In Zanzibar and other tropical regions, iron deficiency, malaria and multiple helminth infections coexist. We addressed the following questions: 1) What are the predictors of low hemoglobin in Zanzibari preschool children? 2) Are indicators of iron status informative in this population? 3) Does malaria modify the relation of iron indicators to hemoglobin? We used multivariate regression to analyze cross-sectional data from a community-based sample of rural Zanzibari children who were not ill (n = 490; 4-71 mo of age) in whom we assessed hemoglobin, serum ferritin (SF), erythrocyte protoporphyrin (EP), serum transferrin receptor (TfR), recent fever, malaria parasitemia and helminth fecal egg counts. Of hemoglobin values, 80% were <100 g/L and 15.5% were <70 g/L. In children <18 mo of age, 40.2% of hemoglobin values were <70 g/L. Our primary findings were as follows: 1) In children <30 mo old, hemoglobin was associated with malaria but not hookworms, whereas in children>>/=30 mo, hemoglobin was related to hookworms but not malaria. In the younger age group, male sex and recent fever also predicted lower hemoglobin. 2) The three iron indicators were informative in this population but did not reflect only iron status. Malaria elevated SF in younger children and TfR and EP in both age groups. Fever elevated SF in older children and EP in both age groups, but not TfR. 3) Malaria modified the relation of all three indicators to hemoglobin. The relation of SF to hemoglobin was weak overall, and absent in malaria-infected children. EP and TfR were strongly related to hemoglobin, but this relation was attenuated by malaria.

We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.

Controlling and reversing the effects of loss are major challenges in optical systems. For lasers, losses need to be overcome by a sufficient amount of gain to reach the lasing threshold. In this work, we show how to turn losses into gain by steering the parameters of a system to the vicinity of an exceptional point (EP), which occurs when the eigenvalues and the corresponding eigenstates of a system coalesce. In our system of coupled microresonators, EPs are manifested as the loss-induced suppression and revival of lasing. Below a critical value, adding loss annihilates an existing Raman laser. Beyond this critical threshold, lasing recovers despite the increasing loss, in stark contrast to what would be expected from conventional laser theory. Our results exemplify the counterintuitive features of EPs and present an innovative method for reversing the effect of loss.

Extracellular and capsular polysaccharides (EPSs and CPSs) are produced by a wide range of bacteria, including important pathogens of humans, livestock, and plants. These polymers are major surface antigens and serve a variety of roles in virulence, depending on the biology of the producing organism. In addition to their importance in disease, some EPSs also have industrial applications as gelling and emulsifying agents. An understanding of the processes involved in the synthesis and regulation of CPSs and EPSs therefore potentially contributes to an understanding of the disease state, surface expression of protective antigens, and modulation of polymer structure to give defined physical properties. Escherichia coli has provided important model systems for EPS and CPS biosynthesis. Here we describe current knowledge concerning assembly of the Group 1 CPSs of E. coli and the conservation of similar mechanisms in other bacteria.

Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1.15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5-20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo-glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.

OBJECTIVE

Lubiprostone alleviates constipation by stimulating intestinal fluid secretion, purportedly through activation of ClC-2-type Cl(-) channels. Intestinal obstruction is also a recurrent cause of distress in cystic fibrosis (CF) patients, caused by loss of CF transmembrane conductance regulator (CFTR) Cl(-) channel activity. Because ClC-2 recruitment might be beneficial to CF patients, we investigated lubiprostone's mode of action.

METHODS

Cl(-) transport was measured in an Ussing chamber, in 3 model systems: (1) T84 colonocytes, (2) intestinal epithelium of wild-type and CF mice, and (3) intestinal epithelium of CF patients and controls.

RESULTS

In T84 monolayers, lubiprostone induced a robust secretory response. Selective permeabilization of the basolateral plasma membrane revealed that lubiprostone activated an apical Cl(-) conductance. The lubiprostone response was attenuated by H89, an inhibitor of the cAMP-dependent protein kinase, and lubiprostone precluded responsiveness to the cAMP agonist forskolin. CFTR blockage by CFTRinh172, but not ClC-2 blockage by CdCl(2), inhibited the lubiprostone response. Lubiprostone induced a CdCl(2)-insensitive secretory response in mouse intestine, but failed to induce intestinal Cl(-) secretion in Cftr-null mice. Correspondingly, lubiprostone induced a secretory response in human intestinal epithelium, but not in tissue of CF patients. The EP(4)-type prostanoid receptor antagonist L-161,982 blocked the lubiprostone response in all 3 models studied. In T84 cells, lubiprostone induced a rise in cAMP levels that was sensitive to EP(4)-receptor blockage.

CONCLUSIONS

Lubiprostone enhances intestinal Cl(-) and fluid secretion via prostanoid receptor signaling, triggering activation of CFTR. Therefore, it is of limited use for treatment of CF-related intestinal disease.

The Asia-Pacific region is home to nearly half of the world's population. The region has seen a recent rapid increase in the prevalence of obesity, type-2 diabetes and cardiovascular disease. The present systematic review summarizes the recent prevalence and trends of Metabolic Syndrome (MetS) among adults in countries of the Asia-Pacific Region.

Data on MetS in Asia-Pacific countries were obtained using a stepwise process by searching the online Medline database using MeSH terms 'Metabolic Syndrome X' and 'Epidemiology/EP'. For the purpose of describing prevalence data for the individual countries, studies that were most recent, nationally representative or with the largest sample size were included. When evaluating secular trends in prevalence in a country we only considered studies that evaluated the temporal change in prevalence between similar populations, prospective studies based on the same population or National surveys conducted during different time periods.

This literature search yielded a total of 757 articles, and five additional article were identified by screening of reference lists. From this total, 18 studies were eligible to be included in the final analysis. Of the 51 Asia-Pacific countries (WHO) we only located data for 15. There was wide between country variation in prevalence of MetS. A national survey from Philippines conducted in 2003 revealed the lowest reported prevalence of 11.9% according to NCEP ATP III criteria. In contrast, the highest recorded prevalence in the region (49.0%) came from a study conducted in urban Pakistan (Karachchi, 2004). Most studies reported a higher prevalence of MetS in females and urban residents. Data on secular trends were available for China, South Korea and Taiwan. An increase in the prevalence of MetS was observed in all three countries.

Despite differences in methodology, diagnostic criteria and age of subjects studied, the Asia-Pacific region is facing a significant epidemic of MetS. In most countries nearly 1/5th of the adult population or more were affected by MetS with a secular increase in prevalence. Strategies aimed at primary prevention are required to ameliorate a further increase in the epidemic and for the reduction of the morbidity and mortality associated with MetS.

Tumor budding is a histological feature that reflects loss of adhesion of tumor cells and is associated with locoregional metastasis of colorectal carcinoma. Although nuclear localization of beta-catenin is associated with tumor budding, the molecular mechanism remains largely elusive. In this study, we hypothesize that the epithelial cell adhesion molecule (Ep-CAM) is involved in tumor budding. In order to address this question, we performed immunohistochemistry on Ep-CAM using three different antibodies (monoclonal antibodies Ber-ep4 and 311-1K1 and a polyclonal antibody) and a double staining on beta-catenin and Ep-CAM. In addition, Ep-CAM mRNA was monitored with mRNA in situ hybridization. Subsequently, we determined the effect of Ep-CAM staining patterns on tumor spread in rectal cancer. In contrast to the tumor mass, budding cells of colorectal carcinoma displayed lack of membranous but highly increased cytoplasmic Ep-CAM staining and nuclear translocation of beta-catenin. mRNA in situ hybridization suggested no differences in Ep-CAM expression between the invasive front and the tumor mass. Importantly, reduced Ep-CAM staining at the invasive margin of rectal tumor specimens (n=133) correlated significantly with tumor budding, tumor grade and an increased risk of local recurrence (P=0.001, P=0.04 and P=0.03, respectively). These data demonstrate abnormal processing of Ep-CAM at the invasive margin of colorectal carcinomas. Our observations indicate that loss of membranous Ep-CAM is associated with nuclear beta-catenin localization and suggest that this contributes to reduced cell-cell adhesions, increased migratory potential and tumor budding.

Evoked potentials (EPs) were recorded directly from 650 frontal and peri-Rolandic sites in 26 subjects during face and/or word recognition, as well as during control tasks (simple auditory and visual discrimination). Electrodes were implanted in order to localize epileptogenic foci resistant to medication, and thus direct their surgical removal. While awaiting spontaneous seizure onset, the patients gave informed consent to perform cognitive tasks during intracerebral EEG recording. The earliest potentials appeared to be related to sensory stimulation, were prominent in lateral prefrontal cortex, and occurred at peak latencies of about 150 and 190 ms. A small triphasic complex beginning slightly later (peak latencies about 200-285-350 ms) appeared to correspond to the scalp N2-P3a-slow wave, associated with non-specific orienting. Multiple components peaking from 280 to 900 ms, and apparently specific to words were occasionally recorded in the left inferior frontal g, pars triangularis (Broca's area). Components peaking at about 430 and 600 ms were recorded in all parts of the prefrontal cortex, but were largest (up to 180 microV) and frequently polarity-inverted in the ventro-lateral prefrontal cortex. These components appeared to represent the N4-P3b, which have been associated with contextual integration and cognitive closure. Finally, a late negativity (650-900 ms) was recorded in precentral and premotor cortices, probably corresponding to a peri-movement readiness potential. In summary, EP components related to early sensory processing were most prominent in lateral prefrontal, to orienting in medial limbic, to word-specific processing in Broca's area, to cognitive integration in ventro-lateral prefrontal, and to response organization in premotor cortices.(ABSTRACT TRUNCATED AT 250 WORDS)

A repeating particle associated with the cristae and the inner membrane of the external envelope has been recognized and characterized in beef heart mitochondria by correlated electron microscopic and biochemical studies. Many thousands (ca. 10(4) to 10(5)) of these particles, disposed in regular arrays, are present in a single mitochondrion. The repeating particle, called the elementary particle (EP), consists of three parts: (1) a spherical or polyhedral head piece (80 to 100 A in diameter); (2) a cylindrical stalk (about 50 A long and 30 to 40 A wide); and (3) a base piece (40 x 110 A). The base pieces of the elementary particles form an integral part of the outer dense layers of the cristae. The elementary particles can be seen in electron micrographs of mitochondria in situ, of isolated mitochondria, and of submitochondrial particles with a complete electron transfer chain. Negative staining with phosphotungstate is only one of several techniques that can be used for reproducible demonstration of the repeating particles and underlying subunit organization of mitochondrial membranes. A particulate unit containing a complete electron transfer chain can be isolated from beef heart mitochondria. The isolated unit approximates in size that of the elementary particle in situ. The molecular weight of the particle in situ is calculated to be 1.3 x 10(6). Evidence is presented for identifying the isolated unit with the elementary particle visualized in situ. The elementary particle of the mitochondrion is believed to be a prototype of a class of functional particles or macromolecular assemblies of similar size found in association with membranes generally.

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(2)AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP(1) receptor, since its endogenous agonist prostaglandin E(2) is abundant in the airway, but its functional implications are poorly defined. Activation of EP(1) failed to elicit ASM contraction in mouse trachea via this G(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), an effect accentuated by EP(1) agonist activation. Thus alone, EP(1) does not appear to have a significant direct effect on airway tone but acts as a modulator of the beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma.

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

In vivo electroporation (EP) is a versatile delivery method for gene transfer which can be applied to any accessible tissue. Delivery of plasmid DNA encoding therapeutic genes or cDNAs with in vivo EP has been tested extensively in preclinical melanoma models. Direct delivery to the tumor has been shown to generate a direct antitumor effect. Delivery to alternative sites may generate additional therapeutic options, for example the production of cancer vaccines, the reduction of tumor angiogenesis, or the induction of tumor cell apoptosis. Several of the preclinical therapies tested have a demonstrated therapeutic effect against melanomas. Two immunotherapies have advanced to melanoma clinical trials. Delivery of a plasmid DNA encoding interleukin-12 (IL-12) or interleukin-2 (IL-2) using electroporation was demonstrated to be a safe with no grade 3 or 4 toxicities reported. Delivery of IL-12 with electroporation resulted in significant necrosis of melanoma cells in the majority of treated tumors and significant lymphocytic infiltrate in biopsies from patients in several cohorts. In addition, clinical evidence of responses in untreated lesions suggested the induction of a systemic response following therapy. This review discusses preclinically tested electroporation gene therapies for melanoma with clinical potential and the conversion of these therapies to clinical trials.

Prostaglandin E(2) (PGE(2)) is both an inflammatory mediator released at the site of tissue inflammation and a neuromodulator that alters neuronal excitability and synaptic processing. The effects of PGE(2) are mediated by four G-protein-coupled <em>EP</em> receptors (<em>EP</em>1-<em>EP</em>4). Here we show that the <em>EP</em>4 receptor subtype is expressed by a subset of primary sensory dorsal root ganglion (DRG) neurons, and that its levels, but not that of the other <em>EP</em>1-3 subtypes, increase in the DRG after complete Freund' adjuvant-induced peripheral inflammation. Administration of both an <em>EP</em>4 antagonist [AH23848, (4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid] and <em>EP</em>4 knockdown with intrathecally delivered short hairpin RNA attenuates inflammation-induced thermal and mechanical behavioral hypersensitivity, without changing basal pain sensitivity. AH23848 also reduces the PGE(2)-mediated sensitization of capsaicin-evoked currents in DRG neurons in vitro. These data suggest that <em>EP</em>4 is a potential target for the pharmacological treatment of inflammatory pain.

HIV-1 integrase (IN) is one of three essential enzymes (along with reverse transcriptase and protease) encoded by the viral pol gene. IN mediates two critical reactions during viral replication; firstly 3'-end processing (3'EP) of the double-stranded viral DNA ends and then strand transfer (STF) which joins the viral DNA to the host chromosomal DNA forming a functional integrated proviral DNA. IN is a 288 amino acid protein containing three functional domains, the N-terminal domain (NTD), catalytic core domain (CCD) and the C-terminal domain (CTD). The CCD contains three conserved catalytic residues, Asp64, Asp116 and Glu152, which coordinate divalent metal ions essential for the STF reaction. Intensive research over the last two decades has led to the discovery and development of small molecule inhibitors of the IN STF reaction (INSTIs). INSTIs are catalytic inhibitors of IN, and act to chelate the divalent metal ions in the CCD. One INSTI, raltegravir (RAL, Merck Inc.) was approved in late 2007 for the treatment of HIV-1 infection in patients with prior antiretroviral (ARV) treatment experience and was recently approved also for first line therapy. A second INSTI, elvitegravir (EVG, Gilead Sciences, Inc.) is currently undergoing phase 3 studies in ARV treatment-experienced patients and phase 2 studies in ARV naïve patients as part of a novel fixed dose combination. Several additional INSTIs are in early stage clinical development. This review will discuss the discovery and development of this novel class of antiretrovirals. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010.

OBJECTIVE

The present study was undertaken to identify potential predictors of and factors associated with early and late progression in acute stroke. We performed secondary analysis of the clinical, biochemical, and radiological data recorded in the acute phase of stroke patients enrolled in the European Cooperative Acute Stroke Study (ECASS) I.

METHODS

Early progressing stroke (EPS) was diagnosed when there was a decrease of>> or = 2 points in consciousness or motor power or a decrease of>> or = 3 points in speech scores in the Scandinavian Neurological Stroke Scale from baseline to the 24-hour evaluation, and late progressing stroke (LPS) was diagnosed when 1 of these decreases occurred between the 24-hour evaluation and the evaluation at day 7. Using logistic regression analyses, we looked for baseline variables that predicted EPS and LPS and for factors measured after the early or late acute phase and associated with the 2 clinical courses.

RESULTS

Of the 615 patients studied, 231 (37.5%) worsened during the first 24 hours after inclusion. The overall incidence of EPS was 37% in the placebo group and 38% in the recombinant tissue plasminogen activator group (P=0.68, Fisher's Exact Test). Focal hypodensity (odds ratio [OR], 1.9; 95% confidence interval [CI], 1.3 to 2.9) and hyperdensity of the middle cerebral artery sign (OR, 1.8; 95% CI, 1.1 to 3.1) on baseline computed tomography, longer delay until treatment (OR, 1.2; 95% CI, 1.1 to 1. 4) and history of coronary heart disease (OR, 1.7; 95% CI, 1.1 to 2. 8) and diabetes (OR, 1.8; 95% CI, 1.0 to 3.1) were independent prognostic factors for EPS. Extent of hypodensity >33% in the middle cerebral artery territory (OR, 2.5; 95% CI, 1.6 to 4.0) and brain swelling (OR, 1.8; 95% CI, 1.1 to 3.2) on CT at 24 hours but not hemorrhagic transformation of cerebral infarct nor decrease in systolic blood pressure within the first 24 hours after treatment were associated with EPS in multivariate analyses. LPS was observed in 20.3% of patients. Older age, a low neurological score, and brain swelling at admission independently predicted late worsening.

CONCLUSIONS

In the setting of a multicenter trial, EPS and LPS are mainly related to computed tomographic signs of cerebral edema. Treatment with recombinant tissue plasminogen activator, hemorrhagic transformation, and moderate changes in systolic blood pressure did not influence the early clinical course.

Cell adhesion receptors (CAMs) are actively involved in regulating various cell processes, including growth, differentiation, and cell death. Therefore, CAMs represent a large group of morphoregulating molecules, mediating cross-talk between cells and of cells with their environment. From this perspective, CAMs do contribute to cells and tissue organization, and in diseased tissue, to the disease development and biological characteristics. Therefore, observed changes in expression patterns of adhesion molecules may contribute to establish a diagnosis. A distinct shift in expression patterns in neoplastic epithelium has been described, for example for cadherins, integrins, and CD44. A relatively novel cell CAM, Ep-CAM, was first reported to be a pan-carcinoma antigen, although it is rather a marker of epithelial lineage. Several antibodies directed to Ep-CAM have been generated, and many epithelial tissues and their neoplastic appendages have been studied. This article outlines the results of these studies. Based on the results of these studies, we conclude that Ep-CAM immunohistochemistry can be a useful tool in the diagnosis of disturbed epithelial tissues.

Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.

Type II secretion systems consist of an assembly of 12-15 Gsp proteins responsible for transporting a variety of virulence factors across the outer membrane in several pathogenic bacteria. In Vibrio cholerae, the major virulence factor cholera toxin is secreted by the Eps Type II secretion apparatus consisting of 14 Eps proteins. One of these, EpsE, is a cytoplasmic putative NTPase essential for the functioning of the Eps system and member of the GspE subfamily of Type II secretion ATPases. The crystal structure of a truncated form of EpsE in nucleotide-liganded and unliganded state has been determined, and reveals a two-domain architecture with the four characteristic sequence "boxes" of the GspE subfamily clustering around the nucleotide-binding site of the C-domain. This domain contains two C-terminal subdomains not reported before in this superfamily of NTPases. One of these subdomains contains a four-cysteine motif that appears to be involved in metal binding as revealed by anomalous difference density. The EpsE subunits form a right-handed helical arrangement in the crystal with extensive and conserved contacts between the C and N domains of neighboring subunits. Combining the most conserved interface with the quaternary structure of the C domain in a distant homolog, a hexameric model for EpsE is proposed which may reflect the assembly of this critical protein in the Type II secretion system. The nucleotide ligand contacts both domains in this model. The N2-domain-containing surface of the hexamer appears to be highly conserved in the GspE family and most likely faces the inner membrane interacting with other members of the Eps system.

OBJECTIVE

The study aim was to establish the incidence and characterize all encapsulating peritoneal sclerosis (EPS) cases in patients treated by peritoneal dialysis (PD).

METHODS

The patient cohort, which started PD from January 1, 2000, to December 31, 2007, was identified from the Scottish Renal Registry (n = 1238). Possible EPS cases were identified by the ten adult Scottish renal units. Patient records were examined to ensure cases met diagnostic criteria.

RESULTS

Forty-six cases were identified; 19 had their first PD exposure after January 1, 2000. The rate was 1.5%, an incidence of 4.9 per 1000 person-years. The incidence increased with PD duration, with rates of 0, 0.6, 2.0, 3.5, 8.1, 8.8 and 5% at <1, 1 to 2, >2 to 3, >3 to 4, >4 to 5, >5 to 6 and >6 yr PD exposure, respectively. The median PD duration of EPS cases was 5.1 yr (interquartile range [IQR] 3.4 to 6.1 yr). At diagnosis, 12 (26%) were on PD and 33 (72%) were diagnosed <2 yr after PD stopped. The cases had a median of 3.3 episodes of peritonitis (range 0 to 20, IQR 1 to 4.5). Thirty (65%) had used 3.86% dextrose dialysate and 45 (98%) had used Extraneal. The mortality was 42% at 1 yr postdiagnosis with a median survival of 149 d (IQR 61 to 408 d).

CONCLUSIONS

The incidence reported in this study may be used to inform patients of the minimum risk of developing EPS on PD.

Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E(2) (PGE(2)) acts on cognate receptor <em>EP</em>4 to promote the migration of A549 lung cancer cells. Treatment with PGE(2) enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE(2)-mediated lung cancer cell migration. PGE(2) affects target cells by activating four receptors named <em>EP</em>1 to <em>EP</em>4. Use of <em>EP</em> subtype-selective ligand agonists suggested that <em>EP</em>4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down <em>EP</em>4 expression. Proximal <em>EP</em>4 effectors include heterotrimeric Gs and betaArrestin proteins. Knockdown of betaArrestin1 expression with shRNA significantly impaired the PGE(2)-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE(2) in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of <em>EP</em>4, betaArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of <em>EP</em>4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer.

Many marine bacteria produce exopolysaccharides (EPS) as a strategy for growth, adhering to solid surfaces, and to survive adverse conditions. There is growing interest in isolating new EPS producing bacteria from marine environments, particularly from extreme marine environments such as deep-sea hydrothermal vents characterized by high pressure and temperature and heavy metal presence. Marine EPS-producing microorganisms have been also isolated from several extreme niches such as the cold marine environments typically of Arctic and Antarctic sea ice, characterized by low temperature and low nutrient concentration, and the hypersaline marine environment found in a wide variety of aquatic and terrestrial ecosystems such as salt lakes and salterns. Most of their EPSs are heteropolysaccharides containing three or four different monosaccharides arranged in groups of 10 or less to form the repeating units. These polymers are often linear with an average molecular weight ranging from 1 x 10(5) to 3 x 10(5) Da. Some EPS are neutral macromolecules, but the majority of them are polyanionic for the presence of uronic acids or ketal-linked pyruvate or inorganic residues such as phosphate or sulfate. EPSs, forming a layer surrounding the cell, provide an effective protection against high or low temperature and salinity, or against possible predators. By examining their structure and chemical-physical characteristics it is possible to gain insight into their commercial application, and they are employed in several industries. Indeed EPSs produced by microorganisms from extreme habitats show biotechnological promise ranging from pharmaceutical industries, for their immunomodulatory and antiviral effects, bone regeneration and cicatrizing capacity, to food-processing industries for their peculiar gelling and thickening properties. Moreover, some EPSs are employed as biosurfactants and in detoxification mechanisms of petrochemical oil-polluted areas. The aim of this paper is to give an overview of current knowledge on EPSs produced by marine bacteria including symbiotic marine EPS-producing bacteria isolated from some marine annelid worms that live in extreme niches.