Intramuscular temperatures during exercise in the heat following pre-cooling and pre-heating

Pre-cooling improves heat tolerance and time to exhaustion in the heat. We tested the possibility that reduced tissue temperatures may explain this phenomenon, using three whole-body treatments: pre-cooling, thermoneutral (control) and pre-heating. Pre-cooling reduced muscle temperature (T_m) by 6.3 °C while pre-heating increased T_m 3.4 °C, relative to control. Despite this offset, T_m climbed towards a common asymptote, with pre-cooling offering no thermal protection beyond 40 min. Following pre-cooling, exercising oesophageal temperature (T_es) initially increased at 0.09 °C min⁻¹, being significantly faster than control (0.05 °C min⁻¹) and pre-heated conditions (0.03 °C min⁻¹). Pre-cooling lowered the sweat threshold and also resulted in a reduced cardiac frequency across the exercise-heat exposure. Our observations do not support the hypothesis that pre-cooling reduces T_m at the end of an exercise-heat exposure, thereby delaying the development of fatigue.     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com     

大麦主栽品种亲缘系数和对叶斑病的抗性分析

为明确我国大麦主栽品种的遗传多样性及其对叶斑病的抗性来源,采用亲缘系数(COP,coefficient of parentage)分析方法对155个主栽大麦品种的遗传系谱进行聚类分析,同时对其中79个供试大麦品种在苗期和成株期分别接种2个强毒性菌株进行抗性鉴定。结果显示,155个品种聚为6个类群,有亲缘关系的品种占全部品种14.77%。在品种间组成的11935个组合中,1763个组合间存在亲缘关系,其COP值变化范围在0~0.7500之间,亲缘系数总和为157.5867,平均值为0.0132。根据系谱分析发现了不同育种单位所育品种的核心亲本,并追溯其主要的祖先亲本。此外,通过对叶斑病的抗性鉴定,发现大多数供试的大麦品种感叶斑病,高抗品种主要集中在垦啤麦系列品种和蒙啤麦3号,部分华大麦和驻大麦系列的品种在苗期或成株期中抗叶斑病。系谱分析及抗性鉴定结果揭示了我国大麦叶斑病抗性基因存在不同来源,分析结果有利于提高抗叶斑病基因筛选效率和缩小筛选范围,也将促进抗叶斑病新基因资源的发掘和利用。     

乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的小鼠免疫应答研究

目的 构建乙型肝炎病毒核心抗原(HBcAg)和Flt3配体(FL)胞外段双表达核酸疫苗,并观察其免疫原性。方法 分别将HBcAg、FL基因克隆入pJW4303载体,获得双表达核酸疫苗,体外转染293T细胞检测目的基因的表达。分组免疫BABL／c小鼠,酶联免疫吸附试验(ELISA)检测小鼠血清抗-HBc IgG效价,酶联免疫斑点试验(ELISPOT)检测HBcAg特异性Th1／Th2型细胞因子的分泌水平。结果 所构建疫苗在体外均能表达HBcAg和FL,当基因位于内部核糖体切入位点(IRES)元件上游时表达水平明显较优。pJW4303／C／FL免疫组产生的抗-HBc IgG效价和Th1／Th2型细胞因子的分泌水平均显著优于pJW4303／C和pJW4303／FL／C组。结论 成功构建双表达核酸疫苗,基因位于上游时表达水平高于下游。FL基因的引入明显增强了HBcAg核酸疫苗的免疫原性。     

Thermoregulatory responses in humans during exercise after exposure to two different light intensities

The effects after exposure to two different light intensities (dim, 50 lx and bright, 5000 lx) on thermoregulatory responses during exercise in a climatic chamber (27 degrees C, 60% relative humidity) were studied in nine untrained female subjects, aged 19-22 years. The subjects were in either the dim or bright light intensities from 0600 hours to 1200 hours. They were then instructed to exercise on a cycle ergometer at an intensity of 60% maximal oxygen uptake from 1200 hours to 1300 hours in a light intensity of 500 Ix. The main results can be summarized as follows. Firstly, exercise-induced increases of core temperature were significantly smaller, after exposure to the bright than after the dim light intensities, although both tests were performed in the same light intensity. Secondly, body mass loss after exercise was significantly greater after exposure to the bright light intensity. Thirdly, an increase in salivary lactic acid during exercise was significantly lower after the bright intensity. Fourthly although the salivary melatonin level was not different between the two light intensities both before and after the exercise, it increased significantly during exercise only after the bright intensity. These results are discussed in terms of the establishment of a lower set-point in the core temperature after exposure to a bright light intensity.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases

The continuous production of chitosan oligosaccharides using a packed-bed enzyme reactor was investigated as to the effects of the operation conditions on the yield of pentamers and hexamers of chitosan oligosaccharides. A column reactor packed with immobilized chitosanases prepared by the multipoint attachment method was used for continuous hydrolysis of chitosan. In this reactor, the decrease of the yield of the target intermediate oligosaccharides due to axial mixing was negligible. The surface enzyme density of the support and flow rate of the substrate solution significantly affected the maximum yield of pentamers and hexamers. These effects were summarized as a correlation with the Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The optimum condition was determined based on Da. Under the optimized condition (Da = 0.12), pentamers and hexamers could be produced continuously for a month with a yield of over 35% (7 kg/m(3) in concentration).     

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.