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Background Arginase plays key roles in methyl jasmonate (MeJA)-mediated quality maintenance in vegetables and fruits. MeJA treatment induced the Arginase 2 (SlARG2) expression, which is one of the most important encoding genes of arginase. In addition, the treatment with MeJA induced resistance to pathogenic infection in many plants. However, the functions of SlARG2 in MeJA-induced defense to Botrytis cinerea are unclear. In our work, control and SlARG2-silenced tomato fruits (Solanum lycopersicum) were treated with 0.05 mmoL L-1 MeJA before storage to assay the roles of SlARG2 in MeJA-induced defense responses to Botrytis cinerea. Results Our results indicated that MeJA treatment induced both pathogenesis-related gene expression (PR1, PR2a, PR2b and PR3b), and the activity of defense-related enzymes, as well as upregulated arginine metabolism. Compared to control fruits, the treatment with MeJA also induced the activity of arginase, arginine decarboxylase (ADC) and ornithine aminotransferase (OAT), and expression of SlARG2, SlADC, ornithine decarboxylase (SlODC) and SlOAT, and consequently increased the accumulation of arginine, proline, glutamate, putrescine and spermine. However, the induction effects by MeJA were significantly reduced in fruits in which SlARG2 was silenced and severe disease symptoms were observed. Conclusion MeJA fumigation could inhibit disease development by inducing pathogenesis-related gene expression (PR1, PR2a, PR2b and PR3b) and defense-related enzymes activity, as well as upregulated arginine metabolism. In addition, SlARG2 silencing could inhibit the functions of MeJA in inducing the accumulation of the above substances. Overall, our study provided strong evidence that SlARG2 was essential for MeJA-induced tomato fruit defense responses to Botrytis cinerea. © 2020 Society of Chemical Industry.

The 2‐acetyl‐1‐pyrroline (2AP) is a key aroma compound in fragrant rice. The present study assessed the γ‐aminobutyric acid (GABA) and nitrogen (N) application induced regulations in the biochemical basis of rice aroma formation. Four N levels, that is, 0, 0.87, 1.75, and 2.61 g/pot, and two GABA treatments, that is, 0 mg/L (GABA0) and 250 mg/L (GABA250), were applied to three fragrant rice cultivars, that is, Yuxiangyouzhan, Yungengyou 14, and Basmati‐385. Results showed that GABA250 increased 2AP, Na, Mn, Zn, and Fe contents by 8.44%, 10.95%, 25.70%, 11.14%, and 43.30%, respectively, under N treatments across cultivars. The GABA250 further enhanced the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT) (both at 15 days after heading (d AH), and diamine oxidase (DAO) (at maturity) by 20.36%, 11.24%, and 17.71%, respectively. Significant interaction between GABA and N for Mn, Zn, and Fe contents in grains, proline content in leaves, GABA content in leaves at 15 d AH and maturity stage (MS), Δ1‐pyrroline‐5‐carboxylic acid (P5C) contents in leaves at 15 d AH, and Δ1‐pyrroline‐5‐carboxylate synthase (P5CS), PDH, and OAT activities in leaves at MS was noted. Moreover, the 2AP contents in grains at MS showed a significant and positive correlation with the proline contents in the leaves at 15d AH. In conclusion, GABA250 enhanced the 2AP, Na, Mn, Zn, and Fe contents, as well as the enzyme activities involved in 2AP biosynthesis. Exogenous GABA and N application improved the 2AP contents and nutrient uptake in fragrant rice.

While pregnancy may accelerate glioblastoma multiforme (GBM) growth, parity and progesterone (P4) containing treatments (ie, hormone replacement therapy) reduce the risk of GBM development. In parallel, low and high doses of P4 exert stimulating and inhibitory actions on GBM growth, respectively. The mechanisms behind the high‐dose P4‐suppression of GBM growth is unknown. In the present study, we assessed the changes in growth and proteomic profiles when high‐dose P4 (100 and 300 µM) was administered in human U87 and A172 GBM cell lines. The xCELLigence system was used to examine cell growth when different concentrations of P4 (20, 50, 100, and 300 µM) was administered. The protein profiles were determined by two‐dimensional gel electrophoresis in both cell lines when 100 and 300 µM P4 were administered. Finally, the pathways enriched by the differentially expressed proteins were assessed using bioinformatic tools. Increasing doses of P4 blocked the growth of both GBM cells. We identified 26 and 51 differentially expressed proteins (fc > 2) in A172 and U87 cell lines treated with P4, respectively. Only the pro‐tumorigenic mitochondrial ornithine aminotransferase and anti‐apoptotic mitochondrial 60 kDa heat shock protein were downregulated in A172 cell line and U87 cell line when treated with P4, respectively. Detoxification of reactive oxygen species, cellular response to stress, glucose metabolism, and immunity‐related proteins were altered in P4‐treated GBM cell lines. The paradox on the effect of low and high doses of P4 on GBM growth is gaining attention. The mechanism related to the high dose of P4 on GBM growth can be explained by the alterations in detoxification mechanisms, stress, and immune response and glucose metabolism. P4 suppresses GBM growth and as it is nontoxic in comparison to classical chemotherapeutics, it can be used as a new strategy in GBM treatment in the future., GBM cell line impedance alterations when different concentrations of Progesterone were administered.

The incidence and mortality of lung cancer ranked the first among all types of cancer in China, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for 85% of all lung cancers. Given that the survival rate of patients with advanced NSCLC is still poor nowadays, identification of novel therapeutic targets and the development of effective therapies are desired for the treatment of NSCLC in clinics. In this study, we reported the upregulation of ornithine aminotransferase (OAT) in NSCLC cells and clinical tumor samples as well as its association with the advanced TNM stage, metastasis, and poor tumor differentiation of lung cancer. Using different NSCLC cell lines, we demonstrated that OAT promoted the proliferation, invasion, and migration, inhibited the apoptosis, and altered cell cycle of NSCLC cells; besides, the involvement of OAT-miR-21-glycogen synthase kinase-3β signaling in the functional role of OAT in NSCLC was also revealed. Importantly, in the absence of OAT, the growth and metastasis of tumor lung cancer xenograft was significantly suppressed in the nude mice. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.

BACKGROUND Small cell lung cancer (SCLC) is highly aggressive and is associated with a dismal prognosis. However, there are no clinically recognized biomarkers for early diagnosis. In this study, we used quantitative proteomics to build differential mitochondrial protein profiles that may be used for early diagnosis and investigated the pathogenesis of lung cancer. METHODS We cultured SCLC cells (NCI-H446) and normal human bronchial epithelial cells (16-HBE); mitochondria were extracted and purified using differential and Percoll density gradient centrifugation. Subsequently, we used Western blot analysis to validate mitochondrial purity and labeled proteins/peptides from NCI-H446 and 16-HBE cells using relative and absolute quantification of ectopic tags. We then analyzed mixed samples and identified proteins using two-dimensional liquid chromatography-tandem mass spectrometry. Additionally, we performed subsequent bioinformatic proteome analyses using the programs ExPASy, GOA, and STRING. Finally, the relationship between ornithine aminotransferase expression and clinicopathological features in lung cancer patients was evaluated using immunohistochemistry. RESULTS One hundred and fifty-three mitochondrial proteins were differentially expressed between 16-HBE and NCI-H446 cells. The expression of 30 proteins between 16-HBE and NCI-H446 cells increased more than 1.3-fold. The upregulation of ornithine aminotransferase was associated with pathological grade and clinical tumor node metastasis stage. CONCLUSION Our experiment represented a promising method for building differential mitochondrial protein profiles between NCI-H446 and 16-HBE cells. Such analysis may also help to identify novel biomarkers of lung cancer.

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased Km value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb.

Water stress is one of the most critical abiotic stresses that restricts growth, development, and alters physiological and biochemical mechanisms of plant. The effects of long-term water shortage-induced oxidative stress on morphophysiological parameters, proline metabolic genes, and artemisinin content were studied in Artemisia annua L. under greenhouse conditions. Plant growth, biomass accumulation, relative water content, and chlorophyll content were reduced under drought. Leaf water potential ranged from -0.3248 MPa to -1.22 MPa in stress conditions. Increased levels of proline accumulation, protein concentration, and lipid peroxidation were detected in water-stressed plants. Stage-dependent increases in activity of antioxidants including superoxide dismutase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were observed. The expression of proline biosynthetic genes including pyrroline-5-carboxylase synthase1, 1-pyrroline-5-carboxylase synthase2, and 1-pyrroline-5-carboxylase reductase was induced, while the ornithine aminotransferase transcript showed a variable response and the expression of proline catabolic genes including proline dehydrogenase1, proline dehydrogenase1, and proline 5-carboxylate dehydrogenase was reduced by water stress. Our results indicate that the glutamine pathway is predominant under drought stress in A. annua and a reduction of catabolic gene expression is adopted as a defense strategy in adverse conditions. Higher expression of biosynthetic genes and lower expression of catabolic genes at the preflowering stage confirmed the important role of proline in flower development. Artemisinin content decreased owing to water stress, but the slightly higher amounts were detected in leaves of severely stressed plants compared with moderately stressed plants. The artemisinin content of A. annua might be regulated by controlling irrigation regimes.

Chronic alcohol consumption causes hepatic steatosis, which is characterized by a considerable increase in free fatty acid (FFA) and triglyceride levels. To identify the possible proteins involved in the progression to alcoholic hepatosteatosis, we performed proteomic analysis on livers of mice exposed to alcohol. 2D-based proteomic analysis revealed that EtOH exposure in mice changed the expression of 43 proteins compared with that in mice fed a normal diet (ND). The most notable protein changes were proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in alcohol-exposed animals. Although non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) seem to share the same molecular processes, the difference between these conditions is still unclear. To address this question, we explored the features of alcoholic hepatosteatosis that were different compared with those of methionine and choline deficient (MCD) diet-induced mice with nonalcoholic liver damage. Although most of the differentially expressed proteins associated with ALD did not significantly differ from those of NAFLD, nine proteins showed considerably different patterns. Of these, ornithine aminotransferase, vitamin D binding protein, and phosphatidylethanolamine-binding protein were considerably upregulated in ALD mice, compared to that in NAFLD and ND mice. However, other proteins including inorganic pyrophosphatase were differentially regulated in MCD mice; however, they did not differ significantly between the alcoholic model and ND control mice. These results suggested that the identified proteins might be useful candidate markers to differentiate ALD from NAFLD. J. Cell. Biochem. 118: 1189-1200, 2017. © 2016 Wiley Periodicals, Inc.

Proline (Pro) is a versatile metabolite playing a role in the protection of plants against environmental stresses. To gain a deeper understanding of the regulation of Pro metabolism under predicted future climate conditions, including drought stress, elevated temperature and CO2, we combined measurements in contrasting grassland species (two grasses and two legumes) at multiple organisational levels, that is, metabolite concentrations, enzyme activities and gene expression. Drought stress (D) activates Pro biosynthesis and represses its catabolism, and elevated temperature (DT) further elevated its content. Elevated CO2 attenuated the DT effect on Pro accumulation. Computational pathway control analysis allowed a mechanistic understanding of the regulatory changes in Pro metabolism. This analysis indicates that the experimentally observed coregulation of multiple enzymes is more effective in modulating Pro concentrations than regulation of a single step. Pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) play a central role in grasses (Lolium perenne, Poa pratensis), and arginase (ARG), ornithine aminotransferase (OAT) and P5CR play a central role in legumes (Medicago lupulina, Lotus corniculatus). Different strategies in the regulation of Pro concentrations under stress conditions were observed. In grasses the glutamate pathway is activated predominantly, and in the legumes the ornithine pathway, possibly related to differences in N-nutritional status.

Background Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism’s drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. Methods Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. Results Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)—belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. Conclusion Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development.

Proline is not only an essential component of proteins but it also has important roles in adaptation to osmotic and dehydration stresses, redox control, and apoptosis. Here, we review pathways of proline biosynthesis in the three domains of life. Pathway reconstruction from genome data for hundreds of eubacterial and dozens of archaeal and eukaryotic organisms revealed evolutionary conservation and variations of this pathway across different taxa. In the most prevalent pathway of proline synthesis, glutamate is phosphorylated to γ-glutamyl phosphate by γ-glutamyl kinase, reduced to γ-glutamyl semialdehyde by γ-glutamyl phosphate reductase, cyclized spontaneously to Δ(1)-pyrroline-5-carboxylate and reduced to proline by Δ(1)-pyrroline-5-carboxylate reductase. In higher plants and animals the first two steps are catalysed by a bi-functional Δ(1) -pyrroline-5-carboxylate synthase. Alternative pathways of proline formation use the initial steps of the arginine biosynthetic pathway to ornithine, which can be converted to Δ(1)-pyrroline-5-carboxylate by ornithine aminotransferase and then reduced to proline or converted directly to proline by ornithine cyclodeaminase. In some organisms, the latter pathways contribute to or could be fully responsible for the synthesis of proline. The conservation of proline biosynthetic enzymes and significance of specific residues for catalytic activity and allosteric regulation are analysed on the basis of protein structural data, multiple sequence alignments, and mutant studies, providing novel insights into proline biosynthesis in organisms. We also discuss the transcriptional control of the proline biosynthetic genes in bacteria and plants.

Ornithine aminotransferase (OAT) and γ-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active-site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer.

The gamma-glutamyl-phosphate reductase (ProA) interlinks both the anabolic and osmostress adaptive proline biosynthetic routes of Bacillus subtilis. Because no paralogous protein to ProA exists in this microorganism, proA mutants should exhibit a tight proline auxotrophic growth phenotype. Contrary to expectations, proA mutants formed microcolonies on agar plates lacking proline and faster growing Pro(+) suppressor mutants arose. These mutants carried alterations in the rocR-rocDEF region encoding enzymes of the arginine degradation pathway and its transcriptional activator RocR. They were of two types: (i) mutants carrying single amino acid substitutions in RocR resulting in partial inducer-independent variants and (ii) mutants carrying single base-pair changes in the vicinity of the SigL/Sig-54-dependent -12/-24 class rocDEF promoter that activate a cryptic SigA-type promoter. Consequently, enhanced rocDEF transcription should lead to increased cellular amounts of the RocD ornithine aminotransferase, an enzyme that synthesizes the same reaction product as ProA, gamma-glutamic-semialdehyde/delta-1-pyrroline-5-carboxylate. This compound can be enzymatically converted into proline. The Pro(+) suppressors also exhibited a new regulatory pattern by allowing enhanced rocDEF transcription in response to proline availability when ammonium is present. Our work provides an example how flexibly bacteria can genetically develop routes to bypass constraints imposed on their biosynthetic networks and evolve new regulatory mechanisms.

BACKGROUND To understand whether arginine catabolism might be involved in hot air (HA)-induced chilling tolerance mechanism in tomato fruit, we investigated the effect of HA treatment on endogenous arginine catabolism in relation to chilling injury. RESULTS Tomato fruit were harvested at mature green stage and treated with HA at 38°C for 12 h and then stored at 2°C for 21 days. The effects of HA treatment on fruit chilling injury and gene expression levels or enzyme activity, and metabolites related to arginine catabolism were evaluated. HA treatment reduced the chilling injury symptoms of tomato fruit and enhanced the accumulation of endogenous polyamines, especially putrescine and proline. This accumulation is associated with the increased transcript levels of genes encoding arginase (LeARG1 and LeARG2), arginine decarboxylase (LeADC), ornithine decarboxylase (LeODC) and ornithine aminotransferase (LeOAT) at most sampling times. However, HA treatment had little effect on nitric oxide synthase activity and nitric oxide concentration. CONCLUSION These results revealed that the reduction in chilling injury by HA treatment may be due to the accumulation of putrescine and proline induced primarily by activating the catabolism of endogenous arginine. © 2013 Society of Chemical Industry

Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped.

The liver plays a major role in the early hypometabolic and later hypermetabolic phases after severe burn injury. Proteomic analysis was used to identify altered proteins in liver during these two phases. Sprague-Dawley rats were subjected to a full-thickness dorsal burn injury covering 40% of the total body surface area. Controls consisted of sham-treated animals. Liver tissues were collected on postburn days 1 and 7. The proteomic data show greater production of positive acute phase proteins on day 1 than on day 7. Many antioxidant enzymes were coordinately downregulated on day 1, including the potent biliverdin reductase. These antioxidants were restored and in some cases upregulated on day 7. This opposite trend in the change of antioxidant proteins corroborated our finding of more pronounced oxidative stress on day 1 than on day 7 as measured via protein carbonyl content. The changes of metabolic enzymes on days 1 and 7 were consistent with hypo- and hyper-metabolic states, respectively. Furthermore, a previously unreported decrease in ornithine aminotransferase on day 7 may be a key contributor to the observed increased urinary urea excretion during the hypermetabolic phase. Overall, the many differences in protein expression observed on postburn days 1 and 7 reflect the dissimilar hepatic metabolic patterns during the acute and flow phases following burn injury.

Acetylornithine aminotransferase (AcOAT) is one of the key enzymes involved in arginine metabolism and catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of $_L$-glutamate. It belongs to the Type I subgroup II family of pyridoxal $5^{\prime}$-phosphate (PLP) dependent enzymes. E. coli biosynthetic AcOAT (eAcOAT) also catalyzes the conversion of N-succinyl-$_L$-2-amino-6-oxopimelate to N-succinyl-$_{L,L}$-diaminopimelate, one of the steps in lysine biosynthesis. In view of the critical role of AcOAT in lysine and arginine biosynthesis, structural studies were initiated on the enzyme from S. typhimurium (sAcOAT). The $K_m$ and $k_{cat}/K_m$ values determined with the purified sAcOAT suggested that the enzyme had much higher affinity for AcOrn than for ornithine (Orn) and was more efficient than eAcOAT. sAcOAT was inhibited by gabaculine (Gcn) with an inhibition constant $(K_i)$ of $7\mu M$ and a second-order rate constant $(k_2)$ of 0.16 $mM^{-1} s^{-1}$. sAcOAT, crystallized in the unliganded form and in the presence of Gcn or $_L$-glutamate, diffracted to a maximum resolution of $1.90 \AA$ and contained a dimer in the asymmetric unit. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop $L_{\alpha k-\beta m}$ in only one subunit (subunit B; the loop from subunit B comes close to the phosphate group of PLP in subunit A). Structural and spectral studies of sAcOAT with Gcn suggested that the enzyme might have a low affinity for PLP-Gcn complex. Comparison of sAcOAT with T. thermophilus AcOAT and human ornithine aminotransferase suggested that the higher specificity of sAcOAT towards AcOrn may not be due to specific changes in the active site residues but could result from minor conformational changes in some of them. This is the first structural report of AcOAT from a mesophilic organism and could serve as a basis for drug design as the enzyme is important for bacterial cell wall biosynthesis.

Today, toxicoproteomics still relies mainly on 2-DE followed by MS for detection and identification of proteins, which might characterize a certain state of disease, indicate toxicity or even predict carcinogenicity. We utilized the classical 2-DE/MS approach for the evaluation of early protein biomarkers which are predictive for chemically induced hepatocarcinogenesis in rats. We were able to identify statistically significantly deregulated proteins in N-nitrosomorpholine exposed rat liver tissue. Based on literature data, biological relevance in the early molecular process of hepatocarcinogenicity could be suggested for most of these potential biomarkers. However, in order to ensure reliable results and to create the prerequisites necessary for integration in routine toxicology studies in the future, these protein expression patterns need to be prevalidated using independent technology platforms. In the current study, we evaluated the usefulness of iTRAQ reagent technology (Applied Biosystems, Framingham, USA), a recently introduced MS-based protein quantitation method, for verification of the 2-DE/MS biomarkers. In summary, the regulation of 26 2-DE/MS derived protein biomarkers could be verified. Proteins like HSP 90-beta, annexin A5, ketohexokinase, N-hydroxyarylamine sulfotransferase, ornithine aminotransferase, and adenosine kinase showed highly comparable fold changes using both proteomic quantitation strategies. In addition, iTRAQ analysis delivered further potential biomarkers with biological relevance to the processes of hepatocarcinogenicity: e.g. placental form of glutathione S-transferase (GST-P), carbonic anhydrase, and aflatoxin B1 aldehyde reductase. Our results show both the usefulness of iTRAQ reagent technology for biomarker prevalidation as well as for identification of further potential marker proteins, which are indicative for liver hepatocarcinogenicity.

The mechanism of pellagrous changes in skin caused by a deficiency of vitamin B6 was studied in respect to neogenesis of proline in skin collagen and glucose metabolism. In vitamin B6 deficiency the insulin/glucagon coefficient in serum decreased significantly from 3.02 to 2.32, indicating a metabolic change towards gluconeogenesis. A deficiency of vitamin B6 caused a decrease in the levels of vitamin B6-dependent enzymes, such as ornithine aminotransferase, alanine aminotransferase, and aspartate aminotransferase, which also contribute to gluconeogenesis. Because the conversion of ornithine to proline via pyrroline-5-carboxylate was suppressed due to the decrease in ornithine aminotransferase activity, the amount of proline in the skin collagen fraction also decreased significantly in vitamin B6-deficient rats compared with the pair-fed control. These results suggest that the pellagrous lesions in vitamin B6-deficiency are caused by an impaired synthesis of proline from ornithine, which results in the suppression of collagen neogenesis in the skin.

Proline and quaternary ammonium compounds (QAC), in addition to being N-rich, are known to accumulate in plants under different environmental stress conditions. The accumulation of N-rich compounds in plants has been shown to confer stress resistance. The aim of our work is two-fold: first, to study the influence of temperature on proline, QAC, and choline metabolism in tomato leaves; and second, to investigate the relationship between N source applied (NO3- or NH4+) and thermal stress resistance in these plants. To do this, experiments were conducted at three different temperatures (10 degrees C, 25 degrees C, 35 degrees C); at each temperature half of the plants received NO3-, and the other half received NH4+. At 35 degrees C the plants had the lowest biomass production with respect to 25 degrees C (optimal temperature) and 10 degrees C (cold stress), suggesting that tomato plants were most affected by heat stress. At 35 degrees C, there were also high levels of choline and proline due to the activation of Delta1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT), and simultaneous inhibition of proline dehydrogenase (PDH) and proline oxidase (PO). However, plants with NH4+ as the N source exhibited reduced growth with respect to the plants fed with NO3-. This is interesting because, under heat stress (35 degrees C), biomass production, as well as proline and choline accumulation, in NH4+ fed plants was higher than in NO3- fed plants. From this, we concluded that tomato plants fed with NH4+ as the N source show higher tolerance to heat stress (35 degrees C) than plants fed with NO3-.

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.

The growth of Helianthus annuus L. calli and plants was reduced in the presence of Na2SO4 (10, 25, 50, and 100 mM). SO42— and Na concentrations increased in stressed calli and plants while NO3—, Cl—, P, K, and Mg decreased in stressed plants and Ca in shoots. Stressed calli showed decreases of NO3—, Ca, K, and Mg concentrations. Calli adapted to 50 mM Na2SO4 accumulated more K and Ca and less ammonium than stressed non-adapted calli. Proline exhibited increases in stressed calli and plants that were accompanied by decreases of proline oxidase activities while pyrroline-5-carboxylate reductase (P5CR) and ornithine aminotransferase (OAT) activities increased. Adapted calli accumulated more proline and had higher P5CR and OAT activities than stressed non-adapted calli. Glutamate concentration decreased with stress, together with a stimulation of cytosolic glutamine synthetase (GS1) and a decrease of plastidal GS (GS2) activity. These data strongly suggest that the increase of P5CR and GS1 activities are responsible for the decrease of glutamate concentration leading, together with the stimulation of OAT and the inhibition of the proline oxidation metabolism, to an increase of proline levels in Na2SO4-stressed sunflower cells. These data also show that salt stress increases the release of endogenous ammonium and suggests that the increase of GS1 activity plays an important role in its elimination. Nahrstoffgehalte und Glutamat- und Prolinstoffwechsel in Sonnenblumenpflanzen und Calli unter Na2SO4-Stress Das Wachstum von Helianthus annuus L. Pflanzen und deren Calli wird in Gegenwart steigender Na2SO4-Belastung (10, 25, 50 bzw. 100 mM) gehemmt. Sowohl in gestressten Pflanzen wie in den Calli waren die SO4- und Na-Konzentrationen erhoht. In den gestressten Pflanzen waren die Konzentrationen von NO3., Cl, P, K und Mg in Wurzel und Spross, die von Ca lediglich im Spross gesenkt, wahrend in gestressten Calli NO3, Ca, K und Mg erniedrigt waren. Calli, die uber 8 Monate an 50 mM Na2SO4 adaptiert waren, akkumulierten unter Salzstress im Vergleich zu nicht-adaptierten mehr K und Ca, aber weniger Ammonium. Am starksten stieg in den Pflanzen sowie Calli unter Salzstress die Prolin-Konzentration, bedingt durch einen Abfall der Prolinoxidase-Aktivitat sowie einen Anstieg der Pyrrolin-5-carboxylat-Reductase (P5CR) und der Ornithin-Aminotransferase (OAT). Die an Salinitat adaptierten Calli akkumulierten mehr Prolin und zeigten hohere Aktivitaten an P5CR und OAT. Bei Salzstress nahm die Konzentration an Glutamat ab. Dies war begleitet von einer Stimulierung der cytosolischen Glutaminsynthetase (GS1), jedoch einem Abfall der plastidaren GS2. Auf Grund der Ergebnisse kann der Anstieg der Prolingehalte nach Salzstress durch die Anstiege in der Aktivitat von P5CR und GS1, die die Absenkung der Glutamatkonzentration bewirken, sowie von OAT und durch die Hemmung der Prolinoxidase erklart werden. Der durch Salzstress ebenfalls geforderten Freisetzung endogenen Ammoniums wirkt die gesteigerte GS1-Aktivitat entgegen.

Summary The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG. It encodes a protein of 160 amino acids with a deduced Mr of 17 117 for the monomer. Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains. Amplification of an arginine (ARG) box-containing sequence results in a 2- to 2.5-fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out. Footprinting experiments using the pure homologous arginine repressor (AhrC) of B. subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC. The protected region contained two tandemly repeated imperfect palindromic 18-nt ARG boxes. The repressor‐operator interaction was confirmed by bandshift experiments of the DNA fragment containing the protected region. By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces. This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well-developed regulatory system of these pathways.

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava (Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.

BACKGROUND Ornithine alpha-ketoglutarate salt efficiently improves the nutritional status of protein-depleted patients. Our aim was to explore the effects of ornithine alpha-ketoglutarate supplementation on intestinal physiology in healthy animals. METHODS Rats were given a nutritive mixture supplemented with ornithine alpha-ketoglutarate (1 g.kg-1 per day) by enteral route for 7 days. Controls received the diet supplemented with casein acid hydrolysate under isoenergetic and isonitrogenous conditions. RESULTS An adaptive hyperplasia of the villi and an increase in the brush-border hydrolase activities were observed in rats receiving ornithine alpha-ketoglutarate. Because of the high ornithine aminotransferase activity, ornithine alpha-ketoglutarate-derived ornithine was extensively transaminated with a concomitant enhancement of ornithine decarboxylation. Surprisingly, with glutamate and putrescine, the products of ornithine transamination and decarboxylation, gamma-aminobutyric acid accumulated (10-fold to 16-fold) dramatically in the intestinal mucosa of rats treated with ornithine alpha-ketoglutarate. Because gamma-aminobutyric acid formation was completely prevented by the diamine oxidase inhibitor aminoguanidine but was not modified after inactivation of ornithine aminotransferase by 5-fluoromethylornithine, it is evident that gamma-aminobutyric acid is formed in the mucosa from ornithine via putrescine as an intermediate. CONCLUSIONS It is assumed that enhanced gamma-aminobutyric acid formation in the intestinal mucosa by ornithine alpha-ketoglutarate treatment might be of physiologic importance in the regulatory processes of cell growth and differentiation.

Light-grown cotyledons of radish (Raphanus sativus L. cv National) subjected to increasing NaCI concentrations displayed a dose-dependent accumulation of proline at the beginning of their differentiation. Neither erythromycin nor DCMU significantly affected the salt-induced proline accumulation, which suggests that plastids may not be involved in proline synthesis. Gabaculine has been shown previously to be a powerful and irreversible inhibitor of ornithine aminotransferase [Hervieu et al. (1993) Phyto-chemistry 34, 231–234]. This inhibitor applied in vivo at a very low concentration (1 mmol m−3) reduced considerably the salt-induced proline accumulation of radish cotyledons. These results indicate that the ornithine pathway contributes, via an increase of ornithine aminotransferase activity, to proline synthesis, as well as the glutamate pathway, in salt-treated cotyledons. The use of transcription and translation inhibitors revealed that the salt-induced increase in ornithine aminotransferase activity may proceed via an activation of the translation of preformed mRNAs. The pre-existing ornithine aminotransferase mRNAs in radish seeds may be an important adaptation to NaCI, leading to proline accumulation at the beginning of seedling growth.

The ornithine aminotransferase (OAT) from pig kidney has been studied on the basis of high-resolution electron microscopy and the morphological appearance of the apoenzyme and holoenzyme have been examined. The quaternary structure of the OAT molecules in the presence of 5 mM pyridoxal 5'-phosphate could be established. The enzyme molecule appears to be built up of two morphological units, called M1. The native holoenzyme, termed morphological unit M2, measures 10.9 nm in length and 5.8 nm in width and its molecular mass is approximately 168 kDa, based on electron microscopical calculations. Since the enzyme is composed of only one type of 45-kDa subunit, the holoenzyme is a homotetramer. Each M1 is composed of two subunits and, as seen in top-view projection, has an oval to triangular shape. Upon tilting to 40 degrees the triangular shape changes into three distinct centers of mass. This morphological differentiation reflects the inner organization of M1, i.e. the shape of the individual subunit deviates from strictly globular proteins. This observation is compatible with the notion that the 45-kDa subunit consists of one large and one small domain. By tilting to 40 degrees, both large domains in M1 represent two of the three centers of mass, while the third center of mass is attributed to the superposition of both small domains. Thus, the four domains of both subunits in M1, in accordance with the triangular top-view projection, are quasi-tetrahedrally arranged. Since the change in shape of M1 upon tilting is only obvious in one of the two halves of the native OAT, it suggests that both morphological units of M2 are oriented asymmetrically relative to one another.

The initial cytogenetic analysis of a biphasic synovial sarcoma revealed complex anomalies involving six different chromosomes: 46,Y,t(X;18;5;4)(p11;q11;p13;q12),t(2;5)(q35;q11). After fluorescence in situ hybridization (FISH) analysis, using chromosome X-specific plasmid library and YAC probes, the situation appeared to be even more complex, with an insertion of part of the X chromosome short arm into the der(5)t(5;18). In spite of these complex chromosomal rearrangements, the Xp11 breakpoint could be mapped to within the ornithine aminotransferase (OAT)L1 cluster, very similar to that reported previously for the standard t(X;18)(p11;q11) in synovial sarcomas. These findings suggest common pathogenetic pathways in these cytogenetically different but morphologically similar tumors. (C) 1994 Wiley-Liss, Inc.

Mice and chicken were given 5-fluoromethylornithine (5FMOrn), a selective inactivator of ornithine aminotransferase (OAT) over extended periods of time. This treatment allowed us to maintain elevated concentrations of ornithine in all tissues. Since gyrate atrophy, an autosomal recessive human disease, is characterized by the absence of OAT, special emphasis was put on the study of the visual system. Ophthalmoscopic and histologic examinations of the eye as well as electroretinograms and locomotor behaviour demonstrated an unimpaired visual system and brain. No toxic effects were observable in the treated mice. Likewise, chick embryo development was normal in spite of highly elevated brain and tissue ornithine concentrations. A likely explanation for the absence of toxic effects of 5FMOrn treatment, disregarding the non-toxicity of ornithine, is the fact that 10-20% of tissue OAT is refractory to inactivation by 5FMOrn. This residual activity may be sufficient to maintain vital functions.

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with α-difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.

Pretreatment of mice with 5-fluoromethylornithine (5FMOrn), a selective inactivator of ornithine aminotransferase, diminishes the accumulation of ammonia in the brain after administration of ammonium acetate, and antagonizes ammonia-induced fatal tonic extensor convulsions. In about 50% of the treated animals the loss of the righting reflex and coma is prevented. Presumably these effects are based on the enhancement of urea formation by the increased liver ornithine concentrations. However, since brain ornithine concentrations are greatly enhanced by 5FMOrn, it is not excluded that ornithine has direct effects on cellular events involved in ammonia-induced seizure generation, even though 5FMOrn had no anticonvulsant properties in a series of established animal seizure models, including N-methyl-D,L-aspartate-induced convulsions. NMDA receptor antagonists are capable of preventing death, but do not protect against the generation of coma and tonic extensor convulsions in ammonium acetate intoxicated mice. Since no evidence was found for ammonia-induced glutamate release from rat hippocampus, there is no convincing evidence for the idea that the tonic convulsions are mediated by NMDA receptors. L-Methionine-D, L-sulfoximine (MSO)-induced seizures can be partially antagonized by pretreatment with 5FMOrn. However, the effect is considerably smaller than against ammonia-induced convulsions, although at the time of seizure onset brain ammonia levels of MSO-intoxicated mice were lower than in the animals receiving ammonium acetate. This suggests that MSO-convulsions are not entirely due to the elevation of brain ammonia concentrations, even though MSO administration mimics effects of ammonia on cortical inhibitory neuronal interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Only one of the four enantiomers of 5-fluoromethylornithine 1 was an irreversible inhibitor of ornithine aminotransferase. The active enantiomer 1a was synthesized from diaminoadipic acid 2 with a chemical diastereomeric separation and an enantiomeric resolution by hydrolysis of a phenylacetamide with benzylpenicillinase.

5-Fluoromethylornithine (5FMOrn) is a selective inactivator of ornithine aminotransferase. Its administration causes a dramatic increase of ornithine concentrations in all tissues. Treatment of mice with 20 mg.kg-1 5FMOrn shortly before or after a lethal dose (600 mg.kg-1, intraperitoneally) of thioacetamide (TAA), followed by a second dose 24 hr later, prevented death of 60% of the mice. Pathologic symptoms of TAA intoxication (liver haemorrhage, elevation of amino acids in blood and tissues, diminution of liver spermidine and spermine concentrations, elevation of the activity of liver enzymes in the plasma) were significantly ameliorated by the treatment. The liver protective action of 5FMOrn is related to the elevation of ornithine concentration, as appears from the fact that other, less selective inactivators of ornithine aminotransferase, also produced some protection against acute intoxication with TAA, but not a structurally related compound with no effect on this enzyme.

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3′- and 5′-untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N-terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln-35 and Gly-36.

Two apoenzymes of crystalline ornithine aminotransferase from rat liver were separated by gel filtration. They could also be separated by ultracentrifugation and sucrose density gradient centrifugation. These enzymes have different molecular weights, specific activities and absorption spectra. Their molecular weights are 177 100 ± 7700 (form I) and 105000 ± 6300 (form II) and their specific activities are 11.7 and 23.3 U/mg respectively. The native ornithine aminotransferase and the reconstituted holo-form I and holo-form II have similar Km values for l-ornithine (0.9 to 1.1 mM), Km values for 2-oxo glutarate (1.4 to 1.8 mM) and Ki; values for l-valine (4.0 to 6.5 mM). In the absence of coenzyme both enzymes are inert, but form I has an absorption shoulder at 330 nm, like the pyridoxamine 5′-phosphate form of enzyme. Apo-form II associates forming a molecule with the same molecular weight as native enzyme of 177000, on addition of the coenzyme, pyridoxal 5′-phosphate. The molecular weights of each enzyme protomer of the native ornithine aminotransferase and of the two apo-forms were all found to be 45000 ± 2000 by dodecylsulfate acrylamide gel disc electrophoresis. The amino acids compositions of form I and form II are very similar, and the total number of SH groups/monomer of the native enzyme, apo-form I and apo-form II were all 5.

Tentative assignments of functional residues in rat liver mitochondrial ornithine aminotransferase have recently been made using the amino acid sequence deduced from a cDNA clone [(1985) J.Biol.Chem. 260, 12993-12997]. Partial sequences obtained using the pure mature protein demonstrate that one of these assignments, that of Lys 292 as the residue that binds the coenzyme pyridoxal phosphate, is correct. However, the identification of the Glu 34-Gln 35 bond as the site of post-translational proteolysis is in error. This cleavage occurs instead at Ala 25-Thr 26.

— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba-mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates. Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.

The concentrations of ten or 12 enzymes involved in the metabolism of DNA, collagen, amino acids, or glucose have been determined in variants of human intestinal and pulmonary tissues. In comparison to nonneoplastic adult colon, normal fetal colon had elevated concentrations of thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, ornithine transcarbamylase, gamma-glutamyl transpeptidase, and ornithine aminotransferase. Raised activities of the first five of these enzymes, and of hexokinase, glucose-6-phosphate dehydrogenase, and pyrroline-5-carboxylate reductase distinguishes neoplastic from nonneoplastic sections of adult colon. Study of a wide range of pulmonary specimens permitted comparisons of different types of tumors, and revealed some subtle differences between lungs of noncancer patients and nonneoplastic portions of host lungs. The concentrations of eight previously identified enzymic indicators were less in moderately or well differentiated than in poorly differentiated pulmonary adenocarcinomas. The latter differed from epidermoid carcinomas (also poorly differentiated) by containing lower concentrations of thymidine kinase (both soluble and particulate) and hexokinase.

A radiometric assay for ornithine aminotransferase was developed using [1-14C]α-ketoglutarate as the labeled substrate and glutamate decarboxylation as a linking step. This assay gives near total measurement of ornithine aminotransferase activities that are, respectively, about 1.5 and 10 times larger than those obtained by the spectrophotometric assay and the radiometric assay using [1-14C]ornithine. It is also the most sensitive of the three assay procedures. Consistent with previous reports, brain ornithine aminotransferase was found to be present predominantly in synaptosomes. Regional distribution of the enzyme correlated with that of the high-affinity uptake of glutamate, but not with the distribution of glutamate decarboxylase. Ornithine aminotransferase may be responsible for the synthesis of glutamate in glutamatergic neurons but it is clearly not localized exclusively in such neurons.

The regional, cellular, and subcellular distribution of some enzymes of the urea cycle in brain is not clearly known. Glia, neurons, and synaptosomes have been prepared from rat cerebral cortex and arginase (EC 3.5.3.1), transamidinase (EC 2.1.4.1), and ornithine aminotransferase (EC 2.6.1.13) have been estimated in order to understand the metabolic and functional role of these enzymes. It has been observed that arginase is predominantly localized in synaptosomes and neurons. The ornithine aminotransferase was found to be high in glial cells and very high in synaptosomes (higher than arginase). Transamidinase was mostly localized in glial cells. The implication of these results has been discussed in relation to a possible role of ornithine acting as a precursor of glutamate in glutametargic nerve endings and its possible participation in the glutamate-glutamine cycle.

Glutamate dehydrogenase in Acanthamoeba castellanii is an NAD-dependent cytosolic enzyme. This is similar to glutamate dehydrogenases in Phycomycetes, but very different from the dual coenzyme-specific enzymes located in mitochondria in animals and in mitochondria and chloroplasts in higher plants. Pyrroline-5-carboxylate (P-5-C) reductase occurs also in the cytoplasm in A. castellanii and has very high affinities for L-P-5-C (Km= 12 μM) and NADH (Km= 15 μM). In contrast, ornithine aminotransferase and proline oxidase are mitochondrial enzymes. No proline-inhibited γ-glutamyl kinase was detected while an active glutamine synthetase was found in the cytosolic compartment. Evidence for a mitochondrial transport system for L-proline was obtained. Two possible pathways for proline biosynthesis in A. castellanii are discussed based on information obtained about activities and subcellular compartmentation of enzymes.

Ornithine aminotransferase was purified from rat liver and crystallized in the presence of ammonium sulphate and poly(ethylene glycol) (PEG 4000). The crystallographic threefold symmetry observed for the resulting two crystal forms stimulated a re-examination of the enzyme's quaternary structure in solution by analytical ultracentrifugation and chemical cross-linking. The results indicate that the oligomeric state or ornithine aminotransferase, under conditions similar to those used in crystallization experiments, is a hexamer (Mr = 256,000) rather than a tetramer or higher oligomers as reported previously. The subunits of the enzyme are identical (Mr = 45,000). Only the hexagonal prismatic crystals obtained with PEG 4000 were suitable for crystallographic studies and diffracted X-rays to a resolution of at least 0.16 nm. However, these crystals contained an unusual element of disorder which was persistent under a variety of conditions and was only noticeably diminished in the presence of the non-ionic detergent octyl beta-glucoside. The crystals apparently belong to the trigonal space group P3(1)12 (or enantiomorph) with axial lengths of a = 19.5 nm, c = 5.9 nm and contain three monomers per asymmetric unit.

We measured L-ornithine oxidation in cultured skin fibroblasts from seven patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (McKusick 23897), and compared it with oxidation by ornithine aminotransferase deficient gyrate atrophy (McKusick 25887) cells and lysinuric protein intolerance (McKusick 22270) cells in which there is an ornithine transport abnormality at the plasma membrane. Net uptake of ornithine is not abnormal in intact HHH cells. Ornithine oxidation was depressed in HHH and gyrate atrophy cells but not in lysinuric protein intolerance cells; the latter finding suggests there is no significant mitochondrial defect in lysinuric protein intolerance cells. Since HHH cells have intact ornithine aminotransferase, impaired oxidation is compatible with deficient penetration of ornithine into mitochondria in this disease. We could not demonstrate a gene dosage effect in oxidation values.

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.

Ornithine aminotransferase catalyzes the reversible transamination of L-ornithine to delta1-pyrroline-5-carboxylate, the immediate precursor of proline. The direction and flux through this pathway in mammalian cells has not been established. Glutamate has generally been considered to be the most important precursor for proline biosynthesis, but recent studies in xiphoid cartilage indicate that a significant fraction of cellular proline is derived from ornithine. Using newly isolated mutant Chinese hamster ovary cells with defined defects in the proline biosynthetic pathways, we now have established that cells can grow at a maximal rate with ornithine as the sole source of proline. Furthermore, we have measured the rate of proline formation from ornithine (1.6 nmol/h/10(6) cells); Future studies with these mutant Chinese hamster ovary cells may offer insight into the regulatory mechanism which coordinates proline biosynthesis from ornithine and glutamate.

100 g rats, submitted for 3 weeks to protein malnutrition (mostly lysine and threonine deficiency) with a diet offered ad libitum but containing 7% gluten as the sole protein, were starved for 24 h and allowed to recover for 3 days on a diet containing 18% casein and 0.3%dl‐methionine. These animals were compared to control rats refed on the gluten diet. The food intake as well as the level of immunoreactive plasma insulin increased immedieately. Among the free amino acids of the plasma, lysine, threonine and tyrosine rose rapidly (4‐ to 6‐fold), others were also significantly elevated while serine, glycine and histidine decreased. In the liver, the concentration of taurine was multiplied 7‐fold, that of threonine tripled, those of lysine, leucine, isoleucine, valine, tyrosine and phenylalanine doubled approximately, while the level of serine was markedly decreased. After 3 days, the total protein and DNA contents had increased by 70% and 2% respectively. Protein concentration on a wet weight basis was slightly higher than that observed in rats maintained on gluten whereas glycogen concentration levelled at a significantly lower (–43%) value. Of the 13 enzymatic specific activities investigated for their contribution to the intermediary metabolism, some did not vary significantly (α‐amylase bound to glycogen, hexokinases 1, 2 and 3, NADP:malate dehydrogenase, ATP:citrate lyase, arginase). Five activities increased (glucokinase 4‐fold, glucose‐6‐phosphate dehydrogenase 6‐fold, 6‐phosphogluconate dehydrogenase 2‐fold, serine dehydrogenase 2‐fold and ornithine aminotransferase 8‐fold) and three activities decreased somewhat (pyruvate kinases 0.60‐fold, glucose‐6‐phosphatase 0.65‐fold and fructose‐1,6‐diphosphatase 0.70‐fold). These rapid modifications might favour in the liver (a), a faster utilization of glucose in the two main glycolytic pathways; (b), a decrease in the hepatic glucose output and (c), the removal of the amino group of amino acids supplied in excess of the needs for protein storage. From these results and previously published experiments it thus appears that within the 3 days following a period of amino acid imbalance, insulin hypersecretion could not only have general effects on protein synthesis and turnover and on mitotic activity but might also be specifically involved in the induction of glucokinase and glucose‐6‐phosphate dehydrogenase and in the repression of glucose‐6‐phosphatase and fructose‐1,6‐diphosphatase. The influx of free amino acids plays the main role in the induction of serine dehydratase and ornithine aminotransferase and could share the responsability with insulin in the induction of glucokinase and glucose‐6‐phosphate dehydrogenase. Copyright © 1969, Wiley Blackwell. All rights reserved

Three Japanese cases of hyperornithinaemia due to a defect in ornithine ketoacid transaminase are described. Different types of disease in responsiveness to vitamin B6 were found.In vivo response to B6 correlated within vitro data.