Ferrous sulfate efficiently kills Vibrio parahaemolyticus and protects salmon sashimi from its contamination.

The primary seafood-borne pathogen Vibrio parahaemolyticus seriously threats the health of consumers preferring raw-fish products, becoming a global concern in food safety. In the present study, we found ferrous sulfate (FeSO4), a nutritional iron supplement, could efficiently induce the death of V. parahaemolyticus. Further, the bactericidal mechanisms of FeSO4 were explored. With a fluorescent probe of Fe2+, a significant influx of Fe2+ was determined in V. parahaemolyticus exposed to FeSO4, and the addition of an intracellular Fe2+ chelator was able to block the cell death. This suggested that cell death in V. parahaemolyticus induced by FeSO4 was dependent on the influx of Fe2+. It was intriguing that we did not observe the eruption of reactive oxygen species (ROS) and lipid hydroperoxides by Fe2+, but the application of liproxstatin-1 (a ferroptosis inhibitor) significantly modified the occurrence of cell death in V. parahaemolyticus. These results suggested FeSO4-induced cell death in V. parahaemolyticus be a ferroptosis differing from that in mammalian cells. Through transcriptome analysis, it was discovered that the exposure of FeSO4 disturbed considerable amounts of gene expression in V. parahaemolyticus including those involved in protein metabolism, amide biosynthesis, two-component system, amino acid degradation, carbon metabolism, citrate cycle, pyruvate metabolism, oxidative phosphorylation, and so on. These data suggested that FeSO4 was a pleiotropic antimicrobial agent against V. parahaemolyticus. Notably, FeSO4 was able to eliminate V. parahaemolyticus in salmon sashimi as well, without affecting the color, texture, shearing force, and sensory characteristics of salmon sashimi. Taken together, our results deciphered a unique ferroptosis in V. parahaemolyticus by FeSO4, and highlighted its potential in raw-fish products to control V. parahaemolyticus.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Electroanalysis of Infection with Methyl Pyruvate.

The discovery of infection enzyme leukocyte esterase (LE) hydrolyzing a mitochondrial substrate methyl pyruvate (MP) was explored in the development of electroanalytical methods for LE in human biofluids. The LE + MP reaction was coupled with alcohol oxidase to produce hydrogen peroxide, which was then reduced at a nitrogen-doped carbon nanotube electrode at -0.20 V, yielding current proportional to the LE content in a sample. The kinetic assays revealed a fast turnover (kcat = 15 s-1) and high specificity constant (kcatKm-1 = 2.3 × 106 M-1 s-1) for the LE-triggered hydrolysis of MP. The analytical assays were short (5 min) and the quantified LE was in the clinically relevant range of 22-300 µg L-1 (R2, 0.985). The immuno-electroanalysis could detect the picomole quantity of LE and yielded linear calibration plots up to 150 µg L-1 of LE with the same slope regardless of the sample matrix (urine, saliva, and phosphate buffer). The spike-and-recovery experiments displayed an LE recovery of 99-104%. The amperometric immunoassay of LE was less laborious than traditional enzyme-linked immunosorbent assay (ELISA) for LE and reduced the required sample incubation time from 4 h (sandwich ELISA) to 30 min (immuno-electroanalysis). The proposed combination of immunosorption with internally calibrated amperometry can also be used for a selective determination of other enzymes, which form enzymatically active immune complexes.

An efficient biocatalytic synthesis of imidazole-4-acetic acid.

OBJECTIVE: To develop a new and efficient biocatalytic synthesis method of imidazole-4-acetic acid (IAA) from L-histidine (L-His). RESULTS: L-His was converted to imidazole-4-pyruvic acid (IPA) by an Escherichia coli whole-cell biocatalyst expressing membrane-bound L-amino acid deaminase (mL-AAD) from Proteus vulgaris firstly. The obtained IPA was subsequently decarboxylated to IAA under the action of H2O2. Under optimum conditions, 34.97 mM IAA can be produced from 50 mM L-His, with a yield of 69.9%. CONCLUSIONS: Compared to the traditional chemical synthesis, this biocatalytic method for IAA production is not only environmentally friendly, but also more cost effective, thus being promising for industrial IAA production.

Kinetics of ventilation-induced changes in diaphragmatic metabolism by bilateral phrenic pacing in a piglet model.

Perioperative necessity of deep sedation is inevitably associated with diaphragmatic inactivation. This study investigated 1) the feasibility of a new phrenic nerve stimulation method allowing early diaphragmatic activation even in deep sedation and, 2) metabolic changes within the diaphragm during mechanical ventilation compared to artificial activity. 12 piglets were separated into 2 groups. One group was mechanically ventilated for 12 hrs (CMV) and in the second group both phrenic nerves were stimulated via pacer wires inserted near the phrenic nerves to mimic spontaneous breathing (STIM). Lactate, pyruvate and glucose levels were measured continuously using microdialysis. Oxygen delivery and blood gases were measured during both conditions. Diaphragmatic stimulation generated sufficient tidal volumes in all STIM animals. Diaphragm lactate release increased in CMV transiently whereas in STIM lactate dropped during this same time point (2.6 vs. 0.9 mmol L-1 after 5:20 hrs; p < 0.001). CMV increased diaphragmatic pyruvate (40 vs. 146 µmol L-1 after 5:20 hrs between CMV and STIM; p < 0.0001), but not the lactate/pyruvate ratio. Diaphragmatic stimulation via regular electrodes is feasible to generate sufficient ventilation, even in deep sedation. Mechanical ventilation alters the metabolic state of the diaphragm, which might be one pathophysiologic origin of ventilator-induced diaphragmatic dysfunction. Occurrence of hypoxia was unlikely.

Apparent rate constant mapping using hyperpolarized [1-(13)C]pyruvate.

Hyperpolarization of [1-13C]pyruvate in solution allows real-time measurement of uptake and metabolism using MR spectroscopic methods. After injection and perfusion, pyruvate is taken up by the cells and enzymatically metabolized into downstream metabolites such as lactate, alanine, and bicarbonate. In this work, we present comprehensive methods for the quantification and interpretation of hyperpolarized 13C metabolite signals. First, a time-domain spectral fitting method is described for the decomposition of FID signals into their metabolic constituents. For this purpose, the required chemical shift frequencies are automatically estimated using a matching pursuit algorithm. Second, a time-discretized formulation of the two-site exchange kinetic model is used to quantify metabolite signal dynamics by two characteristic rate constants in the form of (i) an apparent build-up rate (quantifying the build-up of downstream metabolites from the pyruvate substrate) and (ii) an effective decay rate (summarizing signal depletion due to repetitive excitation, T1-relaxation and backward conversion). The presented spectral and kinetic quantification were experimentally verified in vitro and in vivo using hyperpolarized [1-13C]pyruvate. Using temporally resolved IDEAL spiral CSI, spatially resolved apparent rate constant maps are also extracted. In comparison to single metabolite images, apparent build-up rate constant maps provide improved contrast by emphasizing metabolically active tissues (e.g. tumors) and suppression of high perfusion regions with low conversion (e.g. blood vessels). Apparent build-up rate constant mapping provides a novel quantitative image contrast for the characterization of metabolic activity. Its possible implementation as a quantitative standard will be subject to further studies.

Is selective antegrade cerebral perfusion superior to retrograde cerebral perfusion for brain protection during deep hypothermic circulatory arrest? Metabolic evidence from microdialysis.

OBJECTIVES: This study aimed to investigate whether selective antegrade cerebral perfusion or retrograde cerebral perfusion is a better technique for brain protection in deep hypothermic circulatory arrest by obtaining metabolic evidence from microdialysis. DESIGN: Randomized, animal study. SETTING: Assisted circulation laboratory. SUBJECTS: Eighteen piglets of either sex (9.8 ± 3.1 kg). INTERVENTIONS: Animals were randomly assigned to 40 minutes of circulatory arrest at 18°C without cerebral perfusion (deep hypothermic circulatory arrest group, n = 6) or with selective antegrade cerebral perfusion (selective antegrade cerebral perfusion group, n = 6) or retrograde cerebral perfusion (retrograde cerebral perfusion group, n = 6). Reperfusion was continued for 3 hours. MEASUREMENTS AND MAIN RESULTS: Microdialysis (glucose, lactate, pyruvate, and glycerol) variables in the cortex dialysate were measured every 30 minutes. Intracerebral pressure and serum S-100 levels were also monitored. After 3 hours of reperfusion, cortical tissue was harvested for terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. After 40 minutes of circulatory arrest, the deep hypothermic circulatory arrest group presented marked elevations of intracerebral pressure, and serum S-100 levels were higher in the deep hypothermic circulatory arrest group than in the other two groups (p < 0.001, respectively). The selective antegrade cerebral perfusion group exhibited higher glucose, lower lactate, and lower glycerol levels and a lower lactate-to-pyruvate ratio in comparison to the deep hypothermic circulatory arrest group (p < 0.05, respectively); the retrograde cerebral perfusion group had lower lactate and glycerol levels and a lower lactate-to-pyruvate ratio (p < 0.05, respectively) but similar glucose levels compared to deep hypothermic circulatory arrest alone. Furthermore, selective antegrade cerebral perfusion provided better preservation of energy and cell integrity than retrograde cerebral perfusion with higher glucose and lower glycerol levels (p < 0.05, respectively). After 3 hours of reperfusion, fewer apoptotic neurons were found in selective antegrade cerebral perfusion animals than in the other two groups (p < 0.05, respectively). CONCLUSIONS: Both selective antegrade cerebral perfusion and retrograde cerebral perfusion were superior to deep hypothermic circulatory arrest alone during circulatory arrest. Retrograde cerebral perfusion was a moderate technique that had similar advantages with regard to less cerebral edema, better clearance of metabolic waste, and lower levels of biomarkers of injury than selective antegrade cerebral perfusion, but its capacity for energy preservation, maintenance of cellular integrity, and protection against apoptosis was lower than that of selective antegrade cerebral perfusion.

Products and mechanism of the reactions of OH radicals and Cl atoms with methyl methacrylate (CH2═C(CH3)C(O)OCH3) in the presence of NOx.

The OH radical and Cl atom initiated photodegradation of methyl methacrylate has been investigated in a 1080 L quartz-glass environmental chamber at 298 ± 2 K and atmospheric pressure of synthetic air using in situ FTIR spectroscopy to monitor the reactants and products. The major products observed in the OH reaction were methyl pyruvate (92 ± 16%) together with formaldehyde (87 ± 12%) as a coproduct from the C1-C2 bond cleavage channel of the intermediate 1,2-hydroxyalkoxy radical, formed by the addition of OH to the terminal carbon of the double bond which is designated C1. For the Cl atom reaction, the products identified were chloroacetone (41 ± 6%) together with its coproduct formaldehyde (35 ± 5%) and methyl pyruvate (24 ± 4%) together with its coproduct formylchloride (25 ± 4%). The results show that the fate of the intermediate 1,2-chloroalkoxy radical involves not only cleavage of the C1-C2 bond but also quite substantial cleavage of the C2-C3 bond. The present results are compared with previous studies of acrylates, showing different branching ratios for the OH and Cl addition reactions in the presence of NOx. Atmospheric implications are discussed.

Cerebral energy metabolism during mitochondrial dysfunction induced by cyanide in piglets.

BACKGROUND: Mitochondrial dysfunction is an important factor contributing to tissue damage in both severe traumatic brain injury and ischemic stroke. This experimental study explores the possibility to diagnose the condition bedside by utilising intracerebral microdialysis and analysis of chemical variables related to energy metabolism. METHODS: Mitochondrial dysfunction was induced in piglets and evaluated by monitoring brain tissue oxygen tension (PbtO2 ) and cerebral levels of glucose, lactate, pyruvate, glutamate, and glycerol bilaterally. The biochemical variables were obtained by microdialysis and immediate enzymatic analysis. Mitochondrial function was blocked by unilateral infusion of NaCN/KCN (0.5 mol/L) through the microdialysis catheter (N = 5). As a reference, NaCl (0.5 mol/L) was infused by intracerebral microdialysis in one group of animals (N = 3). RESULTS: PbtO2 increased during cyanide infusion and returned to baseline afterwards. The lactate/pyruvate (LP) ratio increased significantly following cyanide infusion because of a marked increase in lactate level while pyruvate remained within normal limits. Glutamate and glycerol increased after cyanide infusion indicating insufficient energy metabolism and degradation of cellular membranes, respectively. CONCLUSION: Mitochondrial dysfunction is characterised by an increased LP ratio signifying a shift in cytoplasmatic redox state at normal or elevated PbtO2 . The condition is biochemically characterised by a marked increase in cerebral lactate with a normal or elevated pyruvate level. The metabolic pattern is different from cerebral ischemia, which is characterised by simultaneous decreases in intracerebral pyruvate and PbtO2 . The study supports the hypothesis that cerebral ischemia and mitochondrial dysfunction may be identified and separated at the bedside by utilising intracerebral microdialysis.

Detection of radiation-induced lung injury using hyperpolarized (13)C magnetic resonance spectroscopy and imaging.

Radiation-induced lung injury limits radiotherapy of thoracic cancers. Detection of radiation pneumonitis associated with early radiation-induced lung injury (2-4 weeks postirradiation) may provide an opportunity to adjust treatment, before the onset of acute pneumonitis and/or irreversible fibrosis. In this study, localized magnetic resonance (MR) spectroscopy and imaging of hyperpolarized (13)C-pyruvate (pyruvate) and (13)C-lactate (lactate) were performed in the thorax and kidney regions of rats 2 weeks following whole-thorax irradiation (14 Gy). Lactate-to-pyruvate signal ratio was observed to increase by 110% (P < 0.01), 57% (P < 0.02), and 107% (P < 0.01), respectively, in the thorax, lung, and heart tissues of the radiated rats compared with healthy age-matched rats. This was consistent with lung inflammation confirmed using cell micrographs of bronchioalveolar lavage specimens and decreases in arterial oxygen partial pressure (paO2), indicative of hypoxia. No statistically significant difference was observed in either lactate-to-pyruvate signal ratios in the kidney region (P = 0.50) between the healthy (0.215 ± 0.100) and radiated cohorts (0.215 ± 0.054) or in blood lactate levels (P = 0.69) in the healthy (1.255 ± 0.247 mmol/L) and the radiated cohorts (1.325 ± 0.214 mmol/L), confirming that the injury is localized to the thorax. This work demonstrates the feasibility of hyperpolarized (13)C metabolic MR spectroscopy and imaging for detection of early radiation-induced lung injury.

A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate.

A novel replaceable dual-enzyme capillary microreactor was developed and evaluated using magnetic fields to immobilize the alcohol dehydrogenase (ADH)- and lactate dehydrogenase (LDH)-coated magnetic beads at desired positions in the capillary. The dual-enzyme assay was achieved by measuring the two consumption peaks of the coenzyme ß-nicotinamide adenine dinucleotide (NADH), which were related to the ADH reaction and LDH reaction. The dual-enzyme capillary microreactor was constructed using magnetic beads without any modification of the inner surface of the capillary, and showed great stability and reproducibility. The electrophoretic resolution for different analytes can be easily controlled by altering the relative distance of different enzyme-coated magnetic beads. The apparent K(m) values for acetaldehyde with ADH-catalyzed reaction and for pyruvate with LDH-catalyzed reaction were determined. The detection limits for acetaldehyde and pyruvate determination are 0.01 and 0.016 mM (S/N = 3), respectively. The proposed method was successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. The results indicated that combing magnetic beads with CE is of great value to perform replaceable and controllable multienzyme capillary microreactor for investigation of a series of enzyme reactions and determination of multisubstrates.

How the signal-to-noise ratio influences hyperpolarized 13C dynamic MRS data fitting and parameter estimation.

MRS of hyperpolarized (13) C-labeled compounds represents a promising technique for in vivo metabolic studies. However, robust quantification and metabolic modeling are still important areas of investigation. In particular, time and spatial resolution constraints may lead to the analysis of MRS signals with low signal-to-noise ratio (SNR). The relationship between SNR and the precision of quantitative analysis for the evaluation of the in vivo kinetic behavior of metabolites is unknown. In this article, this topic is addressed by Monte Carlo simulations, covering the problem of MRS signal model parameter estimation, with strong emphasis on the peak amplitude and kinetic model parameters. The results of Monte Carlo simulation were confirmed by in vivo experiments on medium-sized animals injected with hyperpolarized [1-(13) C]pyruvate. The results of this study may be useful for the establishment of experimental planning and for the optimization of kinetic model estimation as a function of the SNR value.

[Cerebral microdialysis in the current clinical setting]. / La microdiálisis cerebral en el ámbito clínico actual.

Cerebral microdialysis, introduced in experimental studies 40 years ago, has been used clinically since 1992 for the neurochemical monitoring of patients in intensive care. The principles underlying this technique are closely related to brain metabolism. The study of the metabolites detected at brain interstitial tissue level, through the semipermeable membrane of the device, allows us to assess different physiological pathways in the brain, analyzing the changes that occur when they become less efficient in terms of energy, and also detecting waste products secondary to tissue damage. Despite its current limitations, this technique provides relevant information for research and the clinical management of critical neurological patients.

Intraperitoneal microdialysis in the postoperative surveillance after surgery for necrotizing enterocolitis: a preliminary report.

BACKGROUND/PURPOSE: The aim of the present pilot study was to evaluate the safety and clinical application of intraperitoneal microdialysis (MD) in preterm infants operated on for necrotizing enterocolitis (NEC). METHODS: Fourteen infants underwent MD. Two were excluded from analysis: 1 because of catheter malfunction and 1 because of fatal outcome immediately after surgery. The median MD time was 122 hours. Samples were collected every 4 hours, and the concentration of glucose, lactate, pyruvate, and glycerol was measured. RESULTS: Three infants were reoperated on: 2 because of recurrent NEC and 1 because of ileal stenosis. In the 2 cases with recurrent NEC, changes in MD variables were found. Another had a prolonged postoperative period owing to diffuse fecal peritonitis. The values of MD normalized along with the return of bowel function. In 8 infants, the postoperative course was uncomplicated. The results of peritoneal MD in patients with complications were significantly different from those with an uncomplicated course (lactate/pyruvate ratio and glucose concentration). CONCLUSION: Peritoneal MD is a safe procedure and an applicable method in surveillance of the metabolic and inflammatory changes in the peritoneal cavity after surgery for NEC. Larger series are needed to evaluate the clinical significance and use of this method.

Electrocautery causes more ischemic peritoneal tissue damage than ultrasonic dissection.

BACKGROUND: Minimizing peritoneal tissue injury during abdominal surgery has the benefit of reducing postoperative inflammatory response, pain, and adhesion formation. Ultrasonic dissection seems to reduce tissue damage. This study aimed to compare electrocautery and ultrasonic dissection in terms of peritoneal tissue ischemia measured by microdialysis. METHODS: In this study, 18 Wistar rats underwent a median laparotomy and had a peritoneal microdialysis catheter implanted in the left lateral sidewall. The animals were randomly assigned to receive two standard peritoneal incisions parallel to the catheter by either ultrasonic dissection or electrocautery. After the operation, samples of microdialysis dialysate were taken every 2 h until 72 h postoperatively for measurements of pyruvate, lactate, glucose, and glycerol, and ratios were calculated. RESULTS: The mean lactate-pyruvate ratio (LPR), lactate-glucose ratio (LGR), and glycerol concentration were significantly higher in the electrocautery group than in the ultrasonic dissection group until respectively 34, 48, and 48 h after surgery. The mean areas under the curve (AUC) of LPR, LGR, and glycerol concentration also were higher in the electrocautery group than in the ultrasonic dissection group (4,387 vs. 1,639, P=0.011; 59 vs. 21, P=0.008; 7,438 vs. 4,169, P=0.008, respectively). CONCLUSION: Electrosurgery causes more ischemic peritoneal tissue damage than ultrasonic dissection.

Analyte flux at a biomaterial-tissue interface over time: implications for sensors for type 1 and 2 diabetes mellitus.

OBJECTIVE: The very presence of an implanted sensor (a foreign body) causes changes in the adjacent tissue that may alter the analytes being sensed. The objective of this study was to investigate changes in glucose availability and local tissue metabolism at the sensor-tissue interface in patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). METHOD: Microdialysis was used to model implanted sensors. Capillary glucose and subcutaneous (sc) microdialysate analytes were monitored in five T1DM and five T2DM patients. Analytes included glucose, glycolysis metabolites (lactate, pyruvate), a lipolysis metabolite (glycerol), and a protein degradation byproduct (urea). On eight consecutive days, four measurements were taken during a period of steady state blood glucose. RESULTS: Microdialysate glucose and microdialysate-to-blood-glucose ratio increased over the first several days in all patients. Although glucose recovery eventually stabilized, the lactate levels continued to rise. These trends were explained by local inflammatory and microvascular changes observed in histological analysis of biopsy samples. Urea concentrations mirrored glucose trends. Urea is neither produced nor consumed in sc tissue, and so the initially increasing urea trend is explained by increased local capillary presence during the inflammatory process. Pyruvate in T2DM microdialysate was significantly higher than in T1DM, an observation that is possibly explained by mitochondrial dysfunction in T2DM. Glycerol in T2DM microdialysate (but not in T1DM) was higher than in healthy volunteers, which is likely explained by sc insulin resistance (insulin is a potent antilipolytic hormone). Urea was also higher in microdialysate of patients with diabetes mellitus compared to healthy volunteers. Urea is a byproduct of protein degradation, which is known to be inhibited by insulin. Therefore, insulin deficiency or resistance may explain the higher urea levels. To our knowledge, this is the first histological evaluation of a human tissue biopsy containing an implanted glucose monitoring device. CONCLUSIONS: Monitoring metabolic changes at a material-tissue interface combined with biopsy histology helped to formulate an understanding of physiological changes adjacent to implanted glucose sensors. Microdialysate glucose trends were similar over 1-week in T1DM and T2DM; however, differences in other analytes indicated wound healing and metabolic activities in the two patient groups differ. We propose explanations for the specific observed differences based on differential insulin insufficiency/resistance and mitochondrial dysfunction in T1DM versus T2DM.

Sitting can cause ischaemia in the subcutaneous tissue of the buttocks, which implicates multilayer tissue damage in the development of pressure ulcers.

A better understanding of how pressure ulcers develop in the buttocks will improve prophylactic measures. Our aim was to investigate signs of reduced perfusion and ischaemia in the subcutaneous fat in the buttocks during sitting. A microelectrode was used to quantify oxygen (pO(2)). Metabolites that indicate aerobic or anaerobic metabolism (glucose, lactate, pyruvate, and glycerol) were quantified using microdialysis. Sixteen healthy people were studied while they sat on a wheel chair cushion, and a hard surface. Sitting pressures were mapped, and the thickness of the subcutaneous fatty layer was measured. The results showed that pO(2) and glucose were significantly reduced during sitting, and for pO(2) the effect is significantly more profound during sitting on a hard surface. After loading, both glucose and pO(2) increased significantly. We conclude that the subcutaneous adipose tissue covering the ischial tuberosities becomes ischaemic during sitting. This finding supports the theory that not only is the skin involved in early development of pressure ulcers, but also the deeper tissues.

Acid production by oral strains of Candida albicans and lactobacilli.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42-66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.

Molecular and enzymatic characterization of betaC-S lyase in Streptococcus constellatus.

BACKGROUND/AIMS: Streptococcus anginosus and Streptococcus constellatus are frequently isolated from dental abscesses and other suppurative lesions. We previously reported that betaC-S lyase from a strain of S. anginosus produced significantly more hydrogen sulfide than betaC-S lyases from other streptococci. The purpose of this study was to establish the molecular and enzymatic features of the betaC-S lyase in S. constellatus and to elucidate whether this unique capacity is common to many strains of S. constellatus and S. anginosus. METHODS: The capacity of crude extract to produce hydrogen sulfide was evaluated among 16 strains of S. constellatus, S. anginosus, and Streptococcus gordonii. The lcd gene encoding betaC-S lyase was cloned from the genomic DNA of each strain to compare the deduced amino acid sequences. The recombinant betaC-S lyases of three representative strains were purified and characterized. RESULTS: Incubation of crude extracts from all strains of S. constellatus and S. anginosus with l-cysteine resulted in the production of a large amount of hydrogen sulfide. The primary sequence of betaC-S lyase was very similar among strains of S. constellatus and S. anginosus. The kinetic properties of the betaC-S lyases purified from S. constellatus resembled those for betaC-S lyases purified from S. anginosus. In contrast, the betaC-S lyases of S. constellatus and S. gordonii differed in terms of their hydrogen sulfide production, with the former producing much more. CONCLUSION: A high level of hydrogen sulfide production, which appears to be a common feature in both S. constellatus and S. anginosus, may be associated with their abscess formation.

The O-polysaccharide of Escherichia coli O112ac has the same structure as that of Shigella dysenteriae type 2 but is devoid of O-acetylation: a revision of the S. dysenteriae type 2 O-polysaccharide structure.

The O-antigen structure of Shigella dysenteriae type 2 was reinvestigated using chemical modifications along with high-resolution 2D (1)H and (13)C NMR spectroscopy. The O-antigen was found to contain a pyruvic acid acetal, which was overlooked in an early study, and the following revised structure of the pentasaccharide repeating unit was established: where approximately 70% GlcNAc residues bear an O-acetyl group at position 3. The O-antigen of Escherichia coli O112ac was found to have the same carbohydrate structure but to lack O-acetylation.