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G Griffiths

Publications and source records attributed to G Griffiths.

At least 19 recordsLinked to original sources
PubMed2001

ATP-dependent membrane assembly of F-actin facilitates membrane fusion.

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Actins↗
PubMed2001

Molecular and biochemical characterization of postharvest senescence in broccoli.

Postharvest senescence in broccoli (Brassica oleracea L. var Italica) florets results in phenotypic changes similar to those seen in developmental leaf senescence. To compare these two processes in more detail, we investigated molecular and biochemical changes in broccoli florets stored at two different temperatures after harvest. We found that storage at cooler temperatures delayed the symptoms of senescence at both the biochemical and gene expression levels. Changes in key biochemical components (lipids, protein, and chlorophyll) and in gene expression patterns occurred in the harvested tissue well before any visible signs of senescence were detected. Using previously identified senescence-enhanced genes and also newly isolated, differentially expressed genes, we found that the majority of these showed a similar enhancement of expression in postharvest broccoli as in developmental leaf senescence. At the biochemical level, a rapid loss of membrane fatty acids was detected after harvest, when stored at room temperature. However, there was no corresponding increase in levels of lipid peroxidation products. This, together with an increased expression of protective antioxidant genes, indicated that, in the initial stages of postharvest senescence, an orderly dismantling of the cellular constituents occurs, using the available lipid as an energy source. Postharvest changes in broccoli florets, therefore, show many similarities to the processes of developmental leaf senescence.

Brassica↗
PubMed2001

Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles.

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150(Glued)) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.

Animals↗
PubMed2000

Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin.

Administration of doxorubicin (DXR) formulated in sterically stabilized liposomes, (SL) containing engrafted poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) on their surface, has been shown to increase the therapeutic index of the drug. A further improvement could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. This paper describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of DXR-loaded liposomes by use of a PEG-based heterobifunctional coupling agent. Competitive-binding ELISA of the resulting immunoliposomes (SIL) against the monoclonal anti-idiotype antibody, WN, indicated preserved immunological activity. The pH-sensitive probe, HPTS was used to study the binding of liposomes with target cells. The results showed a 3.8-fold increased cellular association of SIL compared to that of SL and an apparent internalization of SIL into low pH compartments. Addition of an excess of unconjugated free LL2 displaced about 72% of the HPTS-SIL association with cells. Experiments with 125I-labeled free and SIL-bound LL2 showed approximately 50% degradation for both preparations. In vitro MTT cytotoxicity tests against neoplastic B cells gave IC(50) values of 1.6, 2.9 and 0.35 microM for DXR-SIL, DXR-SL and free DXR, respectively. Leakage of drug from the liposomes apparently reduced the specificity of the cytotoxic action of DXR-SIL.

Animals↗
PubMed2000

Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha -UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC.

The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-beta(1,4)-GlcNAc-alpha(1- and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis. Previously, the KfiC protein was shown to be a beta-UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an alpha-UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA. In addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.

Amino Acid Sequence↗
PubMed2000

Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay.

BACKGROUND: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments. METHODS: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation. RESULTS: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane. CONCLUSIONS: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.

Actins↗
PubMed2000

PKC-delta is an apoptotic lamin kinase.

Protein kinase C-delta is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-delta induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-delta in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-delta co-localized with lamin B during apoptosis and activation of PKC-delta by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-delta delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-delta was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-delta is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

Acetophenones↗
PubMed2000

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes.

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Actins↗
PubMed2000

Gut thoughts on the Golgi complex.

The new millennium coincides within 1 year of Camillo Golgi's centennial celebrations. It is quite remarkable that the structure and formation of this organelle is as controversial today as was its mere existence from Golgi's time to the 1950s, when EM approaches were introduced. Since the late 1950s, two opposing models of Golgi structure and function have split the Golgi scientific community, namely vesicular transport versus organelle maturation. Although a few years ago Golgi maturation seemed to be 'out for the count', it has recently seen an almost messianic revival. In this review, I argue that this large-scale desertion from the vesicle transport model to the maturation camp is premature. I propose an alternative, dynamic steady-state model, in which transient tubular connections function in parallel to vesicular transport and that the biosynthetic pathway is made up of three major distinct compartments: the ER, the Golgi and the TGN.

Animals↗
PubMed2000

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV.

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

Actins↗
PubMed2000

Regulation of the Escherichia coli K5 capsule gene cluster: evidence for the roles of H-NS, BipA, and integration host factor in regulation of group 2 capsule gene clusters in pathogenic E. coli.

The expression of Escherichia coli group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20 degrees C. Thermoregulation is at the level of transcription, with no detectable transcription at 20 degrees C. Using the E. coli K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20 degrees C. At 37 degrees C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20 degrees C H-NS functions to repress transcription. The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic E. coli, was shown to play a similar role to H-NS in regulating transcription at 37 and 20 degrees C. The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37 degrees C and IHF binding demonstrated by bandshift assays. The IHF binding site was 3' to the site of transcription initiation, suggesting that sequences in the 5' end of the first gene (kpsF) in region 1 may play a role in regulating transcription from this promoter at 37 degrees C. Two additional cis-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters. These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in E. coli.

Bacterial Capsules↗
PubMed2000

Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon.

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.

Animals↗
PubMed2000

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E.

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

Animals↗
PubMed2000

Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure.

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

Blotting, Western↗
PubMed2000

Reliability of the Camberwell Assessment of Need--European Version. EPSILON Study 6. European Psychiatric Services: Inputs Linked to Outcome Domains and Needs.

BACKGROUND: The five-country European Psychiatric Services: Inputs Linked to Outcome Domains and Needs (EPSILON) Study aimed to develop standardised and reliable outcome instruments for people with schizophrenia. This paper reports reliability findings for the Camberwell Assessment of Need--European Version (CAN-EU). METHOD: The CAN-EU was administered in each country, at two points in time to assess test-retest reliability, and was rated by two interviewers at the first administration. Cronbach's alpha, test-retest reliability and interrater reliability were compared between the five sites. Reliability coefficients and standard errors of measurement for summary scores were estimated. RESULTS: Sites varied in levels and spread of needs. Alphas were 0.48, 0.58 and 0.64 for total, met and unmet needs respectively. Test-retest reliability estimates, pooled over sites, were 0.85 for the total needs, 0.69 for met needs and 0.78 for unmet needs. Pooled estimates for interrater reliability were higher, at 0.94, 0.85 and 0.79 for total, met and unmet needs respectively. There were statistically significant differences in interrater reliability between sites. CONCLUSION: The results confirm the feasibility of using CAN-EU across sites in Europe and its psychometric adequacy.

Adolescent↗
PubMed2000

Preferential association of syntaxin 8 with the early endosome.

Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ((alpha)-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.

Amino Acid Sequence↗
PubMed2000

Lipid hydroperoxide levels in plant tissues.

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and are key intermediates in the octadecanoid signalling pathway in plants. Lipid hydroperoxides (LHPO) were determined spectrophotometrically based on their reaction with an excess of Fe(2+)at low pH in the presence of the dye xylenol orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in Phaseolus: microsomes, senescing potato leaves and in a range of other plant tissues including Phaseolus hypocotyls (26+/-5 nmol g(-1) FW), Alstroemeria floral tissues (sepals 66+/-13 nmol g(-1) FW petals 49+/-6 nmol g(-1) FW), potato leaves (334+/-75 nmol g(-1) FW), broccoli florets (568+/-68 nmol g(-1) FW) and Chlamydomonas cells (602+/-40 nmol g(-1) FW). Relative to the total fatty acid content of the tissues, the % LHPO was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. In order to relate the levels of LHPO to specific signalling pathways, transgenic potato plant lines were used in which lipoxygenase (LOX) (responsible for hydroperoxide biosynthesis) and hydroperoxide lyase (a route of hydroperoxide degradation) activities were largely reduced by an antisense-mediated approach. While the LHPO levels were similar to wild type in the individual LOX antisensed plants, basal LHPO levels, by contrast, were elevated by 38% in transgenic potato leaves antisensed in hydroperoxide lyase, indicating a role for this enzyme in the maintenance of cellular levels of LHPOs.

Chlorophyll↗