Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

The role of Ca2+ and protein kinase C in the differentiation of HL-60 cells induced by 1 alpha,25(OH)2D3 and diltiazem

The roles of calcium (Ca2+) and protein kinase C in the differentiation of HL-60 cells induced by 1 alpha,25(OH)2D3 (D3) and/or a Ca2+ antagonist, diltiazem(D-cis, L-cis), were elucidated. D3 and diltiazem (100 microM) inhibited cell proliferation, and diltiazem enhanced the D3-induced differentiation. There was no difference in potency between the two isomers of diltiazem in the enhancing activity, in spite of their different pharmacological activity. The concentration of free Ca2+ in the HL-60 cells following D3 and/or diltiazem treatment significantly increased. A protein kinase C inhibitor, H-7, inhibited the phenotypic differentiation induced by D3. These results suggest that Ca2+ and protein kinase C play an important role in the differentiation of HL-60 cells induced by D3 and diltiazem.     

A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles

The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA. With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity. The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions. OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+. It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement.     

Preferred temperature of the elderly after cold and heat exposures determined by individual self-selection of air temperature

1. 1. The purpose of the study was to investigate the preferred temperature of the elderly after cold and heat exposures.

2. 2. Eight elderly and 9 young females wearing the same type of clothing were exposed to cold (10°C), moderate (25°C) or hot (35°C) environments for 30 min in the exposure room.

3. 3. Then they moved to the self-control room in which the temperature was set at 25°C, and the room temperature increased or decreased continuously by 0.4°C every minute.

4. 4. The subjects were instructed to operate the switch when they felt uncomfortably warm or cool during a 90-min period.

5. 5. In operating the switch, the changing in room temperature shifted to the opposite direction.

6. 6. The ambient temperature was recorded continuously and analyzed as the preferred temperature, which was defined as the midpoint temperature of the crest and trough of temperature records.

7. 7. The preferred temperatures after the cold exposure were significantly higher than those of other exposure conditions in the elderly.

8. 8. On the other hand, in the young, there was no significant difference in the preferred temperature among the exposure conditions.

9. 9. Although the effect of exposure to cold or hot environments decreased in the latter parts of self-control, the elderly still preferred the higher temperature after cold exposure.

Calcium-Independent Release of Acetylcholine from Stable Cell Lines Expressing Mouse Choline Acetyltransferase cDNA

Abstract: Stably transfected cells expressing mouse choline acetyltransferase (ChAT) cDNA were established, and the synthesis and release of acetylcholine (ACh) were examined. A cDNA clone coding for mouse ChAT was inserted into an expression vector (pEF321) containing a promoter for human elongation factor 1α to construct pEFmChAT. Neuronal (NG108-15, NS20Y, N1E115, and Neuro2A) and nonneuronal cell lines (L cells and NIH3T3) were transfected with pEFmChAT, and the cell lines that stably expressed high ChAT activity were selected. These cells expressed the 66-kDa ChAT protein and accumulated ACh mostly in the cytosol. The concentration of intracellular ACh in the cells increased upon raising the choline level in the medium. The cells continuously released ACh in a Ca²⁺-independent fashion. Neither high K⁺ nor calcium ionophore stimulated release of ACh from the cells.     

Coupled structure change of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and ΔsecG::kan double mutant

An Escherichia coli strain carrying either the secAcsR11 or Δ secG :: kan mutation is unable to grow at low temperature owing to cold-sensitive protein translocation but grows normally at 37°C. However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant. The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor-binding region and was defective in the interaction with the precursor protein. Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37°C, when csSecA was used instead of the wild-type SecA. The insertion of the wild-type SecA was also significantly defective when SecG-depleted membrane vesicles were used in place of SecG-containing ones. No insertion of csSecA occurred into SecG-depleted membrane vesicles. Examination of in vitro protein translocation at 37°C revealed that SecG is essential for csSecA-dependent protein translocation. We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation.     

SecY is an indispensable component of the protein secretory machinery of Escherichia coli

Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.     

Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies.

Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain.