Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Characterization of phosphoinositide kinases in normal and sickle anaemia red cells

PtdIns and PtdInsP kinases from normal erythrocyte (AA) membranes and sickle cell anaemia erythrocyte (SS) membranes have been characterized. PtdIns kinase was studied in native membranes under conditions in which PtdInsP kinase and PtdInsP phosphatase do not express any activity. Kinetic analysis of the AA and SS PtdIns kinases indicate similar K_m values for PtdIns and ATP but higher V_max values for SS PtdIns kinase. PtdInsP kinase was partially purified from erythrocyte ghosts by NaCl extraction. The kinetic parameters of PtdInsP kinase determined under these conditions were similar in AA and SS NaCl extracts. These data suggest the presence of some effector of PtdIns kinase in SS cell membranes, resulting in a greater activity of the enzyme. This leads consequently, to increase the PtdInsP pool and to activate PtdInsP kinase, in agreement with our previous observations of a greater [³²P]Pi incorporation in both polyphosphoinositides in SS cells relatively to AA cells.     

猪链球菌2型Orf207基因缺失菌株的构建及其生物学特性检测

【背景】猪链球菌2型(streptococcus suis type 2, SS2)可引起人、猪的脑膜炎、关节炎及败血症等,不仅给养猪业带来巨大的经济损失,同时严重威胁公共卫生安全。本团队前期通过噬菌体展示文库技术发现Orf207编码蛋白可能参与SS2诱导的脑膜炎发生,然而其在SS2致病过程中的具体作用尚不清楚。【目的】探究Orf207基因对SS2致病性的影响。【方法】采用温敏性自杀质粒介导的同源重组系统,构建SC19 Orf207基因缺失菌株ΔOrf207及其回补菌株CΔOrf207,系统比较缺失菌株与野生株间在生长特性、形态、组织定殖能力、毒力情况、细胞黏附与侵袭及抗巨噬细胞吞噬能力等生物学特性方面的差异。【结果】与野生株相比,缺失菌株链长变短,生长速度略慢;而且Orf207缺失显著增加了小鼠的存活率,降低了细菌在血液、心脏、肝脏、脾脏、肺脏、肾脏、脑组织的定殖能力和对肺组织的病理损伤并显著减弱SS2对HeLa细胞的黏附与侵袭能力及抗巨噬细胞吞噬能力。【结论】Orf207基因可以显著降低SS2对宿主的致病能力,本研究结果不仅丰富了SS2的致病机制,也为SS2疫苗等研发提供了新靶点。     

Induction of small-segment-translocation between wheat and rye chromosomes

A new approach to produce wheat-rye translocation, based on the genetic instability caused by monosomic addition of rye chromosome in wheat, is described. 1 283 plants from the selfed progenies of monosomic addition lines with single chromosome of inbred rye line R12 and complete chromosome complement of wheat cultivar Mianyang 11 were cytologically analyzed on a plant-by-plant basis by the improved C-banding technique. 63 of the plants, with 2n = 42, were found containing wheat-rye translocation or substitution, with a frequency of 4. 91% . Compared with the wheat parent, other 32 plants with 2n = 42 exhibited obvious phenotypic variation, but their com-ponent of rye chromosome could not be detected using the C-banding technique. In situ hybridization with a biotin-la-beled DNA probe was used to detect rye chromatin and to determine the insertion sites of rye segments in the wheat chromosomes. In 20 out of the 32 variant wheat plants, small segments of rye chromosomes were found being inserted into dif     

Identification of cellular prosomatostatin and nonsomatostatin peptides derived from its amino terminus

Rat prosomatostatin was isolated from a somatostatin-producing cell line and was partially microsequenced. This indicated the amino terminal structure of cellular prosomatostatin and implied a 92-amino acid sequence for the somatostatin precursor. Based on the structure for cellular prosomatostatin, a peptide was synthesized and used to develop a radioimmunoassay directed toward the amino terminal portion of prosomatostatin. This assay has revealed two peptides containing the amino-terminal portion of prosomatostatin in a somatostatin-secreting CA-77 rat medullary thyroid carcinoma cell line. These two peptides - MW 4000 and 8000 daltons - lack somatostatin immunoreactivity. Thus, processing of prosomatostatin occurs both at the amino and carboxyl regions. These results open the way for elucidation of the structure, function and metabolism of non-somatostatin peptides derived from the amino terminus of prosomatostatin.     

Effects of antifilarial drugs on the activities of the oxidative enzymes of mitochondria and microsomes of rat liver and kidneys

Centperazine or diethylcarbamazine, administered at various dose levels to rats inhibited the activity of succinate dehydrogenase significantly in 4 hrs in liver. Centperazine also inhibited the activity of cytochrome-c oxidase but stimulated the activity of benzo (a) pyrene hydroxylase in liver. In kidneys, activities of succinate dehydrogenase, cytochrome-c oxidase and aniline hydroxylase were significantly inhibited by centperazine only, however, the activity of benzo (a) pyrene hydroxylase was inhibited by both the drugs. These drugs had no effect on the activity of aminopyrene N-demethylase and cytochrome P-450 contents of liver and kidneys.     

Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in absence of recombinational repair

Near-ultraviolet (NUV) radiation killing of Escherichia coli K-12 can be enhanced by a sub-lethal concentration of hydrogen peroxide. This can be divided into a “RecA-dependent” and “RecA-independent” synergistic killing action. Stationary phase wild-type and 8 closely related repair-deficient mutants were examined for their NUV sensitivities in the presence and absence of H₂O₂. All exhibited the “RecA-independent” synergism; i.e., H₂O₂ enhanced NUV lethality when RecA repair was not operating. The “RecA-independent” synergism did not result from destruction of repair enzymes. Very few DNA—protein crosslinks could be detected following NUV plus H₂O₂ treatment. However, double-strand (DS) DNA breaks were produced, apparently by conversion of closely spaced single-strand (SS) breaks on opposite strands. The correlation between DS-break formation and lethality in wild-type and a polA mutant indicates that the RecA-independent synergistic killing results from the conversion of SS into lethal DS breaks.     

The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum

Maung M. 1978. The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum. International Journal for Parasitology 8: 371–378. Eggs of Ascaris lumbricoides and A. suum were cultured at 28°C and observed daily. Larvae were released by pressure, by artificial hatching with CO₂, and by natural hatching after infection of laboratory mice. The early stages of development in the egg were observed to comprise two moults, one occurring immediately after the other. Both moults were initiated within the egg, but the time of completion of the second moult varied considerably, and in some instances was not completed until the larvae reached the liver of experimentally infected animals.     

Studies on microbiologically influenced corrosion of SS304 by a novel manganese oxidizer,Bacillus flexus

A manganese oxidizing bacterium was isolated from the surface of steel scraps and biochemical tests and 16S rRNA sequencing analysis confirmed the isolate as Bacillus flexus. Potentiodynamic polarization curves showed ennoblement of open circuit potential, increased passive current, a lowering of breakdown potential, active re-passivation potential and enhanced cathodic current in the presence of B. flexus. Adhesion studies with B. flexus on SS304 specimens with different surface treatments demonstrated decreased adhesion on passivated and FeCl₃ treated specimens due to the removal of MnS inclusions. The present study provides evidence that surface treatment of stainless steels can reduce adhesion of this manganese oxidizing bacterium and decrease the probability of microbiologically influenced corrosion.