Rac1 and Toll-IL-1 receptor domain-containing adapter protein mediate Toll-like receptor 4 induction of HIV-long terminal repeat

Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-kappaB activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.     

Macrophages and stromal cells phagocytose apoptotic bone marrow-derived B lineage cells

It has been hypothesized that B cell precursors that undergo programmed cell death due to nonproductive Ig gene rearrangements are cleared from the bone marrow by macrophages. However, a role for macrophages in this process is supported only by micrographs showing their association with apoptotic-appearing, B lineage cells. Functional data demonstrating phagocytosis of apoptotic, bone marrow lymphocytes by macrophages have not been presented, nor have receptors potentially involved in that process been identified. The data in this report demonstrate that macrophages isolated from murine bone marrow efficiently phagocytose apoptotic murine B lineage cells using multiple receptors that include CD14, integrins, class A scavenger receptor, and CD31 (PECAM-1). In addition, the results further reveal a new role for the hemopoietic microenvironment in B cell development in view of data demonstrating that murine bone marrow stromal cells are also capable of clearing apoptotic cells via an integrin-dependent mechanism.     

Case-control and case-only designs with genotype and family history data: estimating relative risk, residual familial aggregation, and cumulative risk

In case-control studies of inherited diseases, participating subjects (probands) are often interviewed to collect detailed data about disease history and age-at-onset information in their family members. Genotype data are typically collected from the probands, but not from their relatives. In this article, we introduce an approach that combines case-control analysis of data on the probands with kin-cohort analysis of disease history data on relatives. Assuming a marginally specified multivariate survival model for joint risk of disease among family members, we describe methods for estimating relative risk, cumulative risk, and residual familial aggregation. We also describe a variation of the methodology that can be used for kin-cohort analysis of the family history data from a sample of genotyped cases only. We perform simulation studies to assess performance of the proposed methodologies with correct and mis-specified models for familial aggregation. We illustrate the proposed methodologies by estimating the risk of breast cancer from BRCA1/2 mutations using data from the Washington Ashkenazi Study.     

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.     

Regulation of gene expression and biochemical changes in small intestine of newborn diabetic rats by exogenous ghrelin

The aim of this study was to investigate (i) the cholecystokinin, somatostatin and apelin mRNA levels, (ii) the changes in levels and localization of these peptides, (iii) relation between these peptides, (iv) antiapoptotic effects and (v) antioxidant effects of ghrelin. The rats were divided into four groups second day after birth. These groups were respectively treated with physiological saline, ghrelin (100μg/kg/day), streptozotocin (100mg/kg), ghrelin and streptozotocin. After four weeks, small intestine and blood samples were taken from rats. Cholecystokinin mRNA and peptide, somatostatin mRNA, release to duodenal lumen of apelin peptide and apelin mRNA signals decreased in ghrelin-treated diabetic rats compared to the diabetic group. There was no statistically significant difference among the four groups for somatostatin and apelin peptides. Caspase-3 signals were not observed only in diabetic group treated with ghrelin. Caspase-8 signals were increased while PCNA signals were decreased in diabetic group given ghrelin compared to diabetic group. Small intestine CAT, SOD, GP(x) and GST activities and GSH levels were decreased and LPO, PC levels were increased in diabetic rats. Administration of ghrelin to diabetic rats caused an increase in intestinal CAT, SOD, GP(x) and GST activities and GSH levels, while PC levels decreased. As a result, we observed positive changes in diabetic rats treated with ghrelin in both microscopic and biochemical studies. We can suggest that ghrelin may be an important hormone for the treatment of diabetes.     

Connecting environment and genome plasticity in the characterization of transformation-induced SOS regulation and carbon catabolite control of the Vibrio cholerae integron integrase

The human pathogen Vibrio cholerae carries a chromosomal superintegron (SI). The SI contains an array of hundreds of gene cassettes organized in tandem which are stable under conditions when no particular stress is applied to bacteria (such as during laboratory growth). Rearrangements of these cassettes are catalyzed by the activity of the associated integron integrase. Understanding the regulation of integrase expression is pivotal to fully comprehending the role played by this genetic reservoir for bacterial adaptation and its connection with the development of antibiotic resistance. Our previous work established that the integrase is regulated by the bacterial SOS response and that it is induced during bacterial conjugation. Here, we show that transformation, another horizontal gene transfer (HGT) mechanism, also triggers integrase expression through SOS induction, underlining the importance of HGT in genome plasticity. Moreover, we report a new cyclic AMP (cAMP)-cAMP receptor protein (CRP)-dependent regulation mechanism of the integrase, highlighting the influence of the extracellular environment on chromosomal gene content. Altogether, our data suggest an interplay between different stress responses and regulatory pathways for the modulation of the recombinase expression, thus showing how the SI remodeling mechanism is merged into bacterial physiology.     

Impact of adrenal versus ovarian androgen ratio on signs and symptoms of polycystic ovarian syndrome

The aim of this study was to examine the effect of adrenal versus ovarian androgen (dehydroepiandrosterone sulfate/total testosterone [DHEAS/TT]) on clinical presentation and related metabolic disturbances in Turkish women with polycystic ovarian syndrome (PCOS). Two hundred eighty PCOS cases were taken into the study. For all cases, the DHEAS/TT ratio was calculated. The median value of this ratio was 4.40. Patients with an androgen ratio lower than 4.40 were included in Group 1 and cases with a ratio higher than 4.40 were Group 2. The two groups were compared in terms of hormonal, biochemical and clinical parameters. Body mass index and waist circumference were lower, the Ferriman-Gallwey score was higher and the cycle length was shorter in Group 2. High DHEAS level was associated with better lipid profiles and lower levels of inflammatory markers, meaning good metabolic control in these women, in spite of increased hirsutism rates. In patients with PCOS, both androgens are usually high in proportion to each other. Therefore, it would be more meaningful to use the DHEAS/TT ratio for an assessment of the metabolic and phenotypic effects of PCOS.     

Takayasu's arteritis is associated with HLA-B*52, but not with HLA-B*51, in Turkey

Introduction

HLA-B*51 and HLA-B*52 are two close human leukocyte antigen (HLA) allele groups with minor amino acid differences. However, they are associated with two different vasculitides (HLA-B*51 in Behçet's disease and HLA-B*52 in Takayasu's arteritis (TAK)) and with major clinical and immunological differences. In this study, we aimed to screen a large cohort of TAK patients from Turkey for the presence of HLA-B*51 and HLA-B*52 as susceptibility and severity factors.

Methods

TAK patients (n = 330) followed at a total of 15 centers were included in the study. The mean age of the patients was 37.8 years, and 86% were women. DNA samples from the patients and healthy controls (HC; n = 210) were isolated, and the presence of HLA-B*51 or HLA-B*52 was screened for by using PCR with sequence-specific primers.

Results

We found a significant association of HLA-B*52 with TAK (20.9% vs HC = 6.7%, P = 0.000, OR = 3.7, 95% CI = 2.02 to 6.77). The distribution of HLA-B*51 did not differ between TAK patients and HCs (22.7% vs 24.8%, OR = 0.9, 95% CI = 0.60 to 1.34). The presence of HLA-B*52 decreased in late-onset patients (> 40 years of age; 12.0%, P = 0.024, OR = 0.43, 95% CI = 0.20 to 0.91). Patients with angiographic type I disease with limited aortic involvement also had a lower presence of HLA-B*52 compared to those with all other disease subtypes (13.1% vs 26%, P = 0.005, OR = 0.43, 95% CI = 0.23 to 0.78).

Conclusions

In this study, the previously reported association of TAK with HLA-B*52 in other populations was confirmed in patients from Turkey. The functional relevance of HLA-B*52 in TAK pathogenesis needs to be explored further.     

Torque generation of kinesin motors is governed by the stability of the neck domain

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules. In stark contrast to kinesin-1, these motors display a wide range of left-handed spiraling around microtubules and thus generate torque during cargo transport. We provide direct evidence that kinesin's neck region determines the torque-generating properties. A model system based on kinesin-1 corroborates this result: disrupting the stability of the neck by inserting flexible peptide stretches resulted in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduced the spiraling of the motor up to the point of protofilament tracking. Finally, we present a model that explains the physical basis of kinesin's spiraling around the microtubule.