Contribution of a Salt Bridge Triad to the Thermostability of a Highly Alkaline Protease from an Alkaliphilic Bacillus Strain*

Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.     

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.     

Metabolism of n-3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells

The metabolism of the linolenic acid family (n-3) of fatty acids, e.g., linolenic, eicosapentaenoic, and docosahexaenoic acids, in cultured smooth muscle cells from rabbit aorta was compared to the metabolism of linoleic and arachidonic acids. There was a time-dependent uptake of these fatty acids into cells for 16 hr (arachidonic greater than docosahexaenoic, linoleic, eicosapentaenoic greater than linolenic), and the acids were incorporated mainly into phospholipids and triglycerides. Eicosapentaenoic and arachidonic acids were incorporated more into phosphatidylethanolamine and phosphatidylinositol plus phosphatidylserine and less into phosphatidylcholine than linolenic and linoleic acids. Docosahexaenoic acid was incorporated into phosphatidylethanolamine more than linolenic and linoleic acids and into phosphatidylinositol plus phosphatidylserine less than eicosapentaenoic and arachidonic acids. Added linolenic acid accumulated mainly in phosphatidylcholine and did not decrease the arachidonic acid content of any phospholipid subfraction. Elongation-desaturation metabolites of linoleic acid did not accumulate. Cells treated with eicosapentaenoic acid accumulated both eicosapentaenoic and docosapentaenoic acids mainly in phosphatidylethanolamine and the arachidonic acid content was decreased. Added docosahexaenoic acid accumulated mainly in phosphatidylethanolamine and decreased the content of both arachidonic and oleic acids. The following conclusions are drawn from these results. The three n-3 fatty acids are utilized differently in phospholipids. The arachidonic acid content of phospholipids is reduced by eicosapentaenoic and docosahexaenoic acids, but not by linolenic acid. Smooth muscle cells have little or no desaturase activity, but have significant elongation activity for polyunsaturated fatty acids.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.     

Alkali Protease and Alcohol Dehydrogenase Immobilized to Weakly Basic Anion Exchange Resins Using 2-Car-boxymethylamino-4,6-dichloro-s-triazine

Alkali protease partially purified from a strain of Bacillus subtilis and yeast alcohol dehydrogenase were immobilized to weakly basic anion exchange resins using a bifunctional reagent, 2-carboxymethyIamino-4,6-dichloro-s-triazine.

Properties of these immobilized enzymes were studied both in batchwise operation and in packed bed reactor systems.     

Effects of rosuvastatin on electronegative LDL as characterized by capillary isotachophoresis: the ROSARY Study

Electronegative LDL, a charge-modified LDL (cm-LDL) subfraction that is more negatively charged than normal LDL, has been shown to be inflammatory. We previously showed that pravastatin and simvastatin reduced the electronegative LDL subfraction, fast-migrating LDL (fLDL), as analyzed by capillary isotachophoresis (cITP). The present study examined the effects of rosuvastatin on the more electronegative LDL subfraction, very-fast-migrating LDL (vfLDL), and small, dense charge-modified LDL (sd-cm-LDL) subfractions. Patients with hypercholesterolemia or those who were being treated with statins (n = 81) were treated with or switched to 2.5 mg/d rosuvastatin for 3 months. Rosuvastatin treatment effectively reduced cITP cm-LDL subfractions of LDL (vfLDL and fLDL) or sdLDL (sd-vfLDL and sd-fLDL), which were closely related to each other but were different from the normal subfraction of LDL [slow-migrating LDL (sLDL)] or sdLDL (sd-sLDL) in their relation to the levels of remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) C-II, and apoE. The percent changes in cm-LDL or sd-cm-LDL caused by rosuvastatin were correlated with those in the particle concentrations of LDL or sdLDL measured as LDL-apoB or sdLDL-apoB and the levels of HDL-C, RLP-C, apoC-II, and apoE. In conclusion, rosuvastatin effectively reduced both the vfLDL subfraction and sd-cm-LDL subfractions as analyzed by cITP.     

Grayanoside C,a new diterpene glucoside from Leucothoe grayana

A new diterpene glucoside has been isolated from Leucothoe grayana. Its structure was elucidated by X-ray diffraction analysis of a dehydrated product of the aglycone and conversion of the same to leucothol A.