The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Comparative evaluation of diagnostic preparations

The diagnostic value of preparations is commonly characterized by sensitivity and specificity. But not all these characteristics make it possible to decide unequivocally which of the preparations to be compared is superior to the other one with respect to its diagnostic value. It is proposed that in the choice of a diagnostic preparation its capacity to provide data for exact diagnosis should be considered, i.e. the additional characteristic indicating the spread of the disease under study. As an example, the comparison of the diagnostic value of conventional methods and the polymerase chain reaction in the diagnostics of helicobacteriosis is presented. The described method for the evaluation of the diagnostic value of the preparation is well-grounded, simple and obvious.     

Effect of a polyoxydonium immunoregulator on the biological properties of microorganisms

The effect of the synthetic immunomodulator polyoxydonium (PO) on some biological properties of pathogenic bacteria (Shigella flexneri, Salmonella enteritidis), opportunistic bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacteroides fragilis, Peptostreptococcus anaerobius, Prevotella melaninogenica, Propionibacterium propionicum, Clostridium difficile) and fungi (Candida albicans), isolated during enteric infections, enteric dysbiosis, pyoinflammatory diseases, was evaluated in a number of in vitro experiments. The study revealed that the recommended therapeutic concentration of PO decreased antilysozyme activity (ALA) and the anticomplement activity in Klebsiella, Shigella, Propionibacterium, Clostridium, bacteroids, fungi of the genus Candida, but increased ALA in nonhemolytic Escherichia. Under the action of PO an increased sensitivity of the microorganisms under study to definite antibiotics of the lincosamide, fluoroquinolon, carbapenem and cephalosporin groups was noted. The data obtained in this study reveal one of the possible mechanisms of the corrective action of PO on the microbiocenosis of the intestine in dysbiosis, enteric infections and pyoinflammatory diseases.     

Evaluation of the effect of medicines on biological properties of Clostridium difficile

The presence of the persistence factors (anti-lysozyme and anti-complement activity) in the vegetative forms of C. difficile was experimentally proved. The effect of different medicines (vitamins B1, B6 and C, prebiotic inulin, probiotics Bifidumbacterin and Enterol) on the persistence factors of C. difficile and microbial resistance to vancomycin, thienam, lincomycin, clindamycin was evaluated. The anti-lysozyme and anti-complement activity of C. difficile was found to decrease under the influence of vitamins B1, B6, C, inulin, exometabolites of bifidobacteria. Under the impact of the preparations used in this study changes in the sensitivity of C. difficile to antibiotics of the lincoamide, carbapenem, glycopeptide groups were found to occur. The data obtained reveal one of the possible mechanisms of the corrective action of the medicines under study on the intestinal microbiocenosis in patients with antibiotic-associated colitis.     

Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions

We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.     

Some mechanisms of Helicobacter pylori persistence

The mechanisms of the pathogenesis of Helicobacter pylori infection, ensuring the prolonged survival of these bacteria in the aggressive medium (gastric mucosa), are considered. A new approach to the systematization of the mechanisms of H. pylori persistence is proposed: the mechanisms ensuring the stability of H. pylori in the presence of aggressive physical and chemical factors, the mechanisms ensuring antagonistic effects in biocenosis and the mechanisms ensuring resistance to the protective factors of the host. Data on the persistence potential of H. pylori in accordance with the proposed classification are presented.