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Biomedical subjects

G Griffiths

Publications and source records attributed to G Griffiths.

At least 73 records · Page 4Linked to original sources
PubMed1997

Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Animals↗
PubMed1997

Molecular requirements for bi-directional movement of phagosomes along microtubules.

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubule-dependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dynein-mediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.

Adenosine Triphosphate↗
PubMed1997

Effect of n-6 polyunsaturated fatty acids on growth and lipid composition of neoplastic and non-neoplastic canine prostate epithelial cell cultures.

BACKGROUND: Polyunsaturated fatty acids (n-6) are reported to selectively kill malignant cells. Most investigations, however, did not compare neoplastic with non-neoplastic cells from the same tissue type. Here we evaluate the effects of n-6 fatty acids on a non-neoplastic epithelium cell line (CAPE) and a spontaneous carcinoma cell line (CPA) derived from the canine prostate. METHODS: Cell lines were cultured in DME in the presence of fatty acids and their effects on cell proliferation monitored by coulter counting. Lipids were extracted and quantitized by gas chromatography. RESULTS: Cell proliferation was reduced more in CAPE. A neoplastic strain (CPA-GLA) tolerant to prolonged culture in 18:3n-6 was isolated. CPA grown in an 18:2n-6 or 18:3n-6 supplemented media accumulated 20:3n-6 and contained little 20:4n-6. CONCLUSIONS: Polyenoic n-6 fatty acids are not specifically inhibitory to neoplastic cells which exhibited a marked alteration in the metabolism of 20:4n-6.

Animals↗
PubMed1997

Vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment.

The use of two-dimensional gel electrophoresis has identified the gene products A14L (p16) and A13L (p8) as abundant membrane proteins of the first infectious form of vaccinia virus, the intracellular mature virus (IMV; O. N. Jensen, T. Houthaeve, A. Shevchenko, S. Cudmore, T. Ashford, M. Mann, G. Griffiths, J. Krijnse Locker, J. Virol. 70:7485-7497, 1996). In this study, these two proteins were characterized in detail. In infected cells, both proteins localize not only to the viral membranes but also to tubular-cisternal membranes of the intermediate compartment, defined by the use of antibodies to either rab1A or p21, which colocalize with rab1A (J. Krijnse Locker, S. Schleich, D. Rodriguez, B. Goud, E. J. Snijder, and G. Griffiths, J. Biol. Chem. 271:14950-14958, 1996). Both proteins appear to reach this destination via cotranslational insertion into the rough endoplasmic reticulum, as shown by in vitro translation and translocation experiments. Whereas p16 probably spans the membrane twice, p8 is inserted into the membrane by means of its single NH2-terminal hydrophobic domain, adopting a topology which leaves the C terminus exposed to the cytoplasm. Combined immunocytochemical and biochemical data show that p16 is a member of the inner of the two IMV membrane layers, whereas p8 localizes to both the inner and the outer membrane. These findings are discussed with respect to our model of IMV membrane assembly.

Blotting, Western↗
PubMed1997

The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.

Animals↗
PubMed1997

Randomised controlled double blind study of role of recombinant erythropoietin in the prevention of chronic lung disease.

AIM: To evaluate the role of recombinant human erythropoietin (R-HuEpo) in reducing iron infusion, which may exacerbate free radical damage, leading to chronic lung disease. METHODS: A multicentre, randomised, placebo controlled, double blind study was carried out in four neonatal intensive care units in Yorkshire. Infants were randomly allocated and received either R-HuEpo (480 U/kg/wk) or placebo by twice weekly subcutaneous injection. The primary outcome measure was the number of days on respiratory support and a secondary outcome the number of blood transfusions required. RESULTS: Forty two very low birthweight (VLBW) infants were randomly allocated. There was little difference in the need for respiratory support one month after randomisation, but subsequently there was a trend towards a reduction in the proportion requiring respiratory support in the R-HuEpo group (difference at three months -0.50, 95% confidence interval -1.00, 0.17). During stay in hospital, the median number of blood transfusions was lower for infants in the R-HuEpo group (difference in medians -2, 95% CI -4, 0). The study was stopped early because of failure to recruit babies at the expected rate. CONCLUSIONS: R-HuEpo seems to reduce the number of days in oxygen for ill VLBW infants. These data could be used to construct a larger multicentre study to evaluate this effect further.

Blood Transfusion↗
PubMed1997

Localization of five annexins in J774 macrophages and on isolated phagosomes.

Annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Although the precise functions of annexins are unknown, there is an accumulating set of data arguing for a role for some of them in vesicular transport and, specifically, in membrane-membrane or membrane-cytoskeletal interactions during these processes. Here we describe our qualitative and quantitative analysis of the localization of annexins I-V in J774 macrophages that had internalized latex beads, both with and without IgG opsonization. Our results show that whereas all these annexins are present on both the plasma membrane and on phagosomes, the localization on other organelles differs. Annexins I, II, III and V were detected on early endosomes, while only annexin V was seen on late endocytic organelles and mitochondria. Annexins I and II distributed along the plasma membrane non-uniformly and co-localized with F-actin at the sites of membrane protrusions. We also investigated by western blot analysis the association of annexins with purified phagosomes isolated at different time-points after latex bead internalization. While the amounts of annexins I, II, III and V associated with phagosomes were similar at all times after their formation, the level of annexin IV was significantly higher on older phagosomes. Whereas annexins I, II, IV and V could be removed from phagosome membranes with a Ca2+ chelator they remained membrane bound under low calcium conditions. In contrast, annexin III was removed under these conditions and needed a relatively high Ca2+ concentration to remain phagosome bound. Because of their purity and ease of preparation we suggest that phagosomes are a powerful system to study the potential role of annexins in membrane traffic.

Animals↗
PubMed1997

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Endosomes↗
PubMed1996

Preformed cytoplasmic nucleocapsids are not necessary for alphavirus budding.

According to the present model for assembly of alphaviruses, e.g. Semliki Forest virus (SFV), the viral genome is first encapsidated into a nucleocapsid (NC) in cytoplasm and this is then used for budding at plasma membrane (PM). The preformed NC is thought to act as a template on which the viral envelope can be organized. In the present work we have characterized two SFV deletion mutants which did not assemble NCs in the cytoplasm but which instead appeared to form NCs at the PM simultaneously with virus budding. The deletions were introduced in a conserved 14 residue long linker peptide that joins the amino-terminal RNA-binding domain with the carboxy-terminal serine-protease domain of the capsid protein. Despite the deletions and the change in morphogenesis, wild-type (wt)-like particles were produced with almost wt efficiency. It is suggested that the NC assembly defect of the mutants is rescued through spike-capsid interactions at PM. The results show that the preassembly of NCs in the cytoplasm is not a prerequisite for alphavirus budding. The apparent similarities of the morphogenesis pathways of wt and mutant SFV with those of type D and type C retroviruses are discussed.

Alphavirus↗
PubMed1996

The role of a 21-kDa viral membrane protein in the assembly of vaccinia virus from the intermediate compartment.

We have recently provided morphological evidence that a key event in the assembly of vaccinia virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521-541). This tightly apposed cisternal domain incompletely surrounds the spherical immature virus that matures into the first of the two distinct infectious forms of vaccinia, the intracellular mature virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH2- and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies against Rab1. Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the IMV inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the IC and membranes of the viral factory.

Amino Acid Sequence↗
PubMed1996

The polyether bistratene A activates protein kinase C-delta and induces growth arrest in HL60 cells.

Bistratene A (BisA) induced growth arrest in G2/M in HL60 cells. In addition, BisA-treated cells (50 nM for 48 h) became adherent and expressed the adhesion molecule CD11c, but did not express the monocyte enzyme alpha-napthyl acetate esterase or phagocytose complement coated yeasts. BisA activated protein kinase C (PKC)-delta and induced translocation of PKC-delta to the nucleus. This suggests that activation of PKC-delta can induce growth arrest and cell adhesion, but is insufficient to mediate full differentiation of HL60 cells. BisA has potential as a new probe for determining the function of PKC isoenzymes, specifically PKC-delta.

Acetamides↗
PubMed1996

A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus.

We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.

Chemical Phenomena↗
PubMed1996

Microtubule-associated protein-dependent binding of phagosomes to microtubules.

In macrophages, phagosome movement is microtubule-dependent. Microtubules are a prerequisite for phagosome maturation because they facilitate interactions between phagosomes and organelles of the endocytic pathway. We have established an in vitro assay that measures the binding of purified phagosomes to microtubules. This binding depends on the presence of membrane proteins, most likely integral to the surface of phagosomes, and on macrophage cytosol. The cytosolic binding factor can interact with microtubules prior to the addition of phagosomes to the assay, suggesting that it is a microtubule-associated protein (MAP). Consistent with this, depletion of MAPs from the cytosol by microtubule affinity removes all binding activity. Microtubule motor proteins show no binding activity, whereas a crude MAP preparation is sufficient to support binding and to restore full binding activity to MAP-depleted cytosol. We show that the activating MAP factor is a heat-sensitive protein(s) that migrates at around 150 kDa by gel filtration.

Animals↗
PubMed1996

Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.)

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

Amino Acid Sequence↗
PubMed1996

Mannose 6-phosphate receptors and ADP-ribosylation factors cooperate for high affinity interaction of the AP-1 Golgi assembly proteins with membranes.

Clathrin coat assembly in the trans-Golgi network, leading to the sequestration of the mannose 6-phosphate receptors (MPRs) into nascent vesicles, requires the ARF-1-dependent translocation of the cytosolic AP-1 Golgi assembly proteins onto the membranes of this organelle. The mechanistic role of the MPRs, i.e. the cargo molecules, in coat assembly is at present unclear. Using a GTP-dependent, brefeldin A-sensitive in vitro AP-1 binding assay, we have determined here the parameters of the AP-1 binding reaction. We demonstrate that, in addition of ARF-1, the MPRs contribute to create high affinity AP-1 binding sites (Kd approximately 25 mM), since their number correlates the number of MPR molecules expressed in MPR-negative cells. The quantitative electron microscopy shows that these high affinity binding sites are present on trans-Golgi network membranes, as expected, and to some extent on early endosomes. The high affinity binding sites are lost when the MPRs or ARF-1 become rate-limiting components. Conversely, GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), which increases the amount of membrane-bound ARF-1, most uncovers low affinity AP-1 binding sites (Kd approximately 150 nM) on trans-Golgi network membranes, normally not detected in its absence. Collectively, these results argue that MPR sorting is highly coupled to the first step of coat assembly and that the MPRs, ARF-1, and possibly other proteins cooperate for high affinity interactions of AP-1.

ADP-Ribosylation Factors↗
PubMed1996

A vaccinia virus core protein, p39, is membrane associated.

We describe herein the characterization of p39, the product of the A4L gene of vaccinia virus. By immunolabelling of thawed cryosections from infected HeLa cells, we show that this protein is initially located in the central region, or viroplasm, of the viral factories, as well as in the immature virions, with very small amounts of labelling observed on the surrounding membranes. The localization of p39 changes dramatically during the transition of the immature virion to the intracellular mature virus (IMV), coincident with the appearance of the core structure in the center of the IMV, with p39 located between this core and the surrounding membranes. Complementary biochemical data, such as partitioning into the Triton X-114 detergent phase and stripping of the viral membranes with Nonidet P-40 and dithiothreitol, suggest that p39 is associated with the innermost of the two membranes surrounding the core. Sodium carbonate treatment also indicates that p39 is associated with membranes, even at the early stages of viral assembly. However, following in vitro translation of p39 in the presence of microsomal membranes, we failed to detect any association of the independently expressed protein with membranes. We also failed to detect any posttranslational acylation of p39 with myristate or palmitate, suggesting that p39 does not achieve its membrane association through lipid anchors. Therefore, p39 is most likely membrane associated through an interaction with an integral membrane protein(s) present in the innermost of the two membranes surrounding the IMV. These data, together with our recent data showing that p39 colocalizes with the spike-like protrusions on the IMV core (N. Roos, M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker, and G. Griffiths, EMBO J. 15:2343-2355, 1996), suggest that p39 may form part of this spike and that it possibly functions as a matrix-like linker protein between the core and the innermost of the two membranes surrounding the IMV.

Cell Membrane↗