Juxtamembrane basic residues in glycoprotein Ibbeta cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex

Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149-154 in the cytoplasmic domain of the GPIbbeta subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbbeta by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbbeta cytoplasmic domain in biosynthesis of the GPIb-IX complex.     

Assessing side-chain perturbations of the protein backbone: a knowledge-based classification of residue Ramachandran space

Grouping the 20 residues is a classic strategy to discover ordered patterns and insights about the fundamental nature of proteins, their structure, and how they fold. Usually, this categorization is based on the biophysical and/or structural properties of a residue's side-chain group. We extend this approach to understand the effects of side chains on backbone conformation and to perform a knowledge-based classification of amino acids by comparing their backbone phi, psi distributions in different types of secondary structure. At this finer, more specific resolution, torsion angle data are often sparse and discontinuous (especially for nonhelical classes) even though a comprehensive set of protein structures is used. To ensure the precision of Ramachandran plot comparisons, we applied a rigorous Bayesian density estimation method that produces continuous estimates of the backbone phi, psi distributions. Based on this statistical modeling, a robust hierarchical clustering was performed using a divergence score to measure the similarity between plots. There were seven general groups based on the clusters from the complete Ramachandran data: nonpolar/beta-branched (Ile and Val), AsX (Asn and Asp), long (Met, Gln, Arg, Glu, Lys, and Leu), aromatic (Phe, Tyr, His, and Cys), small (Ala and Ser), bulky (Thr and Trp), and, lastly, the singletons of Gly and Pro. At the level of secondary structure (helix, sheet, turn, and coil), these groups remain somewhat consistent, although there are a few significant variations. Besides the expected uniqueness of the Gly and Pro distributions, the nonpolar/beta-branched and AsX clusters were very consistent across all types of secondary structure. Effectively, this consistency across the secondary structure classes implies that side-chain steric effects strongly influence a residue's backbone torsion angle conformation. These results help to explain the plasticity of amino acid substitutions on protein structure and should help in protein design and structure evaluation.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Phosphorus limitation on photosynthesis of two dominant understory species in a lowland tropical forest

Aims Elevated nitrogen (N) deposition in tropical regions may accelerate ecosystem phosphorus (P) limitation. However, it is not explicitly addressed that how changes in soil N and P availability affect foliar nutrients and photosynthesis of plants in tropical forests. In this study, we examined the effects of N and P additions on foliar nutrients and net photosynthesis of two dominant understory species, Randia canthioides (R. canthioides) and Cryptocarya concinna (C. concinna) in an N-saturated old-growth tropical forest (>400-year-old) in southern China.Methods A full factorial NP addition experiment (2×2) was established in 2007 and continued through August 2010. Four treatments, including control, N addition (150kg N ha^-1 year^-1), P addition (150kg P ha^-1 year^-1) and NP addition (150kg N ha^-1 year^-1 plus 150kg P ha^-1 year^-1) were set up in this experiment. Photosynthetic traits (maximum photosynthetic CO₂ assimilation (A max), stomatal conductance (g s), leaf transpiration (E), light saturating point, concentrations of chlorophyll a/b and foliar nutrients (N and P) of the two species were measured with standard methods.Important findings Three years of N addition had no significant effects on any measured photosynthetic parameter of either species. However, N addition significantly elevated foliar N and P concentrations of one species (R. canthioides), resulting in lower photosynthetic nitrogen use efficiency (PNUE). N treatments decreased foliar P concentration of the other (C. concinna), resulting in increased photosynthetic phosphorus use efficiency, which was potentially related to N-induced P shortage. In contrast, positive effects of P treatments on g s of R. canthioides, A max and chlorophyll a+b of C. concinna were observed. P treatments also elevated foliar P and PNUE of both species, implying P induced more efficient use of N. Our results suggested a more important role of P than N on influencing photosynthetic traits of these two understory species. Alleviation of P shortage through P addition may enhance photosynthetic performances of some understory species in N-rich tropical forests.     

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp

A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.An erratum to this article can be found at     

Transgenic Plants of Creeping Bent Grass Harboring the Stress Inducible Gene, 9-cis-epoxycarotenoid dioxygenase, are Highly Tolerant to Drought and NaCl Stress

Transgenic lines of creeping bent grass were generated by Agrobacterium-mediated transformation with the VuNCED1 which was cloned from cow pea has a homology to 9-cis-epoxycarotenoid dioxygenase, which is supposed to be involved in abscisic acid (ABA) biosynthesis. ABA, a cleavage product of carotenoids, is involved in stress responses in plants. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. Molecular analyses of transgenic lines as performed by Southern hybridization genomic DNA-PCR revealed integration of the VuNCED1. Challenge studies performed with transgenic plants by exposure to salt stress (up to 10 dS m⁻¹) and water stress (up to 75%) for 10 weeks, revealed that more than 50% of the transgenic plants could survive NaCl and drought stress whereas wild-type was not. ABA levels were measured under drought and normal conditions, endogenous ABA was dramatically increased by drought and NaCl stress in transgenic plants. These results indicate that it is possible to manipulate ABA levels in plants by over expressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. Chenna Reddy Aswath and Sun Hyung Kim - First two authors contributed equally to this work     

Endocrine and exocrine secretion of leptin by the gastric mucosa.

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.     

牛血清白蛋白对小鼠原核期胚胎玻璃化冷冻的影响

以小鼠原核期胚胎为对象,以胚胎的存活率、卵裂率、囊胚率以及囊胚细胞数作为检测指标,在M2液的基础上添加8种浓度(0,2,4,8,16,32,64,96mg/mL)牛血清白蛋白(BSA)配置防冻液,探讨防冻液和玻璃化冷冻后对胚胎发育的影响。BSA防冻液对胚胎发育影响的实验结果表明,8个浓度组间以及与对照组间胚胎的卵裂率、囊胚率以及囊胚细胞数无显著差异(P>0.05),说明在防冻液中加入一定浓度的BSA对小鼠胚胎无毒性作用。防冻液经玻璃化冷冻后对胚胎发育影响的实验表明,8个浓度组间冷冻胚胎复苏后的存活率、卵裂率、囊胚率及囊胚细胞数无显著差异(P>0.05)。表明BSA在这种防冻液中没有明显的保护作用。从经济、实用、生物安全角度考虑,不支持在玻璃化防冻液中添加BSA。     

眼镜蛇及竹叶青蛇咬伤与心肌酶谱研究

目的 探讨眼镜蛇和竹叶青蛇咬伤后心肌酶谱改变的关系,以提高蛇伤的诊治水平.方法 选择在我科住院治疗的眼镜蛇咬伤病人124例及竹叶青蛇咬伤86例,根据其肢体肿胀程度分为轻度、中度、重度3组,所有病人在入院时均查心肌酶谱及心电图,心肌酶谱中还增加了肌红蛋白(Mb)、抗心肌钙蛋白T(cTnT)、肌钙蛋白I(cTnI)检测参数.并对实验室检测的心肌酶谱进行统计分析.结果 124例眼镜蛇咬伤病人中,心肌酶谱异常者占87.8%;86例竹叶青蛇咬伤病人中,心肌酶谱异常者占79.4%.中度肿胀组和重度肿胀组心肌酶谱异常发生率为100%;中度组与轻度组比较,心肌酶谱各项指标明显升高(P＜0.05);重度组与轻度组比较,心肌酶谱各项指标更显著(P＜0.01).眼镜蛇咬伤中、重度肿胀组,其心电图改变有显著性差异(P＜0.05),但竹叶青蛇伤组,心电图变化无特殊临床意义.结论 我国南方常见的眼镜蛇及竹叶青蛇咬伤,心肌酶谱变化显著,且升高与受伤的严重程度呈正相关,可作为诊断和观察病情的敏感指标.