Characterization of Nef-CXCR4 interactions important for apoptosis induction

The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4(+) lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1alpha were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.     

Socioeconomic and Sociodemographic Factors Associated with Asthma Related Outcomes in Early Childhood: The Generation R Study

Rationale

Few studies have analyzed the association of socioeconomic and sociodemographic factors with asthma related outcomes in early childhood, including Fraction of exhaled Nitric Oxide (FeNO) and airway resistance (Rint). We examined the association of socioeconomic and sociodemographic factors with wheezing, asthma, FeNO and Rint at age 6 years. Additionally, the role of potential mediating factors was studied.

Methods

The study included 6717 children participating in The Generation R Study, a prospective population-based cohort study. Data on socioeconomic and sociodemographic factors, wheezing and asthma were obtained by questionnaires. FeNO and Rint were measured at the research center. Statistical analyses were performed using logistic and linear regression models.

Results

At age 6 years, 9% (456/5084) of the children had wheezing symptoms and 7% (328/4953) had asthma. Children from parents with financial difficulties had an increased risk of wheezing (adjusted Odds Ratio (aOR) = 1.63, 95% Confidence Interval (CI):1.18–2.24). Parental low education, paternal unemployment and child''s male sex were associated with asthma, independent of other socioeconomic or sociodemographic factors (aOR = 1.63, 95% CI:1.24–2.15, aOR = 1.85, 95% CI:1.11–3.09, aOR = 1.58, 95% CI:1.24–2.01, respectively). No socioeconomic or gender differences in FeNO were found. The risks of wheezing, asthma, FeNO and Rint measurements differed between ethnic groups (p<0.05). Associations between paternal unemployment, child''s sex, ethnicity and asthma related outcomes remained largely unexplained.

Conclusions

This study showed differences between the socioeconomic and sociodemographic correlates of wheezing and asthma compared to the correlates of FeNO and Rint at age 6 years. Several socioeconomic and sociodemographic factors were independently associated with wheezing and asthma. Child''s ethnicity was the only factor independently associated with FeNO. We encourage further studies on underlying pathways and public health intervention programs, focusing on reducing socioeconomic or sociodemographic inequalities in asthma.     

Mice with dysfunctional TGF-β signaling develop altered intestinal microbiome and colorectal cancer resistant to 5FU

Emerging data show a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant. One potential risk factor is an alteration in the microbiome. Here, we investigated the role of TGF-β signaling on the intestinal microbiome and the efficacy of chemotherapy for CRC induced by azoxymethane and dextran sodium sulfate in mice. We used two genotypes of TGF-β-signaling-deficient mice (Smad4^+/? and Smad4^+/?Sptbn1^+/?), which developed CRC with similar phenotypes and had similar alterations in the intestinal microbiome. Using these mice, we evaluated the intestinal microbiome and determined the effect of dysfunctional TGF-β signaling on the response to the chemotherapeutic agent 5-Fluoro-uracil (5FU) after induction of CRC. Using shotgun metagenomic sequencing, we determined gut microbiota composition in mice with CRC and found reduced amounts of beneficial species of Bacteroides and Parabacteroides in the mutants compared to the wild-type (WT) mice. Furthermore, the mutant mice with CRC were resistant to 5FU. Whereas the abundances of E. boltae, B.dorei, Lachnoclostridium sp., and Mordavella sp. were significantly reduced in mice with CRC, these species only recovered to basal amounts after 5FU treatment in WT mice, suggesting that the alterations in the intestinal microbiome resulting from compromised TGF-β signaling impaired the response to 5FU. These findings could have implications for inhibiting the TGF-β pathway in the treatment of CRC or other cancers.     

Two Antiproliferative Triterpene Saponins from Nematostylis anthophylla from the Highlands of Central Madagascar

Investigation of the endemic Madagascan plant Nematostylis anthophylla (Rubiaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the known triterpene saponin randianin ( 1 ), and the two new bioactive triterpene saponins 2″‐O‐acetylrandianin ( 2 ) and 6″‐O‐acetylrandianin ( 3 ). The structures of the two new compounds were elucidated based on analysis of their 1D‐ and 2D‐NMR spectra, and mass spectrometric data. The three isolated triterpene saponins displayed moderate but selective antiproliferative activities, with IC₅₀ values of 1.2, 1.7, and 2.2 μM , respectively, against the A2780 ovarian cancer, but only weak inhibitions of the proliferation of A2058 melanoma and the H522 lung cancer cell lines.     

Infant birth weight and third trimester maternal plasma markers of vascular integrity: the MIREC study

Background: There is paucity of information on mechanisms constituting adverse birth outcomes. We assessed here the relationship between vascular integrity and adverse birth effects.

Methods and results: Third trimester maternal plasma (n?=?144) from the Maternal-Infant Research on Environmental Chemicals Study (MIREC) was analysed for vascular, inflammatory and oxidative stress markers by HPLC-fluorescence, protein array and EIA method. Analysis of the <25th and >75th percentile birth weight subgroups revealed markers associated with birth weight (ETs, MMP-9, VEGF, and 8-isoPGF-2α) and gestational age (ET-1, MMP-2, and VEGF).

Conclusions: Mechanistic insights into adverse birth outcome pathways can be achieved by integrating information on multiple biomarkers, physiology using systems biology approach.     

Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions

COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.     

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.