Enhanced detection of glycoproteins in polyacrylamide gels

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

HIV promoter activity in primary antigen-specific human T lymphocytes

Human retroviruses, such as the HIV, infects human T cells, and efficient HIV replication occurs primarily in activated T cells rather than resting cells. Increased HIV production is likely caused by the activation of the retroviral promoter, and the HIV promoter may be regulated by intracellular signals induced during immune stimulation. To examine the regulation of retroviral promoter activity in normal, Ag-specific primary T lymphoblasts, a heterogeneous population of primary human T cells was transfected with either the HIV promoter or a promoter from a different retrovirus, Rous sarcoma virus (RSV) by protoplast fusion technique. Transfected T cells responded normally to Ag or mitogen stimulation, and activation of these T cells increased both the HIV and RSV promoter activity. Promoter activity was assessed by using transient expression assays after the T cells were restimulated with Ag, mitogen, or IL-2. In situ hybridization of transfected human T cells showed that 68 to 95% of activated lymphocytes expressed CAT mRNA directed by HIV or RSV. Thus, protoplast transfection of primary T cells was efficient in that the majority of cells expressed CAT message. By deletion of different regions of the HIV promoter, the enhancer region was identified as necessary for effective HIV promoter activity. In addition these deletion studies identified a region that negatively affects HIV promoter activity in primary T cells. Cotransfection of the HIV promoter with the HIV transactivator protein, tat, increases HIV promoter activity in both resting and activated primary human T cells only when the tat target sequences were present.     

Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies

The mutagenic effects of vinyl chloride monomer in man were studied in the lymphocyte culture with 3 methods: the chromosome aberration assay, the micronucleus assay and the sister-chromatid exchange method. Compared with control, values obtained by these tests are increased in workers occupationally exposed to vinyl chloride. In relation to non-smokers, smokers exposed to vinyl chloride show significant increases in sister-chromatid exchange frequencies. The problem of correlating the results of the chromosome aberration assay with micronucleus and sister-chromatid exchange frequencies is discussed.     

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

Insulin binding sites on rat liver nuclear membranes: biochemical and immunofluorescent studies.

Preliminary investigations (Horvat et al., '75) indicated the nucleus of rat liver as a site for specific binding of insulin. In this report these observations are confirmed. Nuclei from rat liver were isolated in a highly purified state as verified by interference contrast and electron microscopy and by chemical analysis. Extensive scanning of the preparations did not reveal the presence of structures resembling plasma membranes. The nuclear envelope was isolated by a modification of the method of Kay et al. ('72). Electron micrographs showed the presence of nuclear "ghosts" and few other recognizable nuclear elements, but no plasma membranes (60--80 A thick) were detected. The preparation was found to contain specific insulin binding activity. Specificity of the binding sites for insulin was demonstrated in competition studies with other polypeptide hormones and a synthetic insulin analog. Scatchard analysis of the binding data indicates the presence of a single class of high affinity receptors. In contrast to findings with plasma membranes the hormone-receptor complex is very stable and the kinetics of the dissociation of bound [125I]-insulin do not indicate negative cooperativity of the binding sites. Immunofluorescent labeling of intact, unfixed nuclei showed a specific fluorescent halo only around those nuclei that have been preincubated with insulin. All other controls were negative.