Cerebral Blood Flow Threshold and Regional Heterogeneity of Heat Shock Protein 72 Induction Following Transient Forebrain Ischemia in Rats

Heat shock proteins (HSPs) induced by brain ischemia may play an important role in neuroprotection from neuronal degeneration. In this study, we examined the cerebral blood flow (CBF) threshold to produce regional differences in HSP72 induction after transient forebrain ischemia in spontaneously hypertensive rats (SHRs). Female SHRs were subjected to 20 min of cerebral ischemia induced by bilateral carotid artery occlusion. The CBF was measured by laser Doppler flowmetry. At forty-eight hours after cerebral ischemia and reperfusion, the rats were decapitated and the brains were removed. Specific areas (hippocampal CA1, CA2-3, dentate gyrus, dorsolateral and ventromedial striatum, and parietal cortex) were thereafter dissected from the brain. The amounts of HSP72 in these samples were determined using Western blot analysis. In the hippocampus, HSP72 was induced when the CBF decreased to less than 18–25% of the resting level. The mean values of HSP72 produced in the CA1 area, CA2-3 area, and the dentate gyrus following ischemia and reperfusion treatment were 4.44 ± 1.43 (±SD) ng/g prtein, 3.51 ± 0.72 ng/g protein and 3.77 ± 1.05 ng/g protein, respectively. In the parietal cortex, the amount of HSP72 induction was less pronounced (2.55 ± 0.40 ng/g protein), while HSP72 was hardly detected at all in the striatum, even under conditions of very severe CBF reduction and reperfusion. We demonstrated the existence of both a CBF threshold (i.e., approximately 20% of the resting level) for HSP72 induction and regional heterogeneity for the induction of HSP72 protein.     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Anti-fungal sesquiterpenoid from the root exudate of Solanum abutiloides

The Solanum abutiloides plant is highly resistant to soil-borne pathogens such as Fusarium oxysporum f. sp. melongenae, Verticillium dahliae, and Ralstonia solanacearum. This species is utilized as a mating source of resistant cultivars and is also used as a rootstock. The root exudate of Solanum abutiloides was extracted from a soil system composed of charcoal and vermiculite. Anti-fungal activity was found in the extract, and an active ingredient was isolated. The chemical structure of the active compound was determined to be 3-beta-acetoxysolavetivone, a new sesquiterpenoid. The anti-fungal activity of 3-beta-acetoxysolavetivone examined by the inhibition of spore germination of Fusarium oxysporum was close to that of lubimin, and higher than that of solavetivone.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.     

Antibacterial activity of hydrolyzable tannins derived from medicinal plants against Helicobacter pylori

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time- and dose-dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN-28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose-dependent membrane-damaging activity. However, it remains to be elucidated whether their membrane-damaging activity directly contributes to their antibacterial action.     

Plant-growth inhibitory activity of heliannuol derivatives

In addition to (+)-, (-)- and (+/-)-heliannuol E, growth-inhibitory activities of five synthetic chromans and four tetrahydrobenzo[b]oxepins were examined against oat and cress. All heliannuol E isomers exhibited similar biological activities against cress, whereas when tested against oat roots, the unnatural optical isomer (+) showed no inhibitory activity. Four brominated chromans and two tetrahydrobenzo[b]oxepin derivatives also showed apparent inhibition against both cress and oat.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

Development of an efficient enzymatic production of gamma-D-glutamyl-L-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial gamma-glutamyltranspeptidase

Gamma-D-Glutamyl-L-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial. Because gamma-D-glutamyl-L-tryptophan has several reactive groups in its molecule, consists of D- and L-amino acids, and is connected by gamma-glutamyl linkage, its chemical synthesis is complicated. An efficient enzymatic method to synthesize gamma-D-glutamyl-L-tryptophan from D-glutamine and L-tryptophan employing bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 50 mM D-glutamine, 50 mM L-tryptophan, and 0.2 U ml(-1) gamma-glutamyltranspeptidase, pH 9-9.5, and incubation at 37 degrees C for 5 h. After a 5 h incubation, 33 mM gamma-D-glutamyl-L-tryptophan was obtained, the conversion rate being 66%. The product was purified by Dowex 1 x 8 column and was considered to be gamma-D-glutamyl-L-tryptophan.