类缺血等多种预处理上调脂多糖应答基因在神经元中的表达

目的:研究体外和体内多种预处理对小鼠神经元细胞内脂多糖应答基因(LRG)表达的影响。方法:利用qRT-PCR和western blot方法检测体外多种预处理(类缺血、高压氧、异氟和脂多糖)对小鼠神经元细胞中LRG的转录水平和蛋白表达水平的影响;运用小RNA干扰方法(siRNA)沉默神经元细胞中LRG的表达,噻唑蓝(MTT)方法检测神经元细胞的生长情况;运用大脑中动脉阻闭法(MCAO)建立多种预处理诱导脑缺血耐受的小鼠试验模型,qRT-PCR和western blot方法检测小鼠脑组织中LRG转录水平和蛋白水平的变化。结果:体外多种预处理明显上调LRG在小鼠脑海马神经元细胞中的表达;沉默LRG在神经元中的表达明显加快类缺血处理细胞的死亡;多种预处理建立的脑缺血耐受小鼠的脑组织中LRG转录水平和蛋白表达水平明显升高。结论:类缺血等多种预处理可上调神经元细胞内LRG的表达,但其在脑缺血再灌注损伤中的作用机制尚不清楚,需进一步的研究。     

Fungal to bacterial biomass ratio in the forests soil profile

The carbon content in microbial biomass (C_mic-MB) was determined in various horizons of the soil profile (sod-podzo, gray, podzol, and rzhavozem) of various forests (oak, spruce archangel, spruce moss, aspen, spruce broadleaf) in the southern taiga of European Russia (Moscow and Kaluga regions) by the substrate-induced respiration (SIR) and direct microscopy (DM) methods. The fungi-to-bacteria ratio was measured by the selective inhibition technique and DM. A quantitative differentiation of the fungal mycelium was suggested. The C_mic-DM / C_mic-SIR in various horizons of the soil profile was about 98%. The fungal contribution to MB was 52–74% and 92–99% according to the SIR and DM methods, respectively. The microbial parameters were associated with the CO₂ and N₂O production by the soils. The contradictory data about the fungal portion in the MB of soils of various ecosystems were discussed.     

Efficient phosphorus solubilization by mutant strain of Xanthomonas campestris using different carbon, nitrogen and phosphorus sources

The phosphate solubilization activity of Xanthomonas campestris was measured in both the wild type and mutant strains using various carbon and nitrogen sources. Glucose was found to be the best in both (wild 39.9%; mutant 67.1%) strains followed by sucrose (46.8%) in the mutant and molasses (36.0%) in the wild type. Ammonium sulphate was the best nitrogen source for both the strains, followed by ammonium nitrate and urea. Dicalcium phosphate (DCP) was solubilized maximally by both the strains followed by tricalcium phosphate (TCP) and rock phosphate (RP) when various concentrations of different phosphate sources were tested.     

Theoretical study of the complementarity in halogen–bonded complexes involving nitrogen and halogen as negative sites

The conformations of trialkylphosphates (alkyl = propyl, butyl, pentyl and hexyl) in various diluents were studied by molecular dynamics simulations. The population density of various conformers of trialkylphosphate in different diluents such as water and n-dodecane was determined. The Helmholtz energy change accompanied by the transition between various conformations was computed. The aggregation behavior of tributylphosphate in water and water-dodecane medium was studied.     

Maillard reaction rate in various glassy matrices

The Maillard Reaction (MR) rate below the glass transition temperature (T(g)) for various model glassy food systems was studied at temperatures between 40 degrees C and 70 degrees C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T(g) was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD(280)), and the MR rate (k(280)) was determined as a pseudo zero order reaction rate from the time course of OD(280). Although k(280) was described by the Arrhenius plot, the temperature dependence of k(280) was almost the same and the intercept was different among the matrices. From the comparison of k(280), it was suggested that the MR rate in glassy matrix was affected not only by the T(g), but also by the hydrogen bonding between MR reactants and glassy matrix.     

Baculovirus expression of mouse lactoferrin receptor and tissue distribution in the mouse

Lactoferrin (Lf) has been shown to have a role in the immune system and in early development of the mouse embryo. A specific receptor for Lf has been suggested to mediate the functions of Lf. We have recently identified a Lf receptor (LfR) in human fetal small intestine. We therefore hypothesized that the mouse homologue of this protein functions as a LfR. We expressed mouse Lf (MLf) and the mouse homologue (MLfR) in a baculovirus-insect cell system. The recombinant MLfR (rMLfR) was purified by immobilized recombinant MLf (rMLf) affinity chromatography, demonstrating an interaction between rMLf and the rMLfR. RT-PCR revealed that MLfR was expressed in various tissues and during embryonic development. Immunohistochemical analysis revealed that the MLfR was localized in various tissues including small intestinal epithelium, stomach, kidney, ovary, and various regions of brain. In summary, the MLfR functions as a receptor for MLf, is expressed and localized in various tissues, and may be involved in the indispensable function of MLf during early embryonic development.     

Transglycosylation and reverse hydrolysis reactions of endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EGCase: EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here transglycosylation and reverse hydrolysis reactions of EGCase from the jellyfish Cynaea nozakii. Various alkyl-GM1 oligosaccharides (alkyl-II(3)NeuAcGgOse4) were synthesized when GM1 ganglioside was treated with the EGCase in the presence of 1-alkanols. Among various 1-alkanols tested, methanol was found to be the most preferential acceptor, followed by 1-hexanol and 1-pentanol. GM1 was the best donor, followed by GD1b and GT1b, when methanol was used as an acceptor. However, neither globoside nor glucosylceramide was utilized by the enzyme as a donor substrate. The enzyme transferred oligosaccharides from various glycosphingolipids to NBD-ceramide, a fluorescent ceramide, producing NBD-labeled glycosphingolipids. In addition to the transglycosylation reaction, the enzyme catalyzed the reverse hydrolysis reaction; lactose was condensed to ceramide to generate lactosylceramide in the presence of the enzyme. These results indicate that the jellyfish enzyme will facilitate the synthesis of various neoglycoconjugates and glycosphingolipids.     

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Reactive extraction of succinic acid with tripropylamine (TPA) in various diluents

Succinic acid is of interest as the raw material of polysuccinate which is a biodegradable polymer. The tripropylamine (TPA) was used as the extraction agent for the reactive extraction of succinic acid in various alcohol diluents. Equilibrium and loading data for various active diluents were obtained at various TPA concentrations. At the same TPA concentration the extractabilities of TPA increased as the number of carbon in alcohols decreased. It was explained by decreasing solubility of acid-amine complex due to decreasing polarity with increasing the number of carbon in alcohols. The type of succinic acid-amine-diluent complex was predicted by the value of loading with various concentration of TPA/various alcohols.     

The flavonoid baicalin counteracts ischemic and oxidative insults to retinal cells and lipid peroxidation to brain membranes

The purpose of the present study was to determine whether the flavonoid, baicalin is effective at blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of various insults to a transformed retinal ganglion cells (RGC-5 cells) in culture. Baicalin was administered intraperitoneally just before and after an ischemic insult to retina of one eye of a rat. Ischemia was delivered by raising the intraocular pressure above the systolic blood pressure for 50min. Seven days after ischemia, retinas were analysed for the localisation of various antigens. Retinal extracts were also analysed for various mRNAs. Moreover, the content of specific proteins was deduced in retinal and optic nerve extracts. Also, RGC-5 cells in culture were given one of three different insults, light (1000lx for 2 days), hydrogen peroxide (200microM H(2)O(2) for 24h) or serum deprivation (48h) where cell survival and reactive oxygen species (ROS) formation was assayed. Moreover, a lipid peroxidation assay was used to compare the antioxidant capacity of baicalin with the flavonoid, epigallocatechin gallate (EGCG). Ischemia/reperfusion to the retina affected the localisation of Thy-1 and choline acetyltransferase (ChAT) and the content of various proteins (optic nerve and retina) and mRNAs (retina). Importantly, baicalin statistically blunted most of the effects induced by ischemia/reperfusion. Only the increase in caspase-8 and caspase-3 mRNAs caused by ischemia/reperfusion were unaffected by baicalin treatment. Baicalin also attenuated significantly the negative insult of light, hydrogen peroxide and serum withdrawal to RGC-5 cells. In the lipid peroxidation studies, baicalin was also found to be equally effective as EGCG to act as an antioxidant. Significantly, the negative insult of serum withdrawal on RGC-5 cell survival was blunted by baicalin but not by EGCG revealing the different properties of the two flavonoids.     

Characterization of<Emphasis Type="Italic">Candida albicans</Emphasis> colony-morphology mutants and their hybrids

The collection wild-type strain of Candida albicans was used to obtain auxotrophic and colony-morphology mutants by 'nitrosoguanidine' treatment. Intraspecific protoplast fusion induced by Ca(2+)-poly(ethyleneglycol) was carried out in various pairings between the auxotrophic strain producing smooth colonies and containing blastospores and the colony-morphology mutants containing a mixture of blastospores and pseudohyphae or only hyphae. Hybrids exhibiting full or partial complementation were obtained when mutants producing smooth colonies and colony-morphology variants of different origins were fused. The mutation responsible for the colony-morphology character (if various types of colony morphomutants were crossed) proved to be recessive or semidominant. Representative hybrids exhibited elevated DNA contents as measured by flow cytometry. To illustrate various cell types, and especially the intermediate one (never observed in natural isolates), a preparation method was further developed for scanning electron microscopic studies.     

Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine

Thrombin, 1-oleoyl-2-acetyl-rac-glycerol (OAG), cis- or trans-octadecadienoic acids (linoleic and linolelaidic acid) and the synergistic combination of octadecadienoic acids plus OAG lead to the activation of gel-filtered human platelets, i.e. aggregation via protein kinase C (PKC). Platelet activation by thrombin was only slightly suppressed by polymyxin B, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporine, all being potent inhibitors of PKC in vitro. The OAG-induced aggregation, however, was strongly inhibited by H-7 or staurosporine but not by polymyxin B. In contrast, octadecadienoic acid-induced aggregation was substantially inhibited only by polymyxin B. Synergistic activation by OAG plus octadecadienoic acids was strongly suppressed by all three PKC inhibitors. Our results indicate (1) that the ability of various compounds to inhibit PKC in vitro does not correlate with their inhibitory effects in intact cells and (2) that platelet activation induced by various PKC activators exhibits differential PKC-inhibitor sensitivity.     

Rapid quantifiable assessment of nutritional parameters influencing pediocin production by Pediococcus acidilactici NRRL B5627

A direct plate bioassay procedure was applied for rapid and quantifiable assessment of the influence of various nutritional parameters on pediocin production by Pediococcus acidilactici NRRL B5627. Solid-state cultivation of the microorganism was done on MRS-based media over 3-and 6-hours incubation periods. Nutritional parameters assessed included the carbon source (glucose, sucrose, fructose, galactose, glycerol), and various salts (NH(4)PO(4), CaCl(2), KH(2)PO(4), MnSO(4).H(2)O). Glucose was found to be the optimal carbon source while glycerol exhibited the most suppressive effect. Using glucose as the carbon source, addition of various salts, in amounts used in liquid media commonly applied in the cultivation of the pediococci, was assessed with respect to bacteriocin production on a per cell basis. Experimental data obtained showed that several nutritional parameters repress pediocin production by P. acidilactici, while the direct plate assay proved to be a good pilot assay prior to conducting more intensive kinetic analysis in liquid cultivation.     

Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineurin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn2+ as the tightly bound metal.     

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.     

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.     

Conditions Permitting Solid Tumor Formation of Teratocarcinoma Cells in Allogeneic Mice

In this report, the growth capacity of murine ascitic teratocarcinomas (embryoid bodies, EBs) was studied in certain strains of allogeneic mice. When EB was intravenously injected into Balb/c mice, various types of tumor colonies were formed with various types of differentiated tissues in the lung, as in the case of syngeneic 129/Sv mice. Tumor colonies in the abdomen of Balb/c and other allogeneic mice formed by the intraperitoneal injection of EB, however, contained merely undifferentiated embryonal carcinoma cells (EC cells) and sometimes some primitive tissues, too. Analysis of lung colonies (LCs) from EBs in Balb/c mice indicated that EC cells are a potent target for being rejected but they can escape this rejection by differentiating into various types of tissues cells.     

Solubilization and Characterization of an Acyl-Coenzyme A : O-LYSOPHOSPHOLIPID ACYLTRANSFERASE FROM THE MICROSOMES OF DEVELOPING SAFFLOWER SEEDS

Acyl-CoA:O-lysophospholipid acyltransferase activity was measured in extracts of developing safflower (Carthamus tinctorius var. UC-1) seeds. Acyltransferase activity was present in all subcellular fractions. The microsomal acyltransferase activity was solubilized with either 30 mm-n-octylglucoside, 1% deoxycholate, or 100 mum lysophosphatidylcholine. The pH optimum of the lysophosphatidylcholine-dependent acyltransferase activity was at 9.0. Among the various acyl-CoA's tested, oleoyl-CoA was the best substrate and with it the enzyme had a K(m) of 9.5 mum. Among the various acyl acceptors tested, lysophosphatidylcholine (oleoyl) gave the highest activity and the optimal concentration was 30 mum. Acyltransferase activity was stimulated by ethanol and inhibited by bovine serum albumin and divalent cations.     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).