Glucose Phosphotransferase System Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci.

Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

Potential of Bacterial Cellulose Chemisorbed with Anti-Metabolites, 3-Bromopyruvate or Sertraline, to Fight against Helicobacter pylori Lawn Biofilm.

Helicobacter pylori is a bacterium known mainly of its ability to cause persistent inflammations of the human stomach, resulting in peptic ulcer diseases and gastric cancers. Continuous exposure of this bacterium to antibiotics has resulted in high detection of multidrug-resistant strains and difficulties in obtaining a therapeutic effect. The purpose of the present study was to determine the usability of bacterial cellulose (BC) chemisorbed with 3-bromopyruvate (3-BP) or sertraline (SER) to act against lawn H. pylori biofilms. The characterization of BC carriers was made using a N2 adsorption/desorption analysis, tensile strength test, and scanning electron microscopy (SEM) observations. Determination of an antimicrobial activity was performed using a modified disk-diffusion method and a self-designed method of testing antibacterial activity against biofilm microbial forms. In addition, bacterial morphology was checked by SEM. It was found that BC disks were characterized by a high cross-linking and shear/stretch resistance. Growth inhibition zones for BC disks chemisorbed with 2 mg of SER or 3-BP were equal to 26.5-27.5 mm and 27-30 mm, respectively. The viability of lawn biofilm H. pylori cells after a 4-h incubation with 2 mg SER or 3-BP chemisorbed on BC disks was ≥4 log lower, suggesting their antibacterial effect. SEM observations showed a number of morphostructural changes in H. pylori cells exposed to these substances. Concluding, SER and 3-BP chemisorbed on BC carriers presented a promising antibacterial activity against biofilm H. pylori cells in in vitro conditions.

High-resolution 1H NMR investigations of the oxidative consumption of salivary biomolecules by oral rinse peroxides.

BACKGROUND: A multicomponent evaluation of the oxidative consumption of salivary biomolecules by a tooth-whitening oral rinse preparation has been performed using high-resolution proton ((1)H) nuclear magnetic resonance spectroscopy (NMR). METHODS: Unstimulated human saliva samples (n = 12) were treated with aliquots of the oral rinse tested and 600 MHz (1)H NMR spectra acquired on these samples demonstrated that hydrogen peroxide (H(2)O(2)) and/or peroxodisulphate (S(2)O(8) (2-)) present in this product gave rise to the oxidative decarboxylation of the salivary electron-donor pyruvate (to acetate and CO(2)), and also oxidized methionine (a precursor to volatile sulphur compounds responsible for oral malodour), and malodourous trimethylamine to methionine sulphoxide and trimethylamine-N-oxide, respectively (reductions observed in the salivary concentrations of each biomolecular peroxide-scavenging agent were all extremely statistically significant, p < 0.005). RESULTS: Experiments conducted on chemical model systems confirmed the consumption of pyruvate by this product, and also revealed that the amino acids cysteine and methionine were oxidatively transformed to cystine and methionine sulphoxide, respectively. CONCLUSIONS: High-field (1)H NMR analysis provides much valuable molecular information regarding the fate of tooth-whitening oxidants in human saliva and permits an assessment of the mechanisms of action of oral healthcare products containing these agents. The biochemical and potential therapeutic significance of the results obtained are discussed.

A multistep enrichment process with custom growth medium improves resuscitation of chlorine-stressed coliforms from secondary sewage effluents.

Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study, we report that the addition of a growth enhancer mix consisting of trehalose, sodium pyruvate, magnesium chloride, and 1× trace mineral supplement improved growth of microorganisms from chlorinated secondary effluent in the base medium with Colilert-18. Improving growth of chlorine stressed microorganisms from secondary effluent is crucial to decreased detection time from 18 to 8 h.

Pretreatment and Detoxification of Acid-Treated Wood Hydrolysates for Pyruvate Production by an Engineered Consortium of Escherichia coli.

The biorefinery concept makes use of renewable lignocellulosic biomass to produce commodities sustainably. A synthetic microbial consortium can enable the simultaneous utilization of sugars such as glucose and xylose to produce biochemicals, where each consortium member converts one sugar into the target product. In this study, woody biomass was used to generate glucose and xylose after pretreatment with 20% (w/v) sulfuric acid and 60-min reaction time. We compared several strategies for detoxification with charcoal and sodium borohydride treatments to improve the fermentability of this hydrolysate in a defined medium for the production of the growth-associated product pyruvate. In shake flask culture, the highest pyruvate yield on xylose of 0.8 g/g was found using pH 6 charcoal-treated hydrolysate. In bioreactor studies, a consortium of two engineered E. coli strains converted the mixture of glucose and xylose in batch studies to 12.8 ± 2.7 g/L pyruvate in 13 h. These results demonstrate that lignocellulosic biomass as the sole carbon source can be used to produce growth-related products after employing suitable detoxification strategies.

Polyol pathway and modulation of ischemia-reperfusion injury in Type 2 diabetic BBZ rat hearts.

We investigated the role of polyol pathway enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH) in mediating injury due to ischemia-reperfusion (IR) in Type 2 diabetic BBZ rat hearts. Specifically, we investigated, (a) changes in glucose flux via cardiac AR and SDH as a function of diabetes duration, (b) ischemic injury and function after IR, (c) the effect of inhibition of AR or SDH on ischemic injury and function. Hearts isolated from BBZ rats, after 12 weeks or 48 weeks diabetes duration, and their non-diabetic littermates, were subjected to IR protocol. Myocardial function, substrate flux via AR and SDH, and tissue lactate:pyruvate (L/P) ratio (a measure of cytosolic NADH/NAD+), and lactate dehydrogenase (LDH) release (a marker of IR injury) were measured. Zopolrestat, and CP-470,711 were used to inhibit AR and SDH, respectively. Myocardial sorbitol and fructose content, and associated changes in L/P ratios were significantly higher in BBZ rats compared to non-diabetics, and increased with disease duration. Induction of IR resulted in increased ischemic injury, reduced ATP levels, increases in L/P ratio, and poor cardiac function in BBZ rat hearts, while inhibition of AR or SDH attenuated these changes and protected hearts from IR injury. These data indicate that AR and SDH are key modulators of myocardial IR injury in BBZ rat hearts and that inhibition of polyol pathway could in principle be used as a therapeutic adjunct for protection of ischemic myocardium in Type 2 diabetic patients.

Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nalecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.

Functional studies on in situ-like mitochondria isolated in the presence of polyvinyl pyrrolidone.

Mitochondria isolated and maintained in sucrose mannitol medium show a large intermembrane space and a condensed matrix unlike the appearance of in situ mitochondria. Mitochondria resembling in situ organelles are obtained when the isolation medium is supplemented with certain macromolecules such as polyvinyl pyrrolidone. We found that the in situ appearance was acquired also by the conventionally isolated mitochondria when they were exposed to 2% polyvinyl pyrrolidone supplemented medium. Paradoxically, however, these in situ looking mitochondria proved functionally inferior in that their brief incubation without substrates led to a marked loss of their ability to respire with subsequently added substrates such as pyruvate, acylcarnitines or glutamate. The oxidation of succinate was, however, not so affected. This phenomenon was shared by heart and skeletal muscle mitochondria of different animal species but not by rat liver mitochondria. The inhibition of respiration could not be related to the failure to oxidize NADH, to the tieing up of mitochondrial free CoASH, or to the increased matrix space of mitochondria that was observed in the presence of polyvinyl pyrrolidone. The polyvinyl pyrrolidone-exposed mitochondria regained their respiratory ability on being freed from polyvinyl pyrrolidone. The same phenomenon was seen also when the medium contained 2% albumin or 20% Ficoll.

Modulating hypoxia-induced hepatocyte injury by affecting intracellular redox state.

Hypoxia-induced hepatocyte injury results not only from ATP depletion but also from reductive stress and oxygen activation. Thus the NADH/NAD+ ratio was markedly increased in isolated hepatocytes maintained under 95% N2/5% CO2 in Krebs-Henseleit buffer well before plasma membrane disruption occurred. Glycolytic nutrients fructose, dihydroxyacetone or glyceraldehyde prevented cytotoxicity, restored the NADH/NAD+ ratio, and prevented complete ATP depletion. However, the NADH generating nutrients sorbitol, xylitol, glycerol and beta-hydroxybutyrate enhanced hypoxic cytotoxicity even though ATP depletion was not affected. On the other hand, NADH oxidising metabolic intermediates oxaloacetate or acetoacetate prevented hypoxic cytotoxicity but did not affect ATP depletion. Restoring the cellular NADH/NAD+ ratio with the artificial electron acceptors dichlorophenolindophenol and Methylene blue also prevented hypoxic injury and partly restored ATP levels. Ethanol which further increased the cellular NADH/NAD+ ratio increased by hypoxia also markedly increased toxicity whereas acetaldehyde which restored the normal cellular NADH/NAD+ ratio, prevented toxicity even though hypoxia induced ATP depletion was little affected by ethanol or acetaldehyde. The viability of hypoxic hepatocytes is therefore more dependent on the maintenance of normal redox homeostasis than ATP levels. GSH may buffer these redox changes as hypoxia caused cell injury much sooner with GSH depleted hepatocytes. Hypoxia also caused an intracellular release of free iron and cytotoxicity was prevented by desferoxamine. Furthermore, increasing the cellular NADH/NAD+ ratio markedly increased the intracellular release of iron. Hypoxia-induced hepatocyte injury was also prevented by oxypurinol, a xanthine oxidase inhibitor. Polyphenolic antioxidants or the superoxide dismutase mimic, TEMPO partly prevented cytotoxicity suggesting that reactive oxygen species contributed to the cytotoxicity. The above results suggests that hypoxia induced hepatocyte injury results from sustained reductive stress and oxygen activation.

Mechanisms of glucose-enhanced extracellular matrix accumulation in rat glomerular mesangial cells.

In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-I), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [3H]leucine and [3H]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-I and C-III accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Extraction, partial purification and functional reconstitution of two mitochondrial carriers transporting keto acids: 2-oxoglutarate and pyruvate.

Bovine heart submitochondrial particles were treated with a medium containing Triton X-114 and cardiolipin. The extract was subjected to hydroxyapatite chromatography. Only a few major polypeptides of similar molecular masses were found in the eluate, as shown by electrophoresis in an SDS-polyacrylamide gel stained with silver. The eluate was reconstituted into liposomes and was shown to catalyse two different transport activities: 2-oxoglutarate-2-oxoglutarate exchange sensitive to phthalonate and phenylsuccinate and pyruvate-pyruvate exchange sensitive to 2-cyano-4-hydroxycinnamate. Since both activities were found to have characteristics similar to those described for intact mitochondria, it was concluded that at least two of the polypeptides found in the hydroxyapatite eluate correspond to the two mitochondrial carriers.

The monocarboxylate carrier from rat liver mitochondria. Purification and kinetic characterization in a reconstituted system.

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Pyruvate oxidation in Charcot-Marie-Tooth disease.

Significant differences were found in pyruvate oxidation of disrupted skin fibroblasts from patients with Charcot-Marie-Tooth disease (CMT) or neuromuscular disease controls and normal controls, but there was a similar oxidative defect in both disease groups. "Heritable heterogeneity in situ" and additional enzyme faults may explain the normal activity of pyruvate dehydrogenase in some explants, and the lack of correlation between enzyme activity and CMT symptoms. Since experimental, histochemical, and microscopic studies support the concept of localized hypoxia in perineural or neural cells in CMT, the peripheral location of the defect suggests an inherited susceptibility to environmental influences in these cells.

Survival in a rat model of lethal hemorrhagic shock is prolonged following resuscitation with a small volume of a solution containing a drag-reducing polymer derived from aloe vera.

Drag-reducing polymers (DRP) increase tissue perfusion at constant driving pressure. We sought to evaluate the effects of small-volume resuscitation with a solution containing a DRP in a rat model of hemorrhage. Anesthetized rats were hemorrhaged at a constant rate over 25 min. In protocol A, total blood loss was 2.45 mL/100 g, whereas in protocol B, total blood loss was 3.15 mL/100 g. Five minutes after hemorrhage, the animals were resuscitated with 7 mL/kg of either normal saline (NS) or NS containing 50 microg/mL of an aloe vera-derived DRP. In protocol B, a third group (CON) was not resuscitated. Whole-body O2 consumption (Vo2) and CO2 production (Vco2) were measured using indirect calorimetry. In protocol A, 5/10 rats in the NS group and 8/10 rats in the DRP group survived for 4 h (P = 0.14). Mean arterial pressure was higher in the DRP-treated group than in the NS-treated group 45 min after resuscitation (89 +/- 8 vs. 68 +/- 5 mmHg, respectively; P < 0.05). In protocol B, survival rates over 2 h in the DRP, NS, and CON groups were 5/15, 1/14, and 0/7, respectively (P < 0.05). Compared with NS-treated rats, those resuscitated with DRP achieved a higher peak Vo2 (9.0 +/- 1.0 vs. 6,3+/- 1.0 mL/kg/min) and Vco2 (9.0 +/- 1.1 vs. 6.0 +/- 1.0 mL/kg/min) after resuscitation. We conclude that resuscitation with a small volume of DRP prolongs survival in rats with lethal hemorrhagic shock.

Normal pyruvate oxidation in Friedreich ataxia and Charcot-Marie-Tooth disease fibroblasts.

Partial defects in activity of the pyruvate dehydrogenase complex have been described by some investigators in cell lines from Friedreich ataxia and Charcot-Marie-Tooth disease patients. Methylene blue was used to stimulate the rate of pyruvate oxidation in two different assay systems of pyruvate dehydrogenase activity in cultured human fibroblasts to determine if such partial defects, if present, could be detected in a stimulated assay system. Cell lines from normal controls, five patients with Friedreich ataxia, six related persons with Charcot-Marie-Tooth disease patients were studied. Although methylene blue (at a concentration of 25 mumol/l) significantly increased pyruvate oxidation in both assay systems and in all cell lines studied, no significant differences in pyruvate oxidation could be demonstrated between the control cells and either the Friedreich ataxia or Charcot-Marie-Tooth cell lines.

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce "variant xanthans". Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.

Pyruvate and acetate metabolism in termite mitochondria.

Intact mitochondria have been successfully prepared from body tissues from the termites Nasutitermes walkeri and Coptotermes formosanus. This is the first report of the successful isolation of mitochondria from termites (Isoptera: Termitidae). Using an oxygen electrode, oxygen consumption by the mitochondria during the oxidation of various respiratory substrates was determined and their properties measured in terms of respiratory control index and ADP/O. ADP/O was as expected for substrates such as pyruvate, acetylcarnitine and acetyl-CoA and carnitine. Pyruvate and acetate were the major respiratory substrates in both species. The total activity of the pyruvate dehydrogenase complex (PDHc) in the mitochondria from N. walkeri and C. formosanus was determined to be 72.87+/-8.98 and 8.29+/-0.42 nmol/termite/h, respectively. Mitochondria isolated in the presence of inhibitors of PDHc interconversion were used to determine that about 60% of the PDHc was maintained in the active form in both N. walkeri and C. formosanus. The sufficient PDHc activity and high rate of pyruvate oxidation in mitochondria from N. walkeri suggest that pyruvate is rapidly metabolised, whereas the low mitochondrial PDHc activity of C. formosanus suggests that in this species more pyruvate is produced than can be oxidised in the termite tissues.

Bite-force development, metabolic and circulatory response to electrical stimulation in the canine and porcine masseter muscles.

The development of fatigue was investigated by electrical stimulation in 15 domestic pigs (1 yr old, 70-90 kg body weight) and seven adult dogs (3 yr old, 45 kg body weight). After anaesthesia, silver electrodes were implanted in the anterior and posterior parts of the right masseter muscles. The contralateral muscle was used as control. The bite force was measured. Muscle biopsies were obtained from the anterior, central and posterior parts, and were immediately frozen in liquid nitrogen. A fluorometrical analysis by enzymatic methods for glycogen, glucose, creatine phosphate, NAD, NADH, lactate and pyruvate was made. Blood flow was measured by 133Xe wash-out; oxygen consumption was monitored with an oxygen electrode. The porcine masseter was continuously stimulated for 60 min (100 V, 4 Hz and 2 ms). The canine masseter was intermittently stimulated (100 V, 20 Hz and 2 ms). The contraction was repeated four times, with a 10-min rest between. The porcine masseter could sustain longer endurance times than the canine masseter, which was easily fatigued. A marked substrate depletion was evident. The precontractional contents of glycogen, glucose and creatine phosphate were reduced. Lactate accumulation was evident (2-4 times more in the porcine and 4-8 times more in the canine masseter). The NADH concentration increased and the NAD content decreased. Blood-flow impairment (80% reduction in the dog, 60% in the pig) was observed. After the contraction phase, there was a hyperaemia (58% elevation of blood flow in the pig masseter, 45% in the canine). The oxygen tension followed in magnitude and time the blood-flow changes. These circulatory variables returned to normal after recovery. The high degree of substrate depletion, blood-flow impairment and a simultaneous decrease in oxygen transport to the contracted muscle, in combination with a prominent lactate accumulation, may induce a decrease in bite-force production.