Cellular localization of facilitated glucose transporter 1 (GLUT-1) in the cochlear stria vascularis: its possible contribution to the transcellular glucose pathway

Immunoreactivity for the facilitated glucose transporter 1 (GLUT-1) has been found in the cochlear stria vascularis, but whether the strial marginal cells are immunopositive for GLUT-1 remains uncertain. To determine the cellular localization of GLUT-1 and to clarify the glucose pathway in the stria vascularis of rats and guinea pigs, immunohistochemistry was performed on sections, dissociated cells, and whole-tissue preparations. Immunoreactivity for GLUT-1 in sections was observed in the basal side of the strial tissue and in capillaries in both rats and guinea pigs. However, the distribution of the positive signals within the guinea pig strial tissue was more diffuse than that in rats. Immunostaining of dissociated guinea pig strial cells revealed GLUT-1 in the basal cells and capillary endothelial cells, but not in the marginal cells. These results indicated that GLUT-1 was not expressed in the marginal cells, and that another isoform of GLUT was probably expressed in these cells. Three-dimensional observation of whole-tissue preparations demonstrated that cytoplasmic prolongations from basal cells extended upward to the apical surface of the stria vascularis from rats and guinea pigs, and that the marginal cells were surrounded by these protrusions. We speculate that these upward extensions of basal cells have been interpreted as basal infoldings of marginal cells in previous reports from other groups. The three-dimensional relationship between marginal cells and basal cells might contribute to the transcellular glucose pathway from perilymph to intrastrial space. This study was supported by a grant-in-aid for scientific research (19570058) from The Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

FasL shedding is reduced by hypothermia in experimental stroke

Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor-mediated pathways, such as that triggered by Fas/FasL. Male Sprague-Dawley rats subjected to 2 h middle cerebral artery occlusion with 2 h intraischemic mild hypothermia (33°C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by ∼ 50–60% at 6 and 24 h post-insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by ∼ 50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane-bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.     

Identification of the extracellular polysaccharide produced by the snow mold fungus Microdochium nivale

A water-insoluble, extracellular polysaccharide was isolated from the culture medium of the snow mold fungus, Microdochium nivale, that had been cultivated in potato/dextrose broth. The polysaccharide consisted of glucose only. Its Fourier transform infrared spectrum showed a beta configuration of the C1 position of glucose. Linkage analysis of the polysaccharide showed that it had a linear structure of -(14)-linked glucose. The polysaccharide was therefore identified as cellulose. This is the first report of extracellular cellulose occurring in fungi.     

Phospholipase A2 products retain a neuron specific gamma isoform of PKC on the plasma membrane through the C1 domain--a molecular mechanism for sustained enzyme activity

To clarify molecular mechanism for sustained activation of gamma protein kinase C (gammaPKC), a neuron-specific subtype, we investigated the involvement of phospholipase A2 (PLA2) products in the membrane association of gammaPKC upon activation of G protein coupled purinoceptors in CHO-K1 and NG 108-15 cells. In addition, the functional domain responsible for PLA2-product mediated retention of gammaPKC on the plasma membrane was determined by simultaneously monitoring two different fluorescence-tagged gammaPKCs and mutants in the same living CHO-K1 cells. Purinoceptor activation by UTP induced a transient translocation of gammaPKC from the cytoplasm to the plasma membrane. Interestingly, PLA2 inhibitors, bromoenol lactone (BEL) and arachidonyl-trifluoromethyl ketone (AACOF3), shortened the retention time of gammaPKC on the plasma membrane in cells treated with UTP, while a DAG kinase inhibitor did not affect it. The C1 domain deficient mutant (DeltaC1-gammaPKC) also showed short membrane association compared with wild type gammaPKC, when cells are treated with UTP or arachidonic acid (AA) plus a Ca(2+) ionophore. However, deletion of C1A or C1B subdomains (DeltaC1A-gammaPKC or DeltaC1B-gammaPKC) did not alter the retention time on the plasma membrane, whereas PLA2 inhibitor shortened the retention times of both mutants. These results indicate that PLA2 products prolong the retention of gammaPKC on the plasma membrane through the C1A and/or C1B subdomain in purinoceptor-stimulated CHO-K1 cells. The importance of PLA2 product and C1 domain for the retention of gammaPKC on the membrane was also confirmed using neuronal cell line, suggesting that these are part of molecular machinery for sustaining enzyme activity in neurons.     

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma

Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34, 840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.