Biomedical subjects

G Griffiths

Publications and source records attributed to G Griffiths.

At least 127 records · Page 7Linked to original sources

PubMed1993

The actin-binding protein comitin (p24) is a component of the Golgi apparatus.

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.

3T3 Cells↗

PubMed1993

Cytoplasmic dynein-dependent vesicular transport from early to late endosomes.

We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.

Animals↗

PubMed1993

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction.

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.

Base Sequence↗

PubMed1993

Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway.

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

Animals↗

PubMed1992

O-glycosylation of the coronavirus M protein. Differential localization of sialyltransferases in N- and O-linked glycosylation.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.

Animals↗

PubMed1992

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.

Animals↗

PubMed1992

Lipid composition of subcellular membranes from larvae and prepupae of Drosophila melanogaster.

Subcellular membranes were analyzed for their lipid composition and protein content at two developmental points representing the third instar wandering larvae and prepupal stages of Drosophila. At both stages, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major constituents with phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) being relatively minor components. In total homogenates and in the nuclear-enriched fraction there was no significant difference in the phospholipid composition of the wandering larvae and prepupae. In mitochondria only a significant increase in the minor component PS was observed in the prepupae. In lysosomal membranes on the other hand, the relative abundance of the major components PE and PC increased in the prepupae although the molar ratios of the two lipids remained almost constant. The fatty acid composition of the phospholipids remained virtually unchanged in all of the fractions examined, including the lysosomes, and there was no evidence of lipid peroxidation. With regard to cellular degeneration and the involvement of lysosomes, we conclude that mechanisms other than gross modification of the lipid and/or lipid/protein ratio of their membranes are involved in the liberation of the acid phosphatase contents.

Animals↗

PubMed1992

Cell biology of viruses that assemble along the biosynthetic pathway.

In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and pox-viruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.

Animals↗

PubMed1992

Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages.

We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA-gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP-binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment.

Animals↗

PubMed1992

Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.

Alpharetrovirus↗

PubMed1992

A block in degradation of MHC class II-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment.

Invariant chain (Ii) associated with MHC class II molecule is processed proteolytically via several distinct intermediates during its intracellular transport through endosomal compartments. Leupeptin added to the culture medium blocks processing of Ii, prevents its dissociation from the class II molecules and leads to an intracellular accumulation of a 22 kDa intermediate form of Ii. We show here that leupeptin has a very general effect on protein transport in the endocytic pathway. When added to Mel Juso cells leupeptin reduces the transport of endocytosed material from multivesicular body-like, endosome carrier vesicles (ECV) to the prelysosomal compartment (late endosome) and leads to a concomitant increase in the number of ECV. Our results argue that one effect of leupeptin, related to antigen processing and presentation, is to block transport of antigen and/or MHC class II molecules to prelysosomal compartments.

Antigen-Presenting Cells↗

PubMed1991

Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.

The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.

Animals↗

PubMed1991

A 14-kDa Schistosoma mansoni polypeptide is homologous to a gene family of fatty acid binding proteins.

The complete nucleotide sequence encoding a Schistosoma mansoni protein termed Sm14 was determined from cDNA clones propagated in bacteriophage lambda gt11 in Escherichia coli. The 14.8-kDa protein bears significant homologies with a family of related polypeptides which bind hydrophobic ligands. Members of this group of cytosolic proteins were originally identified based on their affinity for long chain fatty acids. The purified recombinant protein exhibited an affinity to fatty acids, in contrast to a mutant lacking 16 N-terminal amino acids. Immunofluorescence experiments show that tubercles, which are structures located on the dorsal surface of adult male schistosome and known to contain lipids, are stained using antibodies raised to the beta-galactosidase fusion protein. A regular staining pattern is also evident in the muscle layers as well as in the body of the parasite. As the schistosome cannot synthesize fatty acids de novo and is dependent on the uptake of lipids from serum, the available data support a role for Sm14 in the transport of fatty acids.

Animals↗

PubMed1991

Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells.

The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4-associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells.

CD4 Antigens↗

PubMed1991

Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin.

We have cloned and sequenced beta-COP, a peripheral 110 kd Golgi membrane protein. beta-COP shows significant homology to beta-adaptin. It is present in a membrane-bound form and in a cytosolic complex of 13-14S, with a Stokes radius of approximately 10 nm and an estimated Mr of approximately 550,000. By immunofluorescence labeling, beta-COP is associated with the structures of the Golgi complex. Immunoelectron microscopy has localized beta-COP to non-clathrin-coated vesicles and cisternae of the Golgi complex. These coated vesicles accumulate in rat liver Golgi fractions treated with GTP gamma S and strongly label for beta-COP. Our data suggest that beta-COP is a component of a coat associated with vesicles and cisternae of the Golgi complex.

Amino Acid Sequence↗

PubMed1991

The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding.

Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.

Amino Acid Sequence↗

PubMed1991

Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina.

Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.

Animals↗

PubMed1991

Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

8-Bromo Cyclic Adenosine Monophosphate↗