Nancy P. Y. Chung

Silver nanoparticles fabricated in Hepes buffer exhibit potent cytoprotective and post-infected anti-HIV-1 activities toward Hut/CCR5 cells.

The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl(2)-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 x 10(6) cells/cm(2)) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl(2) at 5-10 microM for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl(2) (0.1-5 microM) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl(2) also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl(2), the presence of testosterone (T) at 1 x 10(-9) M allowed resealing of the inter-Sertoli TJ barrier after CdCl(2) was removed, whereas the presence of 2 x 10(-7) M testosterone even protected Sertoli cells from CdCl(2)-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl(2)-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl(2) treatment. Based on these results, it is apparent that CdCl(2)-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.

Polycomb group (PcG) proteins such as Enhancer of zeste homolog 2 (EZH2) are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. Targets of PcG include cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. Senescence is a characteristic of melanocytic nevi, benign melanocytic proliferations that can be precursors of malignant melanoma. In this study, we report that EZH2, which we find absent in melanocytic nevi but expressed in many or most metastatic melanoma cells, functionally suppresses the senescent state in human melanoma cells. EZH2 depletion in melanoma cells inhibits cell proliferation, restores features of a cellular senescence phenotype, and inhibits growth of melanoma xenografts in vivo. p21/CDKN1A is activated upon EZH2 knockdown in a p53-independent manner and contributes substantially to cell cycle arrest and induction of a senescence phenotype. EZH2 depletion removes histone deacetylase 1 (HDAC1) from the CDKN1A transcriptional start site and downstream region, enhancing histone 3 acetylation globally and at CDKN1A. This results in recruitment of RNA polymerase II, leading to p21/CDKN1A activation. Depletion of EZH2 synergistically activates p21/CDKN1A expression in combination with the HDAC inhibitor trichostatin A. Since melanomas often retain wild-type p53 function activating p21, our findings describe a novel mechanism whereby EZH2 activation during tumor progression represses p21, leading to suppression of cellular senescence and enhanced tumorigenicity.

The lack of a primate model that utilizes HIV-1 as the challenge virus is an impediment to AIDS research; existing models generally employ simian viruses that are divergent from HIV-1, reducing their usefulness in preclinical investigations. Based on an understanding of species-specific variation in primate TRIM5 and APOBEC3 antiretroviral genes, we constructed simian-tropic (st)HIV-1 strains that differ from HIV-1 only in the vif gene. We demonstrate that such minimally modified stHIV-1 strains are capable of high levels of replication in vitro in pig-tailed macaque (Macaca nemestrina) lymphocytes. Importantly, infection of pig-tailed macaques with stHIV-1 results in acute viremia, approaching the levels observed in HIV-1-infected humans, and an ensuing persistent infection for several months. stHIV-1 replication was controlled thereafter, at least in part, by CD8+ T cells. We demonstrate the potential utility of this HIV-1-based animal model in a chemoprophylaxis experiment, by showing that a commonly used HIV-1 therapeutic regimen can provide apparently sterilizing protection from infection following a rigorous high-dose stHIV-1 challenge.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.