Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics.

BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC. AIM: To investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology. METHODS: Male Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs. RESULTS: The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01). CONCLUSION: EA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

Fatal hyperammonemia and carbamoyl phosphate synthetase 1 (CPS1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation.

Fatal hyperammonemia secondary to chemotherapy for hematological malignancies or following bone marrow transplantation has been described in few patients so far. In these, the pathogenesis of hyperammonemia remained unclear and was suggested to be multifactorial. We observed severe hyperammonemia (maximum 475 µmol/L) in a 2-year-old male patient, who underwent high-dose chemotherapy with carboplatin, etoposide and melphalan, and autologous hematopoietic stem cell transplantation for a neuroblastoma stage IV. Despite intensive care treatment, hyperammonemia persisted and the patient died due to cerebral edema. The biochemical profile with elevations of ammonia and glutamine (maximum 1757 µmol/L) suggested urea cycle dysfunction. In liver homogenates, enzymatic activity and protein expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) were virtually absent. However, no mutation was found in CPS1 cDNA from liver and CPS1 mRNA expression was only slightly decreased. We therefore hypothesized that the acute onset of hyperammonemia was due to an acquired, chemotherapy-induced (posttranscriptional) CPS1 deficiency. This was further supported by in vitro experiments in HepG2 cells treated with carboplatin and etoposide showing a dose-dependent decrease in CPS1 protein expression. Due to severe hyperlactatemia, we analysed oxidative phosphorylation complexes in liver tissue and found reduced activities of complexes I and V, which suggested a more general mitochondrial dysfunction. This study adds to the understanding of chemotherapy-induced hyperammonemia as drug-induced CPS1 deficiency is suggested. Moreover, we highlight the need for urgent diagnostic and therapeutic strategies addressing a possible secondary urea cycle failure in future patients with hyperammonemia during chemotherapy and stem cell transplantation.

Low carbamoyl phosphate pools may drive Lactobacillus plantarum CO2-dependent growth phenotype.

Lactobacillus plantarum is often found in nutrient-rich habitats with elevated levels of inorganic carbon (IC), and IC-dependent growth is commonly encountered in natural isolates of this species. High CO(2)-requiring (HCR) prototrophs are unable to grow under conditions of low IC unless arginine and pyrimidines are provided. Prototrophy is restored under high IC conditions, that is in 4% CO(2)-enriched air or bicarbonate-supplemented medium. Bicarbonate is required for the synthesis of carbamoyl phosphate (CP), a precursor of both arginine and pyrimidine biosynthesis. We hypothesize that at low IC levels, intracellular CP pools limit growth through the limitation of arginine and nucleotide supplies. HCR mutants obtained in the laboratory can be classified into 3 functional groups: mutants with impaired CP synthesis, increased CP consumption or increased CP requirements relative to wild type. This classification provides a framework for investigating the origin of the HCR phenotype in natural environmental isolates of Lactobacillus species, and to investigate the hypothesis that a low level of carbamoyl phosphate is a major determinant of the CO(2)-dependent growth phenotype often observed in L. plantarum isolates.

Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).

Purification of ornithine carbamoyltransferase from kidney bean (Phaseolus vulgaris L.) leaves and comparison of the properties of the enzyme from canavanine-containing and -deficient plants.

Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonoacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 degrees C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, L-cysteine and S-carbamoyl-L-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle.

Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.

Reciprocal effects of proline and glutamine on glycogenesis from glucose and ureagenesis in isolated, perfused rat livers.

L-Proline and L-glutamine were used to probe the inverse relationship between glycogenesis and ureagenesis in isolated, perfused livers from 48-h fasted rats. Both amino acids may provide nitrogen in the form of NH+4 for carbamyl-P synthesis. However, one molecule of glutamine may provide additionally for the synthesis of one molecule of the urea cycle substrate L-aspartate, but proline can provide for the synthesis of a molecule of NH+4 or one molecule of aspartate on an either/or basis only. In all perfusates, glucose was initially 30 mM (to favor phosphotransferase activity of glucose-6-phosphatase) and 0.5 mM 3-mercaptopicolinate was present (to inhibit glyconeogenesis from endogenous substrates, from the added amino acids, and via the indirect pathway). Glycogenesis from glucose, perfusate and hepatic urea formation, and levels of hepatic glucose-6-P, citrulline, PPi, and carbamyl-P were measured. The addition of glutamine to the perfusate markedly stimulated the urea cycle, but not glycogenesis. Hepatic urea level, perfusate urea concentration, and hepatic citrulline and PPi increased while carbamyl-P content decreased. In contrast, proline stimulated glycogenesis from glucose, but not ureagenesis. In the proline-supplemented compared with glutamine group, hepatic glycogenesis and carbamyl-P content increased; hepatic glucose-6-P levels showed a tendency toward increase; and hepatic urea formation, hepatic citrulline, and PPi levels were decreased. These observations are interpreted to support an hepatic mechanism whereby the relative availability of carbamyl-P to the urea cycle and as a substrate for glucose phosphorylation via phosphotransferase activity of the glucose-6-phosphatase system preliminary to glycogenesis from glucose is a major metabolic determinant.

Regulation of orotic acid biosynthesis and excretion induced by oral glutamine administration in mice.

Glutamine, the most abundant amino acid in blood and tissues, is degraded by the renal and splanchnic tissues, especially the small intestinal mucosa. Due to the activity of glutaminase, it may be broken down in these tissues and contribute to ammoniagenicity. Glutamine, either directly or through ammonia production, may act as a nitrogenous source for pyrimidine biosynthesis. We have evaluated the effect of glutamine on orotate metabolism in mice, by gavaging (ig) L-glutamine, 1.0 to 4.0 mmol/100 g of body wt/day, during 6 weeks of experimentation. Glutamine at doses of 2.5 to 4.0 mmol/100 g of body wt caused a significant increase in plasma ammonia and urinary orotate. The regulation of the orotic acid biosynthesis and excretion was studied by testing the effects of various inhibitors in mice force-fed with glutamine (4 mmol/100 g of body wt, ig). The orotic aciduria was insensitive to acivicin (1 and 5 mg/100 g of body wt, ip), a specific inhibitor of the cytoplasmic carbamyl phosphate synthetase-II, thus pointing toward the mitochondrion as the principal source of carbamyl phosphate. Cycloheximide (15 and 100 mg/kg of body wt, ip) caused a significant decrease in urinary orotate indicating that the induction of orotate synthesis by glutamine may be associated with the translation of a specific protein. However, orotate excretion was significantly decreased by N-(phosphonoacetyl)-L-aspartate (PALA) (5 mg/100 g of body wt, ip) due to its inhibitory effect on the aspartate transcarbamylase activity. There was a significant increase of urinary orotate following ingestion of adenine supplemented diets (0.1% and 0.2%), suggesting the blockage of the utilization of orotate for nucleotide biosynthesis by glutamine. Since orotate synthesis may also be influenced by ornithine metabolism, we evaluated the effect of glutamine administration on various ornithine-metabolizing enzymes. There was a decrease in hepatic ornithine decarboxylase activity with no change in hepatic ornithine aminotransferase activity following the administration of glutamine. This observation indicates that an increased metabolic utilization of ornithine is not responsible for the increase in orotate excretion, which may be caused principally through an effect of glutamine on mitochondrial carbamyl phosphate synthesis.

Characterization of the 31P NMR spectrum of the schistosome vector Biomphalaria glabrata and of the changes following infection by Schistosoma mansoni.

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.

Effect of selected dietary additives on the incidence of dental caries in the rat.

Weanling rats fed a cariogenic diet supplemented with carbamyl phosphate, in combination with egg shell meal and trace elements, showed a striking reduction in the incidence of caries. The cariostatic efficacy of carbamyl phosphate is significant because it stimulates fluid movement within odontoblasts via the hypothalamic-parotid gland endocrine axis.