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Biomedical subjects

M Levine

Publications and source records attributed to M Levine.

At least 487 records · Page 27Linked to original sources
PubMed1983

Expression of herpes simplex virus beta and gamma genes integrated in mammalian cells and their induction by an alpha gene product.

The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed alpha, beta, and gamma, whose synthesis is regulated in a cascade fashion. alpha products are synthesized first during infection, and they are required for synthesis of beta and gamma proteins. To examine the expression of several HSV-1 beta and gamma genes in the absence of alpha functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only beta and gamma genes (0.315 to 0.421 map units). We found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the beta and gamma classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 alpha gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 beta and gamma genes can be transcribed in the absence of alpha functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the alpha gene product ICP4.

Animals↗
PubMed1983

Studies of the biosynthesis and metabolism of rat testicular galactoglycerolipids.

1-O-Alkyl-2-O-acyl-3-O-beta-D-(3'-sulfatoxygalactopyranosyl)-sn- glcerol (SGG) and its nonsulfated analog (GG) are the major glycolipids of rat testis. Aspects of the biosynthesis and metabolism of these two lipids have been investigated by determining their specific activities at various times after injection of [16-14C]palmitic acid, [1-14C]cetyl alcohol, and D-[1-14C]galactose into the testes of the adult rats. Evidence was obtained from studies with each of these three radioactive compounds that is consistent with the interpretation that GG exhibits a precursor relationship to SGG in vivo. The turnover time of GG, as estimated from the use of each of the three precursors, ranged between 21 and 69 h. In contrast, a slow increase of radioactivity in SGG was observed following injection of each of the three precursors, a plateau value being reached between 72 and 168 h. The radioactivity in the acyl, alkyl, and galactosyl moieties of SGG thereafter remained quite constant for another 21 days. Small amounts of monoalkylmonoacylglycerol were detected in rat testis. Radioactive studies indicated that this compound could be a precursor of GG and (or) monoalkyldiacylglycerol, another lipid that was also detected in rat testis. The results are consistent with the concept that the synthesis of SGG occurs primarily at an early stage of spermatogenesis and that the various moieties of this lipid exhibit almost complete metabolic stability during the subsequent complex stages of this process.

Animals↗
PubMed1982

Method or madness: on the alienation of the professional.

Burnout has affected a significant number of professionals in research and in clinical practice. The Marxian theory of alienation is insufficient to account for the phenomenon among professional workers. A critical factor in professional alienation is, paradoxically, the very factor which distinguishes the professional from the layman, namely the reliance on method and concept to develop knowledge. The alienating character of professional method may be observed in varying degrees in all human service professions. Method alienates the professional from the subject matter of interest, and to some degree from his or her own perceptions and interests. In the course of attempting to solve problems, too often the emphasis changes from understanding the substantive problem to checking and correcting artifacts of method, losing the focus on the original substantive problem, as the work turns in on itself. The cure requires professional social support for studies better suited to illuminate the human condition and to enlighten and intrigue the professional worker.

Humans↗
PubMed1982

Study of behavioral factors associated with psychiatric rehospitalization.

Archival data from daily logs maintained at a private proprietary home were tracked from four to 29 weeks prior to psychiatric rehospitalization for ten deinstitutionalized adult females and ten nonhospitalized controls. Three raters coded behavioral incidents into ten categories. The rehospitalized women had significantly more incidents than did the controls, including more affective disturbances (depression, anxiety, anger, and interpersonal concerns) and more attempts to secure psychiatric rehospitalization. The rehospitalized groups also exhibited symptomatic behavior for a greater number of weeks than did the control group, suggesting that the symptom pattern was probably detectable long before rehospitalization was necessary. Peaks in incidence frequency often occurred several weeks prior to rehospitalization.

Adult↗
PubMed1982

Immunological method for mapping genes on Drosophila polytene chromosomes.

A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly, (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.

Animals↗
PubMed1982

Detection, isolation and characterization of cell free HLA A and B antigens from human amniotic fluid.

Cell-free HLA A and B antigens, paternal and maternal, have been detected in human amniotic fluids obtained from women at 16-18 weeks' gestation. Fractionation of amniotic fluid samples on sephadex showed that HLA A and B antigen activity was mainly in the 40,000 to 70,000 mol. wt. fractions. Lentil lectin sepharose 4B affinity chromatography of 30,000 to 70,000 sephadex molecular weight fraction of amniotic fluids isolated proteins which gave 2 bands, M and N, on discontinuous polyacrylamide gel electrophoresis (disc PAGE without SDS). SDS disc PAGE of the LcH bound glycoproteins recovered from amniotic fluids indicated 4 main protein bands approximately 12,900, 32,000, 52,600 and 78,500 mol. wt. Schiff's stain of the SDS gels showed that all the proteins except the 12,900 mol. wt. polypeptide contained carbohydrate. It was suggested that the 12,900 and 32,000 mol. wt. proteins obtained on disc PAGE gels represented the 2 subunits of the HLA A and B antigen molecule, i.e., beta 2-microglobulin and the allospecific heavy chains, similar to the papain-solubilized moiety of membrane antigens. In the absence of SDS the 2 polypeptides migrate in polyacrylamide gels as 1 protein band M, representing the intact (undissociated) antigen molecules. Enzyme linked immunosorbent assays were proposed for the estimation of cell-free HLA antigens.

Amniotic Fluid↗
PubMed1982

Developmentally regulated gene expression in Drosophila larval fat bodies.

During third-instar larval development of Drosophila melanogaster, the fat body tissue synthesizes six major methionine-containing polypeptides, three of which are the alpha, beta, and gamma subunits of the hexameric larval serum protein LSP-1, a fourth is the single subunit of the hexameric larval serum protein LSP-2, and the other two are polypeptides P6 and P1. Genomic DNA clones of the six structural genes for the polypeptides were isolated and characterized. Each gene maps by in situ hybridization at a single chromosomal site and appears to be present as a single copy in the genome. The LSP-1 and LSP-2 genes show striking regulatory similarities: The LSP-1 beta and gamma transcripts are first detected in fat bodies within an hour after the second molt, and the LSP-1 alpha and LSP-2 transcripts a few hours later; the four transcripts are subsequently maintained at high levels during most of the third instar and rapidly decrease shortly before pupariation. Ecdysterone increases the levels of at least three of the four LSP transcripts in the fat bodies when ecdysterone-deficient larvae from the temperature-sensitive mutant ecd1 are supplemented with the hormone. The regulatory characteristics of the P6 and P1 genes differ in several ways from those of the LSP genes. Expression of the P6 and P1 genes begins later than the LSP genes, and the levels of the transcripts remain high at the end of the third instar after the LSP transcripts have markedly decreased. Ecdysterone increases the level of the P1 transcript, but not of the P6 transcript, in ecdysterone-deficient ecd1 larvae.

Adipose Tissue↗
PubMed1981

Constancy of somatic DNA organization in developmentally regulated regions of the Drosophila genome.

The purpose of this study was to test for the occurrence of changes in the organization, modification, or selective amplification of six developmentally regulated regions of genomic DNA during Drosophila development. Five of the regions contain structural genes, each of which maps at a single chromosomal site; the sixth region contains a dispersed segment that maps at about 30 different sites and appears to be transposable. The RNA transcripts and encoded proteins of the structural genes are major components of the fat body tissue in late third-instar larvae, in contrast to other larval tissues and young embryos in which the transcripts and proteins are hardly detectable. The dispersed segment shows the reverse developmental regulation: the amount of transcript is relatively high in young embryos and low in larval fat bodies. The test for changes in genomic DNA associated with the regulated expression of the six regions was based on comparative restriction mapping of the DNA from these sources. Genomic clones containing the transcribed and also flanking regions of DNA were used as probes to determine the positions of the complementary restriction fragments after electrophoresis in agarose gels. Each test, which involved digesting the genomic DNA samples with one of the endonucleases and probing with one of the clones, produced identical restriction maps for the different samples. The tests are capable of detecting changes in the organization of the cloned regions resulting from addition or removal either of endonuclease cleavage sites or of segments of DNA located between cleavage sites. Because the activities of several of the endonucleases, and probably all, are sensitive to methylation of one of the bases in the DNA recognition sequence, certain modifications of the cloned regions by methylation could also be detected. Although it is not certain that genomic reorganization or modification would have been detected by these tests, the number of endonucleases used was sufficient to provide persuasive evidence that such changes did not occur. Furthermore, the quantitative as well as qualitative similarities of the restriction maps in each test indicated that there was no significant selective amplification of the DNA within the cloned regions.

Adipose Tissue↗
PubMed1981

Method for induction of mutations in physically defined regions of the herpes simplex virus genome.

A procedure was developed for inducing mutations in isolated restriction enzyme fragments of herpes simplex virus type 1 (HSV-1) DNA with nitrous acid. The mutations were then transferred to the viral genome by genetic recombination during cotransfection of rabbit kidney cells with the mutagenized fragments and intact HSV-1 DNA. The HpaI restriction enzyme fragments LD, B, LG, I, and J were mutagenized. Temperature-sensitive mutants were found at frequencies of 1 to 5% among the progeny of the transfections. Syncytial mutants also were found at high frequency when fragment B or LD was used for mutagenesis. Fifteen of these mutants, 11 temperature sensitive and 4 syncytial, were used for further studies, including complementation analysis, DNA synthesis, and marker rescue. Marker rescue data presented here and in the accompanying publication (A. L. Goldin, R. M. Sandri-Goldin, M. Levine, and J. C. Glorioso, J. Virol. 38: 50-58, 1981) confirm the map position of some of the newly isolated mutants.

DNA Restriction Enzymes↗
PubMed1981

Cloning of herpes simplex virus type 1 sequences representing the whole genome.

Sequences representative of the whole genome of herpes simplex virus type 1 (HSV-1) strain KOS were cloned in the plasmid vector pBR325 in the form of EcoRI-generated DNA fragments. The cloned fragments were identified by digestion of the chimeric plasmid DNA with restriction enzymes EcoRI or EcoRI and BglII followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested HSV-1 virion DNA. The cloned fragments showed the same migration patterns as the corresponding fragments from restricted virion DNA, indicating that no major insertions or deletions were present. The presence of HSV-1 sequences in the chimeric plasmids was confirmed by hybridization of plasmid DNA to HSV-1 virion DNA. Additionally, some of the cloned fragments were shown to be biologicaly active in that they efficiently rescued three HSV-1 temperature-sensitive mutants in cotransfection marker rescue experiments.

Cloning, Molecular↗
PubMed1981

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

Animals↗