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Biomedical subjects

G Griffiths

Publications and source records attributed to G Griffiths.

At least 145 records · Page 8Linked to original sources
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

The arguments for pre-existing early and late endosomes.

The past decade has seen the elucidation of many of the events and processes responsible for receptor-mediated endocytosis. However, a fundamental question about the endocytic pathway remains unresolved: do early endosomes mature into late endosomes, or are these two distinct and pre-existing cellular organelles? General opinion tends to favour the former possibility, to the point where one poster session at the recent American Society for Cell Biology meeting was entitled 'Maturation of Endosomes'. This article draws together new data arguing in favour of pre-existing early and late endosomes, between which transport occurs by vesicle budding and fusion.

Journal Article↗
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↗
PubMed1991

Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain.

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.

Biological Transport↗
PubMed1991

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

Animals↗
PubMed1991

Comparative behavior of lysosomes and the pre-lysosome compartment (PLC) in in vivo cell fusion experiments.

Interspecies cell fusion was used to compare protein intermixing within the mannose 6-phosphate receptor (MPR)-enriched pre-lysosome compartment (PLC) and within the MPR-negative lysosomal compartment. Both compartments were positive for lysosomal glycoprotein (lgp) membrane markers but were morphologically distinct. In most experiments, rat-mouse cell syncytia were formed by u.v.-inactivated Sindbis virus-mediated fusion. By immunogold electron microscopy of syncytia, extensive intermixing of species-specific lysosomal membrane proteins was observed in both lysosomes and PLC. At 3 h post cell fusion, multiple-label immunogold studies showed that 82% of the lysosome-like structures positive for the rat lysosomal membrane protein LIMP-I were also positive for the mouse lysosomal membrane protein mLAMP-1. By immunofluorescence, LIMP-I and mLAMP-1 co-localized with a t1/2 of 30 min after cell fusion; although the lgp-positive organelle populations had evidently interchanged their proteins, the lysosomal structures remained small, punctate bodies distributed throughout the syncytoplasm as observed in single cells. In contrast, the initially separate units of the PLC congregated with a t1/2 of 1 h to form large, pre-lysosome complexes associated with individual nuclear clusters. At the electron-microscope level, gold markers endocytized by the rat and mouse parent cells in a 1 h uptake followed by a 16-20 h chase co-localized in these extended PLC complexes, as did the membrane markers mLAMP-1 and LIMP-I. The density of labeling for rat MPR in the extended PLCs was markedly decreased, consistent with membrane fusions and dilution of the antigen upon congregation of the PLC compartments from the donor cells. The extended PLC complex behaved as a late endocytic compartment, as shown by co-localization of the MPR and rhodamine-dextran following a 10 min dextran uptake and a 50 min chase. These differences in behavior between lysosomes and the PLC in rat-mouse cell syncytia suggest that the pathway(s) of protein intermixing with respect to the two organelles may be different.

Animals↗
PubMed1990

A mitochondrial import receptor for the ADP/ATP carrier.

We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor.

Animals↗
PubMed1990

A T. cruzi-secreted protein immunologically related to the complement component C9: evidence for membrane pore-forming activity at low pH.

Protozoan parasite T. cruzi invades cells within acidic vacuoles, but shortly afterward escapes into the cytosol. Exit from the phagosome is blocked by raising the pH of acidic compartments, suggesting that a previously described acid-active hemolysin secreted by T. cruzi might be involved in the membrane disruption process. Here we show that T. cruzi supernatants are cytotoxic for nucleated cells at pH 5.5 and contain a protein reactive with antibodies against reduced and alkylated human C9 (the ninth component of complement). The C9 cross-reactive protein (TC-TOX) copurified with the cytolytic activity, and the active fractions induced conductance steps characteristic of transmembrane ion channels in planar phospholipid bilayers. Immunocytochemical studies using antibodies against purified TC-TOX showed that the protein was localized to the luminal space of parasite-containing phagosomes. We postulate that TC-TOX, when secreted into the acidic environment of the phagosome, forms pores in the membrane, which contribute to its disruption.

Animals↗
PubMed1990

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.

Cytochromes c1↗
PubMed1990

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

Actin Cytoskeleton↗
PubMed1990

Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells.

The structure of a late endosomal compartment, which contains the bulk of the cation-independent mannose 6-phosphate receptor (MPR) in NRK cells, is documented using immunocytochemistry and cryo-sections, as well as conventional Epon sections. This compartment, which we refer to as the prelysosomal compartment (PLC), has a complex three-dimensional structure consisting of tubuloreticular domains in continuity with vesicular parts. The latter are characterized by a high density of internal membranes, which may be either tubular or sheet-like, that label extensively for the MPR. This structural organization was also maintained after fractionation in sucrose gradients. The amount of MPR immunolabelling was then quantitated with respect to the membrane surface areas of the four compartments where it is found: namely, the plasma membrane, early endosomes, the trans Golgi network and the PLC. The results showed that in NRK cells 90% of the labelling for the receptor was found in the PLC, with the rest distributed over the other three compartments. Cytochemical studies indicated that the PLC is the first structure along the endocytic pathway that gives a significant reaction for acid phosphatase. However, the PLC is clearly distinct from the MPR-negative lysosomes, which are also acid phosphatase-positive, since the two organelles could be physically separated from each other after fractionation on Percoll gradients.

Animals↗
PubMed1990

Ultrastructural localization of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase to subcellular compartments active in endocytosis and recycling of membrane receptors.

The subcellular distribution of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase (PK-A) was analyzed at the electron-microscopical level using thawed cryo-sections of Madin Darby Bovine Kidney (MDBK) cells. The highest density of labelling for RII was found on membranes of the prelysosomal compartment (PLC; marked with the cation-independent mannose 6-phosphate receptor, MPR) and the trans-Golgi network, TGN (at 20 degrees C, marked with the G protein of vesicular stomatitis virus, VSV), as well as in coated buds on the latter. Significant labelling was also localized to the cytoplasmic surface of the plasma membrane, including clathrin-coated pits and microvilli, and to early endosomes (identified using internalized HRP). In contrast, no significant label was seen over the Golgi compartments proximal to the TGN, the endoplasmic reticulum (ER) or over lysosomes. From these results we conclude that PK-A type II is associated with the membranes of precisely those subcellular compartments that are active in endocytosis and recycling of cell surface receptors. We believe these findings to be related to the well-established role of cyclic AMP in signal transduction. In particular, we propose that activation of PK-A in endocytic compartments may contribute to regulation (via phosphorylation) of the subcellular distribution of internalized surface receptors or their functional coupling to effector systems involved in signal propagation.

Animals↗
PubMed1990

Endoscopic retrograde cholangiopancreatography using a mobile image intensifier.

A method of performing endoscopic cholangiopancreatography using a mobile intensifier in a theatre or endoscopy unit setting is described. A total of 140 ERCPs were attempted in 132 patients. The examination was diagnostically successful in 114 (85%) of patients and endoscopic sphincterotomy was performed in 54 (38.5%). There were 10 (18%) complications after sphincterotomy and one postoperative death after a retroperitoneal leak. With increasing expertise, it is hoped to match the figures from specialist centres, and this method is suggested as an alternative way to expand this service where X-ray department facilities are not readily available.

Adolescent↗
PubMed1989

MOM19, an import receptor for mitochondrial precursor proteins.

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit beta. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor.

Animals↗
PubMed1989

Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis.

The cation-independent mannose 6-phosphate receptor (Cl-MPR) sorts newly synthesized lysosomal enzymes in the Golgi and endocytoses extracellular lysosomal enzymes. To determine the role of the 163 amino acid cytoplasmic domain of the Cl-MPR in these functions, receptor-deficient mouse L cells were transfected with normal bovine Cl-MPR cDNA or cDNAs mutated in the cytoplasmic domain. The normal Cl-MPR functioned in sorting and endocytosis. Mutant receptors with 40 and 89 residues deleted from the carboxyl terminus of the cytoplasmic tail functioned normally in endocytosis, but were partially impaired in sorting. Mutant receptors with larger deletions leaving only 7 and 20 residues of the cytoplasmic tail were defective in endocytosis and sorting. A mutant receptor containing alanine instead of tyrosine residues at positions 24 and 26 was defective in endocytosis, and partially impaired in sorting. Receptors deficient in endocytosis accumulated at the cell surface. These results indicate that the cytoplasmic domain of the Cl-MPR contains different signals for rapid endocytosis and efficient lysosomal enzyme sorting.

Amino Acid Sequence↗