Induction of cell cycle arrest and apoptosis by the proteasome inhibitor PS-341 in Hodgkin disease cell lines is independent of inhibitor of nuclear factor-kappaB mutations or activation of the CD30, CD40, and RANK receptors.

PURPOSE: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor kappaB (NF-kappaB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-kappaB and modulating critical cellular regulatory proteins, but its activity in cells carrying IkappaBalpha gene mutations has not been reported previously. Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot. RESULTS: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IkappaBalpha and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G(2)-M phase. PS-341 not only inhibited nuclear localization of NF-kappaB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2. CONCLUSIONS: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.     

Downregulation of human kallikrein 10 (KLK10/NES1) by CpG island hypermethylation in breast, ovarian and prostate cancers.

OBJECTIVE: The human kallikrein 10 (KLK10)/normal epithelial cell-specific-1 (NES1) gene is highly expressed in normal mammary, ovary and prostate cells, but its expression is dramatically decreased in cancer cell lines. Recently, it has been shown that CpG island hypermethylation of the KLK10 gene is responsible for the tumor-specific loss of KLK10 gene expression in certain breast cancer cell lines. METHOD: We examined the role of CpG island hypermethylation in the tumor-specific loss of KLK10 expression in breast, ovarian and prostate cancers. We treated cells with the demethylating agent 5-aza-2'-deoxycytidine (dC) and monitored changes in KLK10 mRNA by RT-PCR and secreted hK10 protein expression by ELISA. The following cell lines were used: MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-1, T-47D and BT-474 (breast); BG-1, MDAH-2774, HTB-75, HTB-161, PA-1 and ES-2 (ovary), and LNCaP and PC-3 (prostate). RESULTS: Upregulation of KLK10 mRNA levels, which was accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2'-dC. Genomic sequencing of sodium-bisulfite-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. CONCLUSION: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested.     

Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.     

Phleboviruses encapsidate their genomes by sequestering RNA bases

Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible α-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with “reading” by the viral polymerase.     

Health challenges for older women: Some implications for sexual health

Premature ejaculation (PE) seems to be the most common male sexual dysfunction. Our knowledge about the aetiology (causative and contributing factors) and neurophysiology of the disorder is still incomplete, and there is a lack of diagnostic criteria that are accepted universally. It seems that both biogenic and psychogenic-relationship factors might be implated and interact; however, there is a need for more research to clarify the issue. The treatment of PE is also a controversial issue, and the long-term outcome of therapy is often poor. The present paper looks at these issues, discusses medical, non-medical and combined approaches and reviews the relevant literature.     

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1

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