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Biomedical subjects

M Marsh

Publications and source records attributed to M Marsh.

At least 73 records · Page 4Linked to original sources
PubMed1995

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.

Animals↗
PubMed1995

A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.

Amino Acid Sequence↗
PubMed1994

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes.

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6-phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti-phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.

Amino Acid Sequence↗
PubMed1994

Umbilical vessel wall fatty acids after normal and retarded fetal growth.

In a prospective observational study, the fatty acid content of human umbilical artery and vein wall phospholipids was determined in fetuses classified according to their change in abdominal circumference during the third trimester. Three groups were identified: appropriate for gestational age (AGA; 24 infants) and small for gestational age (SGA; 38 infants) with normal antenatal growth rate, and SGA with fetal growth retardation (22 infants). The venous linoleic acid (18:2 omega 6) content (expressed as a percentage of the total fatty acids identified) was greater in growth retarded SGA fetuses (3.5 (0.6)%) than in SGA fetuses with a normal growth rate (3.1 (0.5)%) and AGA fetuses (3.0 (0.5)%), whereas the venous contents of eicosatrienoic (20:3 omega 6) and docosahexaenoic acid (22:6 omega 3) were lower. In growth retarded SGA fetuses, the venous and arterial 20:3 omega 6 content correlated with the change in abdominal circumference. In SGA fetuses with a normal growth rate, lower contents of arterial 18:2 omega 6 and 22:6 omega 3 were associated with a smaller change in abdominal circumference and birth weight. Different metabolic derangements appear to underly normal and subnormal growth rate in SGA fetuses, suggesting that different strategies of dietary intervention may be required to aid fetal growth and reduce the sequelae of fetal growth retardation.

Abdomen↗
PubMed1993

Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting.

The phorbol ester phorbol myristate acetate (PMA) induces a rapid downregulation of CD4 from the surface of T cells and lymphocytic cell lines, as well as from CD4-transfected nonlymphoid cells. Here we have studied the mechanisms of this phorbol ester-induced CD4 modulation. Using HeLa-CD4 or NIH-3T3-CD4 cells, in which the endocytosis of CD4 is not influenced by the protein tyrosine kinase p56lck, we show that PMA enhanced the uptake of CD4, increasing the rate of CD4 endocytosis three to five-fold, and doubling the proportion of CD4 found inside the cells. Trafficking of a CD4 mutant lacking the major portion of the cytoplasmic domain, as well as fluid phase endocytosis were not affected by PMA treatment. Studies in which clathrin-coated pits were disrupted through the use of hypertonic media indicated that both the constitutive and PMA-induced CD4 uptake occurred through coated vesicles. Electron microscopy demonstrated directly that PMA increases the association of CD4 with coated pits. Immunofluorescent staining of internalized CD4 showed that PMA also diverted CD4 from the early endosome-plasma membrane recycling pathway to a mannose 6-phosphate receptor-containing late endosomal compartment. In lymphoid or p56lck-expressing transfected cells, these effects were preceded by the PMA-induced dissociation of CD4 and p56lck, which released CD4 and made possible increased endocytosis and altered intracellular trafficking. Together these results indicate that phorbol esters have multiple effects on the normal endocytosis and trafficking of CD4, and suggest that phosphorylation may influence the interaction of CD4 with coated pits.

3T3 Cells↗
PubMed1993

Expression of HIV-1 envelope glycoproteins by Semliki Forest virus vectors.

We have used Semliki Forest virus (SFV) vectors to express both the human immunodeficiency virus type 1 (HIV-1) envelope precursor gp160 and the cleaved external portion gp120. Expression of the foreign gene in this system is by transfection of recombinant SFV RNA, or by infection with a recombinant SFV virus that has a wide host range. pSFV1-gp120 or pSFV1-gp160 were expressed in baby hamster kidney (BHK) cells and two human cell lines: HeLa cervical carcinoma and MOLT-4 CD4+ T cells. After SFV1-gp120 infection of HeLa cells, 3.3 micrograms of gp120 was secreted into the media by 1 million cells in a 24-hr period. The secreted envelope glycoprotein was recognized by anti-gp120 monoclonal antibodies directed against both linear and conformation-dependent epitopes in different regions of the molecule. The recombinant gp120 also bound to a soluble form of the CD4 receptor. Syncytium formation was observed when MOLT-4 cells were infected with SFV1-gp160. The gp160 expressed by BHK cells induced syncytia during cocultivation with C8166 CD4+ T cells. These data indicate that SFV vectors can be used to produce the HIV-1 envelope glycoproteins to high levels, and that these proteins are correctly processed, folded, and transported to the cell surface. Furthermore, they exhibit functional activity as indicated by their ability to bind to soluble receptor and induce cell-to-cell fusion.

Animals↗
PubMed1992

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits.

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

3T3 Cells↗
PubMed1992

The prognostic value of fetal ultrasonography before induction of labour.

OBJECTIVE: To investigate whether ultrasound examination of the fetus and amniotic fluid before induction of labour can provide useful prognostic information about the course of labour and mode of delivery. DESIGN: A prospective observational study. SETTING: The delivery suite of St Mary's Hospital, London. SUBJECTS: 101 women undergoing induction of labour. MEASURED VARIABLES: Biparietal diameter, head circumference, abdominal circumference, occipital position, amniotic fluid volume and umbilical artery Doppler velocimetry waveforms. MAIN OUTCOME MEASURES: The outcome of labour was assessed in terms of the induction delivery interval, the mode of delivery and the incidence of abnormal cardiotocograms in labour. RESULTS: Seven women were delivered by emergency caesarean section early in the first stage of labour because of a significant abnormality of the fetal heart trace and these pregnancies were characterized by small fetal abdominal circumference measurements. Marked oligohydramnios was also noted in five of these seven subjects. Small or average size babies who were surrounded by an adequate volume of amniotic fluid tended to be born spontaneously, whereas the majority of operative deliveries for failure to progress in labour were associated with fetal abdominal circumference measurements > or = 340 mm. The mean Bishop score of women before labour who delivered spontaneously was not significantly different from the scores of women who had a forceps delivery or caesarean section. Abnormal cord Doppler waveforms were observed in only one instance. CONCLUSIONS: In women undergoing induction of labour, the measurement by ultrasonography of two variables (fetal abdominal circumference and amniotic fluid volume) may allow the prospective identification of pregnancies at increased risk of fetal distress or dystocia. If these findings can be replicated in early spontaneous labour than more rational utilization of resources may be possible in hospital based obstetric practice.

Amniotic Fluid↗
PubMed1992

Update on endocrinology.

Endocrinology covers diverse groups of organs and diseases. In this article, advances in the investigation of thyroid dysfunction, the management of Paget's disease of bone and the investigation and management of polycystic ovary syndrome are discussed.

Calcitonin↗