Specificity of action on neuropeptides of an endopeptidase from the synaptosomal membranes of guinea pig brain

An endopeptidase was solubilized and highly purified from the synaptosomal membrane fraction of guinea pig brain, and its specificity of action on various neuropeptides was investigated. It hydrolyzed specifically the Pro10-Tyr11 bond of neurotensin and showed a marked specificity toward Pro-X bonds present in the interior parts of various neuropeptides and related peptides. No cleavage, however, was observed at the first and second peptide bonds from the NH2-termini or from the COOH-termini of the peptides examined, suggesting that the enzyme requires both NH2- and COOH-terminal extentions of at least 3 residues from the scissile bond for its action. In addition, a limited number of other peptide bonds were cleaved, indicating that the enzyme is not strictly specific to Pro-X bonds. These results suggest the possible implication of this enzyme in the specific degradation of neurotensin and other peptide neurotransmitters in the synaptic cleft.     

Changes in Lysozyme due to Interaction with Vaporized Hexanal

In order to elucidate the mechanism of the alteration of proteins induced by vaporized aldehydes, unmodified and chemically-modified lysozymes were exposed in the solid state to vaporized hexanal at 50°C and 5.8 or 75% relative humidity (RH). On exposure at 75%RH, the unmodified lysozyme exhibited polymerization, browning, loss of solubility, fluorescence production and impairment of lysine, tryptophan and methionine residues. Methionine residues seemed to be oxidized to methionine sulfoxide residues. The polymerization did not proceed at 5.8RH. All the above alterations were almost completely prevented by the removal of oxygen from the reaction cells. Acetylation of lysozyme retarded these alterations fairly well except that the impairment of tryptophan residues was unaffected.

On the basis of all the results it is suggested that at the first step the concerned reaction essentially requires hexanal derivatives such as peroxyhexanoic acid and/or related radicals induced through the reaction with oxygen. The second step seems to consist at least of two routes which are independent of each other and require water. One route is assumed to be an amino-carbonyl reaction involving lysine residues. The other route seems responsible for the attack on tryptophan and methionine residues through oxidation involving the radicals.     

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.     

Increased Solubility of High-Molecular-Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of l -[³⁵S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.     

Cytosolic acidification leads to Ca mobilization from intracellular stores in single and populational parietal cells and platelets

Regulatory relationship and gain control between cytosolic free Ca²⁺ concentration (Ca_i) and cytosolic pH (pH_i) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca²⁺-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Ca_i. In both populational cell types, an initial change in pH_i during agonist stimulation occurred almost simultaneously with the mobilization of Ca²⁺; an initial transient decrease in pH_i was followed by a slower increase in pH_i above the prestimulation level. When populational platelets were preloaded with the Ca²⁺ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Ca_i was apparently inhibited, whereas the pH_i decrease induced by thrombin was not altered. This suggests that the initial Ca_i change is not a prerequisite for the pH_i change. The effect of pH_i on Ca_i was examined next. In both single and populational cell types, application of the K⁺-H⁺ ionophore nigericin, which induced a transient decrease in pH_i, led to the release of Ca²⁺ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pH_i preceded the rise in Ca_i after stimulation with nigericin. A decrease in pH_i, and an increase in Ca_i occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na⁺ with N-methyl- -glucamine (NMG⁺), which also induced a decrease in pH_i, resulted in repetitive Ca²⁺ spike oscillations. The source of Ca²⁺ utilized for the Ca²⁺ oscillation that was induced by NMG⁺ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pH_i, led to an increase in Ca_i. Cytosolic acidification may be a part of the trigger for Ca²⁺ mobilization from intracellular stores in both parietal cells and platelets.     

Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.

A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.