Cytopathic variants of an attenuated isolate of human immunodeficiency virus type 2 exhibit increased affinity for CD4.

Naturally occurring isolates of human immunodeficiency virus (HIV) have been described which are deficient in their ability to fuse with and kill CD4+ target cells. Although the molecular basis for their attenuation has not yet been defined, several lines of evidence point toward the viral envelope gene as a key determinant of viral pathogenicity. In the present article, we report the biological characterization of two highly cytopathic variants derived by repeated cell-free passage of an attenuated isolate of HIV type 2 (HIV-2), termed HIV-2/ST. Unlike the parental virus, the cytopathic variants were found to infect Sup-T1 cells with great efficiency and to induce both cell fusion and profound killing in these cultures. To determine whether changes in the viral envelope gene were responsible for the observed phenotypic differences, we examined the CD4 binding affinity of these viruses using a novel assay designed to quantitate the binding of fluoresceinated CD4 to viral envelope in its native configuration on the cell surface. The results demonstrated that the affinity of parental HIV-2/ST envelope for CD4 was 2 orders of magnitude reduced, while the cytopathic variants exhibited a high CD4 binding affinity, comparable to that of cytopathic HIV-1 and HIV-2 isolates. From these data, we conclude that the cytopathic potential of HIV depends, at least in part, on its receptor-binding affinity. In addition, our study documents strong selection pressures for viruses with increased CD4 affinity during propagation in immortalized T-cell lines, thus emphasizing the need to study HIV envelope biology in natural target cells.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.      

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

Molecular signatures of human induced pluripotent stem cells highlight sex differences and cancer genes

Although human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct. Loss of XIST expression is strongly correlated with upregulation of X-linked oncogenes, accelerated growth rate in?vitro, and poorer differentiation in?vivo. Whereas differences in X-inactivation potential result in epigenetic variability of female hiPSC lines, male hiPSC lines generally resemble each other and do not overexpress the oncogenes. Neither physiological oxygen levels nor HDAC inhibitors offer advantages to culturing female hiPSC lines. We conclude that female hiPSCs may be epigenetically less stable in culture and caution that loss of?XIST may result in qualitatively less desirable stem cell lines.     

Structure-based design of eugenol analogs as potential estrogen receptor antagonists

Eugenol is an essential oil mainly found in the buds and leaves of clove (Syzygium aromaticum (L.) Merrill and Perry), which has been reported to have activity on inhibition of cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. This biological activity is correlated to its activity as an estrogen receptor antagonist. In this article, we present the construction and validation of structure-based virtual screening (SBVS) protocols to identify the potent estrogen receptor α (ER) antagonists. The selected protocol, which gave acceptable enrichment factors as a virtual screening protocol, subsequently used to virtually screen eugenol, its analogs and their dimers. Based on the virtual screening results, dimer eugenol of 4-[4-hydroxy-3-(prop-2-en-1- yl)phenyl]-2-(prop-2-en-1-yl)phenol is recommended to be developed further in order to discover novel and potent ER antagonists.