Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.     

Interferon regulatory factor-1 (IRF-1) suppression and derepression during endometrial tumorigenesis and cancer progression

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene presumed to be involved in the control of cellular proliferation and transformation. Given that the IRF-1 is consistently expressed in the normally cycling endometrium, the question was raised of the possible role of IRF-1 in the genesis of endometrial adenocarcinoma. A series of 25 normal and 86 malignant endometria was investigated using immunohistochemical techniques and the anti-IRF-1 polyclonal antibody c-20. Normal endometrial glands were, indeed, consistently reactive with IRF-1. Excluding the invading tumor front, malignant endometria were deprived of IRF-1 reactivity, as 81 of the 86 cases (94.2%) were negative for this antigen. At the invading tumor front, however, IRF-1 was derepressed in tumor cells in 35% of the cases. This phenomenon was independent of the extent of lymphocytic response, but it was associated with thymidine phosphorylase (TP) expression. Furthermore, TP up-regulation and host's lymphocytic response in the area were directly associated. IRF-1 derepression by invading tumor cells was associated with poor prognosis, independently of FIGO stage. It is concluded that down-regulation of IRF-1 is a constant finding in endometrial tumorigenesis. However, derepression of IRF-1 may occur in a subset of tumors, and this event is associated with TP up-regulation and aggressive tumor behavior.     

Lipid peroxidation and inguinal hernia repair. Tension-free vs. Andrews technique

AIM OF THE STUDY: To evaluate the early effect of inguinal hernia repair by the tension-free method compared to the conventional Andrew's technique on lipid peroxidation. PATIENTS-METHODS: Thirty-four patients subjected to elective hernia repair were enrolled in the study divided in two groups. Group A (n=18) underwent hernia repair by the tension-free method using a polypropylene mesh. Group B (n=16) underwent hernia repair by the Andrew's technique (i.e. a modification of the Bassini's technique). Venous blood samples were drawn preoperatively and at 12, 24 and 48 h postoperatively. Malondialdehyde (MDA) was estimated by the thiobarbiturate assay. RESULTS: Neutrophil counts were significantly higher in patients of group B compared to group A at 12 and 48 h postoperatively. Concentrations of fibrinogen were similar between the two groups. MDA was significantly higher in patients of group B hours compared to group A at 12, 24 and 48 h postoperatively. Positive correlation was found between neutrophil counts and MDA at 12 h (r: +0.43, P: 0.015) and 48 h (r: +0.496, P: 0.005) but not at 24 h. No correlation was found between serum fibrinogen and MDA. CONCLUSION: Hernia repair by the Andrews's technique elicits a sustained triggering of lipid peroxidation, compared to the tension-free method.     

Myxopyronin B analogs as inhibitors of RNA polymerase, synthesis and biological evaluation

A series of myxopyronin B analogs has been prepared via a convergent synthetic route and were tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against E. coli and S. aureus. The parent lead compound proved to be very sensitive to even small changes. Only the achiral desmethyl myxopyronin B (1a) provided enhanced potency.     

Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s^–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s^–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. This work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen or IgG and does not involve the S. aureus adhesins clumping factor A or protein A. adhesion; red blood cell     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.     

Short-term effects of selected exercise and load in contrast training on vertical jump performance

The present study examined the short-term effects of loaded half squats (HSs) and loaded jump squats (JSs) with low and moderate loads on the squat jump (SJ) and the countermovement jump (CMJ) performance using a contrast training approach. Ten men (mean +/- SD age, 23 +/- 1.8 years) performed the HS and JS exercises twice with loads of 30% of 1 repetition maximum (1RM) (HS30% and JS30%, respectively) and 60% of 1RM (HS60% and JS60%, respectively). On each occasion, 3 sets of 5 repetitions with 3 minutes of rest were performed as fast as possible. Vertical jump performance was measured before exercise, 1 minute after each set, and at the fifth and 10th minutes of recovery. The CMJ increased significantly after the first and second set (3.9%; p < 0.05) compared with preexercise values following the JS30% protocol and 3.3% after the second and third sets of the JS60% protocol. Following the HS60% protocol, CMJ increased after the first and the second sets (3.6%; p < 0.05) compared with preexercise values, whereas SQ increased only after the first set (4.9%; p < 0.05) in this condition. These data show that contrast loading with the use of low and moderate loads can cause a short-term increase in CMJ performance. The applied loads do not seem to present different short-term effects after loaded JSs. When the classic form of dynamic HS exercise is performed, however, at least a moderate load (60% of 1RM) needs to be applied.     

A criticality-based framework for task composition in multi-agent bioinformatics integration systems

MOTIVATION: During task composition, such as can be found in distributed query processing, workflow systems and AI planning, decisions have to be made by the system and possibly by users with respect to how a given problem should be solved. Although there is often more than one correct way of solving a given problem, these multiple solutions do not necessarily lead to the same result. Some researchers are addressing this problem by providing data provenance information. Others use expert advice encoded in a supporting knowledge-base. In this paper, we propose an approach that assesses the importance of such decisions with respect to the overall result. We present a way of measuring decision criticality and describe its potential use. RESULTS: A multi-agent bioinformatics integration system is used as the basis of a framework that facilitates such functionality. We propose an agent architecture, and a concrete bioinformatics example (prototype) is used to show how certain decisions may not be critical in the context of more complex tasks.     

Tight regulation of expression of two Arabidopsis cytosolic Hsp90 genes during embryo development

The spatial and temporal distribution of expression of two cytosolic members of the AtHsp90 gene family was assessed during early development. In stressed transgenic plants bearing the AtHsp90-3 promoter, beta-glucuronidase (GUS) activity was strong in meristematic tissues. Expression was also detected in vascular tissues, leaf veins, siliques, and in pollen sacs. The promoter induced gene expression after heat shock in a time-course dependent manner. AtHsp90-1 promoter activity was low throughout the early stages of embryo development but high just before embryo maturation, with expression most prominent in cotyledons. AtHsp90-3 promoter activity was almost constant and restricted to the root and the cotyledon tips of the embryo. This highly specific spatial distribution of GUS activity changed when the tissues were heat-stressed. Both promoters were also active in unstressed mature pollen grains and during pollen germination. The results shown here indicate that different regulatory and developmental mechanisms control and differentiate the expression of the two cytosolic members of the Arabidopsis AtHsp90 gene family under normal conditions. The developmental and restricted pattern of expression of the AtHsp90-1 and -3 gene promoters in unstressed transgenic plants suggest prominent and distinctive roles of these two genes during different developmental processes.