Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.     

Degradation of L-Ascorbic Acid and Mechanism of Non-enzymic Browning Reaction

In the presence of oxygen, L-ascorbic acid sol ution (0.05 M) browned more intense1 y than dehydro-L-ascorbic acid solution (0.05 M) during storage for longer period.

The mixed solution of L-ascorbic acid (ASA) and dehydro-L-ascorbic acid (DHA) with the ratio of 1:1 or 1:3 in concentration gave more intense browning than DHA solution during storage at 38°C for about 3 weeks. Essentially the same type of browning was observed in case of the mixture of ASA and DHA with D-glucose. Browning of partially oxidized ASA solution also showed substantially the same results as those mentioned above.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Salicylic acid-mediated cell death in the Arabidopsis len3 mutant

The Arabidopsis lesion initiation 3 (len3) mutant develops lesions on leaves without pathogen attack. len3 plants exhibit stunted growth, constitutively express pathogenesis-related (PR) genes, PR-1, PR-2, and PR-5, and accumulate elevated levels of salicylic acid (SA). Furthermore, len3 is a semidominant, male gametophytic lethal mutation with partial defects in female gametophytic development. To determine the signaling pathway activated in len3 plants, we crossed the len3 plants with nahG, npr1-1, and pad4-1 plants and analyzed the phenotypes of the double mutants. The len3-conferred phenotypes, including cell death and PR-1 expressions, were suppressed in the double mutants. Thus SA, NPR1, and PAD4 are required for the phenotypes. However, none of these double mutants could completely suppress the len3-conferred stunted growth. This result suggests that an SA-, NPR1-, and PAD4-independent pathway is also involved in the phenotype. Treatment with BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid), an SA analog, induced cell death in len3 nahG plants but not in len3 npr1 or len3 pad4 plants, suggesting the involvement of the PAD4-dependent but SA-independent second signal pathway in cell death in len3 plants.     

Biosynthesis of poly(ɛ- l-lysine)s in two newly isolated strains of Streptomyces sp.

The biosynthesis of poly(ɛ-l-lysine) (ɛ-PL) in the two newly isolated strains of Streptomyces lydicus USE-11 (USE-11) and Streptomyces sp. USE-51 (USE-51) was studied by a newly developed two-stage culture method of cell growth at pH 6.8 and ɛ-PL production at pH 4.5. USE-11 synthesized ɛ-PL consisting of about 28 residues at a high production level, whereas USE-51 did the polymer with 15 ones at a low level. The secreted ɛ-PLs in culture media were digested in a neutral pH range with a peptide hydrolase(s) produced by the ɛ-PL producers. The optimum production levels were presumed to be dependent upon the inherent ɛ-PL synthesis machinery of each producer. The production in USE-51 was sharply dependent upon cell density as was often observed in the production of antibiotics, whereas that in USE-11 was scarcely affected by the density. The was found to be essential for the ɛ-PL production in both strains. This might suggest the involvement of a thiol group in the polymerization reactions including the activation of l-lysine. This study indicates that USE-11 is a most suitable strain for the exploration of the ɛ-PL biosynthesis at the molecular level as well as for the technical applications.Electronic supplementary material Electronic supplementary material is available if you access this article at and is accesible for authorized users.An erratum to this article can be found at     

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris-HCl, 7 mm 2-mercaptoethanol, 7 mm MnCl₂ and 50 mm NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5′-GACGTC-3′, cuts between T and C and produces a 3′-tetranucleotide extension in the presence of MnCl₂, as it does in the presence of MgCl₂.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.