(1/935) Regulation of de novo purine biosynthesis in human lymphoblasts. Coordinate control of proximal (rate-determining) steps and the inosinic acid branch point.
Purine nucleotide synthesis de novo has been studied in a permanent tissue culture line of human splenic lymphoblasts with particular attention to coordination of control of the proximal (rate-determining) steps with the distal branch point of the pathway. An assay was used which permits simultaneous determination of the overall rate of labeling of all intracellular purines with sodium [14C]formate, as well as the distribution of isotope into all intracellular guanine- and adenine-containing compounds. The guanine to adenine labeling ratio was used as an index of IMP branch point regulation. It was found that exogenous adenine and guanine produce feedback-controlling effects not only on the first step in the de novo pathway, but also on the IMP branch point. Concentrations of adenine which produce less than 40% inhibition of the overall rate of de novo purine synthesis do so by selectively inhibiting adenine nucleotide synthesis de novo by 50 to 70% while stimulating guanine nucleotide synthesis de novo by up to 20%. A reciprocal effect is seen with exogenous guanine. The adenosine analog 6-methylmercaptopurine ribonucleoside selectivity inhibits adenine nucleotide synthesis via the de novo pathway but not from exogenous hypoxanthine. Thus, the reactions of purine nucleotide interconversion, in particular adenylosuccinate synthetase, may be regulated differently in cells deriving their purine nucleotides solely from de novo synthesis than when deriving them via "salvage" of preformed hypoxanthine. (+info)
(2/935) Formate-induced inhibition of photoreceptor function in methanol intoxication.
Formic acid is the toxic metabolite responsible for the retinal and optic nerve toxicity produced in methanol intoxication. Previous studies in our laboratory have documented formate-induced retinal dysfunction and histopathology in a rodent model of methanol intoxication. The present studies define the time and concentration dependence of formate-induced retinal toxicity in methanol-intoxicated rats. Retinal function was assessed 24, 48, and 72 h after the initial dose of methanol by flicker electroretinographic measurements. Retinal histopathology was assessed at the same time intervals. Rod- and cone-mediated electroretinogram (ERG) responses were attenuated in a formate concentration- and time-dependent manner, and both retinal sensitivity and maximal responsiveness to light were diminished. Attenuation of UV-cone-mediated responses was temporally delayed in comparison to the functional deficits observed in the 15 Hz/510 nm responses, which have a rod-mediated component and occurred at significantly higher formate concentrations. Both 15 Hz/510 nm and UV-cone-mediated ERG responses were undetectable by 72 h; however, if light intensity was increased, a retinal ERG response could be recorded, indicating that photoreceptor function was profoundly attenuated, but not abolished, under these intoxication conditions. Functional changes preceded structural alterations. Histopathological changes were most pronounced in the outer retina with evidence of inner segment swelling, photoreceptor mitochondrial disruption, and the appearance of fragmented photoreceptor nuclei in the outer nuclear layer. The nature of both the functional and structural alterations observed are consistent with formate-induced inhibition of mitochondrial energy production, resulting in photoreceptor dysfunction and pathology. (+info)
(3/935) Structure and transcriptional regulation of the gene encoding pyruvate formate-lyase of a ruminal bacterium, Streptococcus bovis.
The gene (pfl) encoding pyruvate formate-lyase (Pfl) from Streptococcus bovis was sequenced. The deduced amino acid sequence of Pfl was similar to Streptococcus mutans Pfl, and included the conserved regions necessary for free-radical formation and a catalytic site. The Pfl of S. bovis appeared to be a free-radical-containing enzyme because of its dioxygen sensitivity and its amino acid sequence similarity with the Escherichia coli enzyme. The pfl mRNA of S. bovis was approximately 2.3 kb and was transcribed in a monocistronic fashion. When cells were grown in batch culture at pH 6.9, the level of pfl transcript increased as the growth phase changed from exponential growth to stationary phase. This result was in constrast to the previous observation that the level of lactate dehydrogenase (Ldh) mRNA decreased during the later stages of growth. Continuous culture experiments conducted at pH 6.9 under glucose-limited and ammonia-limited conditions revealed that pfl mRNA was decreased by an excess supply of glucose, as well as by a high growth rate. On the contrary, ldh mRNA increased when excess glucose was supplied and the growth rate was high. The amount of pfl mRNA in cells was lower at pH 4.5 than pH 6.9, whereas the level of ldh mRNA was higher at pH 4.5. This result was consistent with the amounts of Pfl and Ldh in cells and the proportion of formate and lactate produced. These results support the hypothesis that S. bovis regulates Pfl and Ldh synthesis at the transcriptional level in response to growth conditions. (+info)
(4/935) Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons.
Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes). Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium. Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes. In E. coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD). In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate. Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate. Nitrate-dependent enhancement of transcription of narX-lacZ was completely abolished in a modE mutant. Nitrate-response by narG-lacZ and narK-lacZ was reduced by about 50% in a modE mutant. DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate. ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis. DNA sequences (5' TAYAT 3' and 5' GTTA 3') found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5' GTTA-7 bp-CATAT 3') and narX-narK intergenic region (5' GTTA-7 bp-TACAT 3'). Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhlA and NarL, respectively. (+info)
(5/935) Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover.
In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus. (+info)
(6/935) D-tagatose has low small intestinal digestibility but high large intestinal fermentability in pigs.
The digestibility of D-tagatose, its effect on the digestibility of macronutrients and the metabolic response of the microbiota of the gastrointestinal tract to the ingestion of this carbohydrate were studied in pigs. Eight pigs were fed a low fiber diet comprising 15% sucrose (control group). Another eight pigs were fed a similar diet except that 100 g sucrose per kg diet was replaced by D-tagatose (test group). After 18 d, the pigs were killed and the gastrointestinal contents removed for analysis. The digestibility of D-tagatose was 25.8 +/- 5.6% in the distal third of the small intestine. The small intestinal digestibilities of dry matter (86.9 +/- 1.3 vs. 92.9 +/- 0.9%), gross energy (74.4 +/- 1.6 vs. 80.7 +/- 1.8%) and sucrose (90.4 +/- 2.5 vs. 98.0 +/- 0.5%) were lower (P < 0. 05) in the pigs fed D-tagatose. Digestibilities of starch, protein and fat did not differ between groups. D-Tagatose, sucrose and starch were fully digested in the large intestine. The fecal digestibilities of energy, dry matter and fat did not differ between the two groups, whereas D-tagatose reduced the fecal digestibility of protein (91.1 +/- 0.6 vs. 93.5 +/- 0.7%, P < 0.05). D-Tagatose served as a substrate for the microbiota in the cecum and proximal colon as indicated by a reduced pH, and a greater ATP concentration, adenylate energy charge (AEC) ratio and concentration of short-chain fatty acids. In particular, the increase in the concentrations of propionate, butyrate and valerate suggests possible health benefits of this monosaccharide. (+info)
(7/935) Vanadate-induced activation of activator protein-1: role of reactive oxygen species.
The present study was undertaken to test the hypothesis that the toxicity and carcinogenicity of vanadium might arise from elevation of reactive oxygen species leading to activation of the transcription factor activator protein-1 (AP-1). The AP-1 transactivation response has been implicated as causal in transformation responses to phorbol esters and growth factors. To investigate the possible activity of vanadium in the activation of AP-1, we treated mouse epidermal JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid with various concentrations of vanadate. This resulted in concentration-dependent transactivation of AP-1. Superoxide dismutase (SOD) and catalase inhibited AP-1 activation induced by vanadate, indicating the involvement of superoxide anion radical (O2-*), hydroxyl radical (*OH) and/or H2O2 in the mechanism of vanadate-induced AP-1 activation. However, sodium formate, a specific *OH scavenger, did not alter vanadate-induced AP-1 activation, suggesting a minimal role for the *OH radical. NADPH enhanced AP-1 activation by increasing vanadate-mediated generation of O2-*. N-acetylcysteine, a thiol-containing antioxidant, decreased activation, further showing that vanadate-induced AP-1 activation involved redox reactions. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited activation of AP-1, demonstrating that PKC is involved in the cell signal cascades leading to vanadate-induced AP-1 activation. Electron spin resonance (ESR) measurements show that JB6 P+ cells are able to reduce vanadate to generate vanadium(IV) in the presence of NADPH. Molecular oxygen was consumed during the vanadate reduction process to generate O2-* as measured by ESR spin trapping using 5,5-dimethyl-L-pyrroline N-oxide as the spin trapping agent. SOD inhibited the ESR spin adduct signal, further demonstrating the generation of O2-* in the cellular reduction of vanadate. These results provide support for a model in which vanadium, like other classes of tumor promoters, transactivates AP-1-dependent gene expression. In the case of vanadium, AP-1 transactivation is dependent on the generation of O2-* and H2O2, but not *OH. (+info)
(8/935) Characterization of the anaerobic propionate-degrading syntrophs Smithella propionica gen. nov., sp. nov. and Syntrophobacter wolinii.
A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen. nov., sp. nov. The strain was enriched from an anaerobic digestor and isolated. Initial isolation was as a monoxenic propionate-degrading co-culture containing Methanospirillum hungateii JF-1T as an H2- and formate-using partner. Later, an axenic culture was obtained by using crotonate as the catabolic substrate. The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO2 and methane from propionate. However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 16S rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter. (+info)