Biomedical subjects

G Griffiths

Publications and source records attributed to G Griffiths.

At least 109 records · Page 6Linked to original sources

PubMed1995

Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN.

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

Animals↗

PubMed1995

Treatment of the failing implant: case reports.

Three case reports of treatment of the failing implant are presented. The implants were immobile but had lost a significant amount of osseous support. The cause of failure was determined to be a combination of bacterial and occlusal traumatogenic insult. The defects were debrided and the implant surface was detoxified with tetracycline. Decalcified freeze-dried bone allograft was implanted in the osseous defects and covered with expanded polytetrafluoroethylene material in accordance with principles of guided tissue regeneration. The barrier membrane was removed 6 to 8 weeks after placement. Eight months to 1 year posttreatment, all sites demonstrated a substantial reduction in probing depth, a gain in clinical attachment, and bone fill of the defects adjacent to the implant.

Aged↗

PubMed1994

Molecular characterization of phagosomes.

The transformation of newly formed phagosomes into mature phagolysosomes is a process that involves a complex series of interactions between phagosomes and other vacuolar organelles. The machinery required by phagosomes to mediate these interactions is poorly understood. In this study, we allowed human and various rodent cells to take up latex beads whose density facilitates a simple purification of phagosomes using discontinuous sucrose gradients. With this system, we initiated a systematic study of phagosome proteins using two-dimensional gel electrophoresis and the currently available two-dimensional gel protein data bases. By this approach, we were able to recognize a group of polypeptides associated with mouse J774 phagosomes-phagolysosomes including annexin II, annexin VI, the beta-1 and beta-2 subunits of trimeric G proteins, and a group of actin-binding proteins. While the amount of annexin II associated to phagosomes was similar at all times of latex internalization, the levels of annexin VI were higher on late phagosomes. Phospholipid analysis of J774 phagosomes isolated at early and late time points during phagolysosome formation also revealed significant differences in their lipid composition. In the human phagosomes, we resolved over 200 polypeptides on the two-dimensional gels. These included the proteins described in the mouse, as well as 32 polypeptides that were found to be highly enriched in phagosomes, 15 of which are not present in the current data bases. The results demonstrate that the use of latex bead phagosomes is a powerful system to identify key molecules involved in phagolysosome biogenesis.

Animals↗

PubMed1994

The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif.

We have identified a new human Ca(2+)-binding protein that is specifically localized in the endoplasmic reticulum (ER). The protein is termed ERC-55, i.e. ER calcium-binding protein of 55 kDa. ERC-55 is a single copy gene and is encoded by an approximately 1900-base mRNA, which shows a ubiquitous expression pattern. The ERC-55 protein comprises an amino-terminal signal sequence followed by six copies of the EF-hand Ca2+ binding motif. Ca2+ binding was demonstrated directly for recombinant ERC-55 using the 45Ca2+ overlay technique. The carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL) is required for retention of ERC-55 in the ER. Deletion of HDEL results in slow secretion into the medium. In pulse-chase experiments, approximately 50% of the HDEL deletion mutant is secreted, whereas no detectable secretion is observed with the wild-type protein. This represents the first example of an endogenous human protein that is retained in the ER by an HDEL rather than Lys-Asp-Glu-Leu (KDEL) carboxyl-terminal tetrapeptide. Comparative sequence analysis indicates that ERC-55, together with the recently identified protein reticulocalbin (Ozawa and Muramatsu, 1993), constitute a new subfamily of the EF-hand superfamily of Ca(2+)-binding proteins that are specifically located in the ER.

Amino Acid Sequence↗

PubMed1994

Purification and biochemical characterization of acetylcholinesterase (AChE) from the excretory/secretory products of Trichostrongylus colubriformis.

Acetylcholinesterase (AChE) has been purified from the excretory/secretory (ES) products of Trichostrongylus colubriformis (using edrophonium chloride linked to epoxy-activated Sepharose) with yields of 40-50%. Purity was confirmed by polyacrylamide gel electrophoresis (using silver [protein] and Karnovsky [activity] stains) and measurement of specific AChE activity. Further analysis of the purified fractions by gel filtration and sucrose density gradient techniques revealed the existence of 2 forms of hydrophilic AChE (M(r) 189 and 80 kDa). From the data we deduce these to be the globular monomer and dimer, G1 and G2 forms of AChE. Inhibition studies using BW284C51, iso-OMPA and excess substrate, along with substrate specificity studies, show both forms to be true acetylcholinesterases. We are currently assessing the protective immunogenicity of purified AChE in sheep.

Acetylcholinesterase↗

PubMed1994

Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step.

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

Acetylgalactosamine↗

PubMed1994

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus.

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.

Animals↗

PubMed1994

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.

Animals↗

PubMed1994

Vaccination against gastrointestinal nematodes of sheep using purified secretory acetylcholinesterase from Trichostrongylus colubriformis--an initial pilot study.

Purified secretory acetylcholinesterase (sAChE) from Trichostrongylus colubriformis was used as a candidate vaccine against mixed T. colubriformis, Haemonchus contortus and C. oncophora challenge infections of sheep. Cross species protection was achieved with an average reduction in worm burden of all species of 31%, rising to 58% in individual cases. There was no consistent reduction in faecal egg counts and increases in anti-T. colubriformis sAChE IgG antibody levels following vaccination were modest. We suggest that improved antigen delivery systems will result in increased host protection.

Acetylcholinesterase↗

PubMed1994

Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network.

During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins VV-p37, VV-p42, VV-p21, and VV-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular VV-p37, VV-p21, and VV-p42 were in the TGN, whereas VV-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.

Animals↗

PubMed1994

Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.

Amino Acid Sequence↗

PubMed1994

Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin)

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.

Amino Acid Sequence↗

PubMed1994

Homeless & hungry: the evidence from Liverpool.

Much research has established the link between low incomes and poor nutritional standards. A research team from the Centre for Consumer Education & Research at Liverpool John Moores' University recently found that 30% of all families with children in Britain today are spending less on food than what is required to achieve a dietary which adheres, at minimum cost, to the Department of Health's Dietary Recommended Values (DRVs). But very little, if any, research has investigated the nutritional implications of a particularly extreme form of material deprivation--homelessness. This pilot study therefore sets out to study the dietaries of a number of homeless families in Liverpool--homeless as defined by living in Bed & Breakfast accommodation. Not only do such families have to contend with dependency upon welfare benefits when purchasing their foodstuffs; they also have to labour under inadequate cooking facilities. The study has involved these families keeping a dietary diary of all food and drink consumed. This information has then been analysed for its nutrient composition, using the Microdiet computer programme at Liverpool JMU. The results will show that, in every single case, the dietaries of these homeless families fall substantially short of the government's own nutritional guidelines and are, without doubt, unhealthy in the extreme. This paper is thus an examination of the nature and extent of the problem, using the science of nutrition and dietetics: not a policy prescription (although this is obvious) not a policy analysis. A study of the dietary implications of homelessness for 100 individuals (the largest ever undertaken) on Merseyside will be undertaken between September 1993 and June 1994.

Adolescent↗

PubMed1994

Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment.

We have investigated the interactions occurring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992) J. Cell Biol. 116, 95-112). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes.

Animals↗

PubMed1993

Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor.

In mammalian cells, clathrin-coated vesicles mediate transport of the lysosomal enzyme receptors from the trans-Golgi network to the endocytic pathway. A critical step of this process is the recruitment of Golgi-specific adaptors onto Golgi membranes for efficient clathrin polymerization. An in vitro assay was used here to quantitate this event in streptolysin-O-permeabilized NRK cells. At 37 degrees C, these interactions are cytosol- and energy-dependent, sensitive to GTP gamma S (guanosine 5'-O-(thiotriphosphate)) and brefeldin A. We report that Golgi-specific adaptor binding is enhanced in mannose 6-phosphate/insulin-like growth factor II (IGF II) receptor-overexpressing cells and reduced in mannose 6-phosphate receptor-deficient cells. Furthermore, adaptor binding is partially inhibited after addition of soluble cytoplasmic domains of the mannose 6-phosphate/IGF II receptor. Almost complete inhibition is only observed when this domain is phosphorylated on serines 2421 and 2492, a major modification acquired during exit of the receptor from the Golgi. These results show that the mannose 6-phosphate/IGF II receptor is part of the components that recruit the Golgi-specific adaptors and that its phosphorylation is an important feature for high affinity interactions with sorting components.

Animals↗

PubMed1993

Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor.

The major subunit of the human asialoglycoprotein receptor contains signals for efficient endocytosis and specific basolateral expression in polarized Madin-Darby canine kidney cells, both of which are located within its 40-residue cytoplasmic domain. The aromatic residue in this segment, tyrosine 5, which is necessary for efficient clustering into clathrin-coated pits at the plasma membrane, is also necessary for exclusive basolateral delivery. Mutation of this residue to alanine resulted in a nonpolar expression of the protein. Replacement of tyrosine 5 with phenylalanine yielded almost wild-type rates of endocytosis as well as specific basolateral expression, indicating that tyrosine phosphorylation is not essential for either sorting step. The close similarity between the two sorting signals was further corroborated by deletion mutants showing that the amino-terminal 10 residues of the cytoplasmic domain are sufficient for basolateral polarity and efficient endocytosis. The kinetics of appearance of newly synthesized wild-type and mutant receptor protein at the apical and basolateral surfaces indicate that these proteins are sorted intracellularly and are transported directly to the respective domains. Mutants affected in basolateral sorting lost polarity, i.e, appeared to similar extents on both surfaces, indicating that there is no significant apical sorting information elsewhere in the protein. The close correlation between endocytosis and basolateral polarity suggests common recognition mechanisms at the plasma membrane and in the trans-Golgi network.

3T3 Cells↗

PubMed1993

Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo.

Microinjection of antibodies against a synthetic peptide of a non-clathrin-coated vesicle-associated coat protein, beta-COP, blocks transport of a temperature-sensitive vesicular stomatitis virus glycoprotein (ts-O45-G) to the cell surface. Transport is inhibited upon release of the viral glycoprotein from temperature blocks at 39.5 degrees C (endoplasmic reticulum [ER]) and 15 degrees C (intermediate compartment), but not at 20 degrees C (trans-Golgi network). Ts-O45-G is arrested in tubular membrane structures containing p53 at the interface of the ER and the Golgi stack. This is consistent with inhibition of acquisition of endoglycosidase H resistance of ts-O45-G in injected cells. Secretion of endogenous proteins and maturation of cathepsin D are also inhibited. These data provide in vivo evidence that beta-COP has an important function in biosynthetic membrane traffic in mammalian cells.

Animals↗

PubMed1993

The immunofluorescent era of membrane traffic.

In recent years immunofluorescence microscopy has been increasingly used to study membrane traffic. In this article seven electron microscopists, all with considerable experience in using light microscopy, take a critical look at the immunofluorescence approach and argue that results obtained with this method are often overinterpreted.

Journal Article↗