Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

Effect of dietary pectin on the production of immunoglobulins and cytokines by mesenteric lymph node lymphocytes in mouse colitis induced with dextran sulfate sodium

The present study explores the dietary effect of pectin on the MLN lymphocyte functions of mice with dextran sulfate sodium (DS)-induced colitis. We found that the immunoglobulin (Ig)A level in mesenteric lymph node (MLN) lymphocytes was high, while the IgE level was lower, in mice fed with pectin than in those fed with cellulose. Interestingly, the fecal IgA concentration of the pectin-fed mice was significantly higher than that of the cellulose-fed mice. The concentrations of interferon-gamma and interleukin (IL)-2 treated with concanavalin A (ConA) were significantly higher in the pectin-fed group than in the cellulose-fed group. Although dietary pectin did not affect the IL-4 and IL-10 levels, the activation-induced IL-4 and IL-10 secretion was lower in MLN cells of the pectin-fed mice than of the cellulose-fed mice following DS-induced colitis. Based on these findings, we propose that the effect of dietary pectin on mice with DS-induced colitis is mediated by the manipulation of Th1 cells. Furthermore, the inhibitory effect of IL-4 and IL-10 by dietary pectin may play an important role in promoting a change in Th1/Th2 balance toward Th1-dominant immunity.     

A Second Tubulin Binding Site on the Kinesin-13 Motor Head Domain Is Important during Mitosis

Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.     

Enhanced aerobic granulation and nitrogen removal by the addition of zeolite powder in a sequencing batch reactor

The aim of this study was to evaluate the impact of zeolite powders on feasibility of rapid aerobic granulation in the column-type sequencing batch reactors. After 90 days' operation, aerobic granular sludge was formed in both reactors by altering influent chemical oxygen demand/nitrogen (COD/N) ratios. R1 with zeolite powders had better removal capabilities of COD and total nitrogen than R2, which was without zeolite powders. Mixed liquor volatile suspended solid concentrations of the two reactors were 7.36 and 5.45 g/L, while sludge volume index (SVI₃₀) values were 34.9 and 47.9 mg/L, respectively. The mean diameters of aerobic granular sludge in the above two reactors were 2.5 and 1.5 mm, respectively. Both reactors achieved the largest simultaneous nitrification and denitrification (SND) efficiency at an influent COD/N ratio of 8; however, R1 exhibited more excellent SND efficiency than R2. The obtained results could provide a novel technique for rapid aerobic granulation and N removal simultaneously, especially when treating nitrogen-rich industrial wastewater.     

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.