Biochemical changes in the sublingual and submandibular glands after interruption of chorda tympani

The left chorda tympani nerve was interrupted through meatus acusticus externus in ten dogs. In total, 40 dog salivary glands (20 submandibular and 20 sublingual) innervated via chorda tympani were examined. Twenty glands (10 submandibular and 10 sublingual) on the left side were deprived of parasympathetic innervation by chordectomy, whereas contraleteral glands, on the right side, served as controls. Biochemical analysis showed that the interruption of chorda tympani did not cause any significant changes in the concentrations of eight enzymes investigated, i.e. lactate dehydrogenase, alkaline phosphatase, acid phosphatase, amylase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase and creatine kinase. There were no significant changes in the concentrations of most important extracellular ions (sodium, potassium, chloride and phosphorus) in the right glands, but the loss of parasympathetic innervation in the left glands was found to cause a statistically significant decrease in the concentration of potassium as intracellular cation and of phosphorus as extracellular anion.     

Progressive loss of lambda prophage recombinogenicity in UV-irradiated Escherichia coli: the role of RecBCD enzyme

RecBCD enzyme is involved in the radiation-induced process known as prophage inactivation. The process leads to the inability of lambda prophage to excise itself from the Escherichia coli chromosome via site-specific recombination. In this work we sought to further characterize the role of RecBCD enzyme in this process. In addition, we examined the ability of irradiated prophage to recombine with the infecting homologous phage. We used several E. coli mutants differentially altered in RecBCD's activities. The results showed that in the mutants carrying either recB2109 or recD1903, which do not exhibit significant nuclease activities, the prophage progressively loses its capacity for both site-specific and general recombination. In the recB268 null mutant, however, prophage recombinogenicity remained fully preserved. We also showed that the prophage unable to recombine retained its ability to complement the mutant infecting phage and that the recombination frequencies in phage x phage crosses were not affected by postirradiation incubation. Our results suggest that the helicase activity of RecBCD is responsible for the progressive loss of prophage recombinogenicity. This loss is most probably a consequence of the unsuccessful RecBCD-dependent recombinational repair of double-stranded breaks in the cell chromosome, during which some structures unsuitable for further recombination reactions may be produced.     

Distinct pathways for constitutive endocytosis of fully conformed and non-conformed L(d) molecules

PROBLEM: To characterize the constitutive internalization of major histocompatibility complex (MHC) class I molecules, we have studied the expression of completely conformed (full) and unconformed (empty) L(d) molecules on non-polarized murine P815 cells. METHODS OF STUDY: Spontaneous endocytosis of L(d) molecules was induced by cycloheximide, an inhibitor of protein synthesis, and their disappearance from the cell surface was determined by flow cytometry. In order to investigate the mechanism of internalization, a palette of inhibitors of endocytosis and vesicular transport was used. RESULTS: Inhibitors of clathrine endocytosis did not influence the internalization of L(d) molecules. Inhibitors of caveolar endocytosis and inhibitors of endolysosomal degradation prevented down-regulation of empty, but not of full L(d) molecules. CONCLUSIONS: Empty L(d) molecules are internalized mostly by caveolar endocytosis and full L(d) molecules use a different pathway, neither clathrine-mediated nor caveolar. After internalization, full L(d) molecules are probably degraded and empty L(d) molecules recycle between endosomal compartment and the cell surface before they enter into the degradation compartment.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.