Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974,Biochimie54, 907–924). When carbonylcyanide-p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide-p-trifluoromethoxyphenylhydrazone.     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

Interactions among morphotype,nutrition, and temperature impact fitness of an invasive fly

Invasive animals depend on finding a balanced nutritional intake to colonize, survive, and reproduce in new environments. This can be especially challenging during situations of fluctuating cold temperatures and food scarcity, but phenotypic plasticity may offer an adaptive advantage during these periods. We examined how lifespan, fecundity, pre‐oviposition periods, and body nutrient contents were affected by dietary protein and carbohydrate (P:C) ratios at variable low temperatures in two morphs (winter morphs WM and summer morphs SM) of an invasive fly, Drosophila suzukii. The experimental conditions simulated early spring after overwintering and autumn, crucial periods for survival. At lower temperatures, post‐overwintering WM lived longer on carbohydrate‐only diets and had higher fecundity on low‐protein diets, but there was no difference in lifespan or fecundity among diets for SM. As temperatures increased, low‐protein diets resulted in higher fecundity without compromising lifespan, while high‐protein diets reduced lifespan and fecundity for both WM and SM. Both SM and WM receiving high‐protein diets had lower sugar, lipid, and glycogen (but similar protein) body contents compared to flies receiving low‐protein and carbohydrate‐only diets. This suggests that flies spend energy excreting excess dietary protein, thereby affecting lifespan and fecundity. Despite having to recover from nutrient depletion after an overwintering period, WM exhibited longer lifespan and higher fecundity than SM in favorable diets and temperatures. WM exposed to favorable low‐protein diet had higher body sugar, lipid, and protein body contents than SM, which is possibly linked to better performance. Although protein is essential for oogenesis, WM and SM flies receiving low‐protein diets did not have shorter pre‐oviposition periods compared to flies on carbohydrate‐only diets. Finding adequate carbohydrate sources to compensate protein intake is essential for the successful persistence of D. suzukii WM and SM populations during suboptimal temperatures.     

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.     

556例多发伤患者的急诊救治临床分析

目的探讨多发伤患者的救治策略。方法回顾分析我科2000年1月至2008年5月急诊抢救的556例多发伤患者的临床资料。结果 16例患者经抢救无效死亡,死亡率2.88%;其余患者均经紧急抢救及行必要实验室检查,病情稳定,好转率达97.12%。平均抢救时间为(1.37±1.05)h。结论强化多发伤的急诊科早期救治,树立创伤急救"黄金1 h"观念,是提高多发伤患者生存率及降低死亡率的关键。     

Multiple loci are associated with white blood cell phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.     

Life history and host range of the leaf blotcher Eucosmophora schinusivora: a candidate for biological control of Schinus terebinthifolius in the USA

The host range of Eucosmophora schinusivora Davis and Wheeler (Lepidoptera: Gracillariidae) was studied to assess its suitability as a biological control agent of Schinus terebinthifolius Raddi (Anacardiaceae), a serious environmental and agricultural weed in the USA and elsewhere in the world. The life history of this insect species and its host range were determined in the laboratory with adult no-choice oviposition and larval development tests. This species has five instars, the first three are sap-feeding miners and the last two are tissue feeding. Total development time was 31.7 days. To examine specificity of this species, 10 plant species in Anacardiaceae were selected based on taxonomic relatedness to S. terebinthifolius, economic importance, and availability. In the laboratory, except for Anacardium occidentale and Cotinus obovatus, all of the tested species were accepted for oviposition with a marked preference for the weed S. terebinthifolius, Schinus molle, Rhus copallinum, Rhus sandwicensis and Pistacia chinensis. Complete development, from egg to adult, was achieved only on S. terebinthifolius, S. molle, R. copallinum, P. chinensis and Metopium toxiferum. In conclusion, E. schinusivora will not be considered for the biological control of S. terebinthifolius in the continental USA. However, the utilisation of this species in other infested areas such as Hawai'i and Australia should be considered.     

Sporogenesis in Eusporangiate Ferns: II. Polyplastidic Meiosis in Ophioglossum (Ophioglossales)

Ophioglossum petiolatum . Unlike Angiopteris (Marattiales), which is monoplastidic, Ophioglossum undergoes polyplastidic meiosis like members of the fern-seed plant clade. The meiotic spindle is distinctly multipolar in origin and is consolidated into a bipolar spindle that is variously twisted and curved to accommodate the large number of chromosomes. Although a phragmoplast forms after first meiosis, no wall is deposited. Instead, an organelle band consisting of intermingled plastids and mitochondria is formed in the equatorial region between the dyad domains. Following second meiosis, a complex of phragmoplasts forms among sister and non-sister nuclei. Cell plates are deposited first between sister nuclei and then in the region of the organelle band resulting in a tetrad of spores each with a equal allotment of organelles. Received 30 January 2001/ Accepted in revised form 24 April 2001