allelic expression ratio. Together, the results confirm that X inactivation is coordinately controlled and is stable across the early- to mid-adult life span.