Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.     

Complications of Gastroesophageal Reflux

An edited summary of an Interdepartmental Conference arranged by the Department of Medicine of the UCLA School of Medicine, Los Angeles. The Director of Conferences is William M. Pardridge, MD, Professor of Medicine.Several specialists have recently recognized that gastrointestinal reflux causes complications resulting in significant disease. It causes discomfort, indigestion, esophagitis, Barrett''s esophagus, and carcinoma of the esophagus. Pediatricians attribute many early pulmonary problems, and even some sudden deaths in infants, to the reflux of gastric contents. Otolaryngologists now recognize that many cases of nonbacterial, nonspecific pharyngitis and laryngitis are due to the reflux of gastrc acid secretions. Contact granuloma and cancer of the larynx may, in some instances, be secondary to nocturnal reflux. Thoracic surgeons and pulmonologists believe chronic tracheobronchitis and some cases of pulmonary disease are attributable to recurrent bathing of the respiratory epithelium by aspirated gastric contents. An awareness of the many complications of gastrointestinal reflux should lead to a multidisciplined attack on the factors responsible for these diseases.     

Reconstitution of ATP-dependent calcium transport from streptococci

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.     

Dilute acid hydrolysis of paper birch: kinetics studies of xylan and acetyl-group hydrolysis

Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18M and temperatures of between 100 and 170 degrees C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based on the existence of reactive and resistant xylan portions. The resulting rate equation predicts the experimental xylan concentrations in the residue to within 10%. Hydrolysis of xylan-associated acetyl groups was found to occur at the same rate as that of xylan, except at 100 degrees C, where acetyl is released preferentially. No effect of acid concentration on the rate of acetyl removal relative to that of xylan was evident.     

Pharmacological investigation of the mechanisms of platelet-activating factor induced mortality in the mouse

Although the platelets of the mouse are refractory to the direct effects of platelet-activating-factor (PAF), tail vein injection of 10-150 micrograms/kg PAF produces lethal anaphylactic shock. Sensitivity varies with strain and source: Swiss Webster mice show a range of sensitivity and DBA/2 (complement C5-deficient) mice are very resistant. At lethal doses of PAF, animals show labored respiration and general depression; death occurs within 15-45 min. Dexamethasone administered at least 1.5 hr prior consistently protects, whereas the cyclooxygenase inhibitors do not. Antihistamines, adrenergic antagonists, and methysergide have no effect, but cyproheptadine is partially protective at near lethal doses. Calcium entry blockers and calcium chelators, tetracycline and chlortetracycline are partially protective at very high doses consistent with non-specific effects on calcium dependent processes. The arachidonic acid lipoxygenase inhibitors BW755c, phenidone, nordihydroguaiaretic acid and diphenyldisulfide provide nearly complete protection after oral administration of 50-200 mg/kg. Phosphodiesterase inhibitors and dapsone are also effective orally. The leukotriene antagonist FPL55712 administered intraperitoneally (10 mg/kg) 5 min. prior to PAF challenge provides almost complete protection. PAF-induced mortality in the mouse represents a small animal model of systemic anaphylaxis particularly useful for the systemic testing of arachidonic acid lipoxygenase inhibitors and leukotriene antagonists.     

Characterization of phosphate:hexose 6-phosphate antiport in membrane vesicles of Streptococcus lactis

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a Kt of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a Kt of 26 microM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to loss of internal 32Pi without transfer of 32P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (Kiapp): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 microM) greater than fructose 6-phosphate (150 microM) greater than glucosamine 6-phosphate (420 microM) greater than alpha-methylglucoside 6-phosphate (740 microM). Stoichiometry for phosphate:2-deoxyglucose 6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phosphate.     

Changes in Gut Microbiota in Rats Fed a High Fat Diet Correlate with Obesity-Associated Metabolic Parameters

The gut microbiota is emerging as a new factor in the development of obesity. Many studies have described changes in microbiota composition in response to obesity and high fat diet (HFD) at the phylum level. In this study we used 16s RNA high throughput sequencing on faecal samples from rats chronically fed HFD or control chow (n = 10 per group, 16 weeks) to investigate changes in gut microbiota composition at the species level. 53.17% dissimilarity between groups was observed at the species level. Lactobacillus intestinalis dominated the microbiota in rats under the chow diet. However this species was considerably less abundant in rats fed HFD (P<0.0001), this being compensated by an increase in abundance of propionate/acetate producing species. To further understand the influence of these species on the development of the obese phenotype, we correlated their abundance with metabolic parameters associated with obesity. Of the taxa contributing the most to dissimilarity between groups, 10 presented significant correlations with at least one of the tested parameters, three of them correlated positively with all metabolic parameters: Phascolarctobacterium, Proteus mirabilis and Veillonellaceae, all propionate/acetate producers. Lactobacillus intestinalis was the only species whose abundance was negatively correlated with change in body weight and fat mass. This species decreased drastically in response to HFD, favouring propionate/acetate producing bacterial species whose abundance was strongly correlated with adiposity and deterioration of metabolic factors. Our observations suggest that these species may play a key role in the development of obesity in response to a HFD.