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.     

Effects of the supply levels and ratios of nitrogen and phosphorus on seed traits of Chenopodium glaucum

全球氮沉降不仅改变土壤氮和磷的有效性, 同时也改变氮磷比例。氮磷供应量、比例及其交互作用可能会影响植物种子性状。该研究在内蒙古草原基于沙培盆栽实验种植灰绿藜(Chenopodium glaucum), 设置3个氮磷供应量水平和3个氮磷比例的正交实验来探究氮磷供应量、比例及其交互作用对灰绿藜种子性状的影响。结果发现氮磷供应量对种子氮浓度、磷浓度和萌发率影响的相对贡献(15%-24%)大于氮磷比例(3%-7%), 而种子大小只受氮磷比例的影响。同时氮磷供应量和比例之间的交互作用显著影响种子氮浓度和磷浓度。同等氮磷比例情况下, 低量养分供应提高种子氮浓度、磷浓度和萌发率。氮磷比例只有在养分匮乏的环境中才会对种子大小和萌发率产生显著影响。总之, 灰绿藜种子不同性状对氮或磷限制的敏感性不同, 同时种子性状也对养分限制表现出适应性和被动响应。     

Focal contacts of spreading platelets with the substratum

Contacts with glass substratum formed by the spreading rabbit platelets were examined by an antibody-exclusion method; monoclonal antibodies against 80 kD bovine serum protein were used. It was found that platelets form focal contacts in the course of spreading. The size of the largest focal contacts formed by platelets is smaller than that of the contacts formed by fibroblasts. The antibody-exclusion method revealed focal contacts of platelets much more clearly than interference reflection microscopy (IRM). The similarity of reactions involved in spreading platelets and of large nucleus-containing tissue cells is discussed.     

ROCKing cytokine secretion balance in human T cells

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.     

Design and synthesis of 3-isoxazolidone derivatives as new Chlamydia trachomatis inhibitors

Chlamydia trachomatis (Ct) is a bacterial human pathogen responsible for the development of trachoma, the worldwide infection leading to blindness, and is also a major cause of sexually transmitted diseases. As iron is an essential metabolite for this bacterium, iron depletion presents a promising strategy to limit Ct proliferation. The aim of this study is to synthesize 3-isoxazolidone derivatives bearing known chelating moieties in an attempt to develop new bactericidal anti-Chlamydiaceae molecules. We have investigated the paths by which these new compounds affect Ct serovar L2 development in HeLa cells, in the presence or absence of exogenously added iron. The iron-chelating properties of these molecules were also determined. Our data reveal important bactericidal effects which are distinguishable from those due to iron chelation.     

The occurrence and frequency of 2n pollen in 2x, 4x,and 6x wild,tuber-bearing Solanum species from Mexico,and Central and South America

Summary The occurrence of 2n pollen-producing plants was investigated in 187 plant introductions (PIs) of 38 wild species of tuber-bearing Solanum. These 2x, 4x, and 6x species are from Mexico, and Central and South America. The determination of 2n pollen-producing plants was conducted using acetocarmine glycerol. Plants with more than 1% large-size pollen were regarded as 2n pollen-producing plants. 2n pollen-producing plants were identified in the following species: 10 out of 12 Mexican 2x species, seven of nine South American 2x species, seven of seven Mexican and Central American 4x species, five of five South American 4x species, and five of five Mexican 6x species. The frequency of 2n pollen-producing plants varied among species at the same ploidy level, but the range of frequency, generally between 2 and 10% among species, was similar over different ploidy levels. The general occurrence of 2n pollen in both 2x and polyploid species, which are evolutionarily related, is evidence that the mode of polyploidization in tuber-bearing Solanums is sexual polyploidization. Furthermore, the frequencies of 2n pollen-producing plants in autogamous disomic polyploid species were not markably different from those of their related diploid species. It is thought that the frequent occurrence of 2n gametes with autogamy tends to disturb the fertility and consequently reduce fitness of polyploids. Thus, we propose that the breeding behavior of polyploids and the occurrence of 2n gametes may be genetically balanced in order to conserve high fitness in polyploid species in tuberbearing Solanum.Paper No. 3114 from the Laboratory of Genetics. Research supported by the College of Agriculture and Life Sciences; International Potato Center; USDA, SEA, CGRO 84-CRCR-1-1389; and Frito Lay, Inc.     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.     

Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen‐sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)‐mediated activation of Notch and repression of Wnt/β‐catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF‐mediated support for Notch signalling may decline while the β‐catenin‐directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation.