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G Griffiths

Publications and source records attributed to G Griffiths.

At least 19 recordsLinked to original sources
PubMed2005

Pilot study of the effects of local pressure on microvascular function in the diabetic foot.

AIM: The aim of the study was to determine whether areas of the diabetic foot that experience high pressures during normal activity also demonstrate reductions in cutaneous microvascular flow and/or endothelial function. METHODS: Sixteen patients with diabetes mellitus and eight healthy, age-matched control subjects were recruited. Maps of dynamic pressure on the plantar aspect of both feet were recorded during a normal gait cycle, and the skin microvascular blood flow response to the endothelium-dependent vasodilator acetylcholine was assessed at the sites of highest and lowest plantar pressure over the metatarsal heads. RESULTS: Patients with diabetes had higher plantar pressures than control subjects (P = 0.002), but there were no significant differences in basal skin blood flow or acetylcholine response between the groups. In the patients, baseline flow was increased (P = 0.041) but the acetylcholine response reduced (P = 0.03) at the high-pressure compared with the low-pressure site; this was most apparent in those who were particularly at risk of ulceration due to high plantar pressures. CONCLUSIONS: Chronically raised plantar pressure in the diabetic foot is associated with increased basal skin blood flow, compared with lower pressure areas on the same foot. Further work is required to determine whether, and under what conditions, this additional hyperaemia is protective or maladaptive. In addition, high-pressure areas have a reduced responsiveness to an endothelium-dependent vasodilator, although the clinical significance of these changes is not clear.

Acetylcholine↗
PubMed2003

White cell scanning for inflammatory bowel disease: are biochemical markers useful referral criteria?

The aim of this study was to determine whether biochemical markers for inflammation could prove effective in identifying the most appropriate patients with suspected inflammatory bowel disease (IBD) for labelled white cell scanning. One hundred and twenty-five patients referred for 99mTc-HMPAO labelled white cell scans were investigated. The values of C-reactive protein (CRP), antichymotrypsin (ACT) and acid glycoprotein (AGP) were measured in 73 patients, AGP and CRP in 10 and CRP only in a further 42. Sensitivity and specificity of each test were calculated using the white cell scan result as the 'gold standard'. ACT had the highest specificity (1.0), but the lowest sensitivity (0.27) of the three markers. CRP (using specified limits) had the lowest specificity (0.67) and the highest sensitivity (0.79). The corresponding values for AGP are 0.87 and 0.48. The low sensitivity of ACT and AGP preclude them from being useful referral criteria. CRP (using specified limits) is the most sensitive marker, but not sensitive enough to be useful as a referral indicator. However, by lowering the upper limit of normal to 5 mg.l-1, the sensitivity of the test increases to 0.96. Using this threshold to select the patients, 30% would not have been scanned and only one patient out of the 22 with IBD would have been missed. Where there is high demand for white cell scans this may provide a useful strategy for rationalizing the requests with minimal consequence on clinical management.

Adolescent↗
PubMed2002

Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections.

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test-point lattices on cell profiles, design-based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test-line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds microm(-2) or membranes (golds microm(-1)), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi-squared analysis to test if the observed distribution differs significantly from random and, if it is non-random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.

Chi-Square Distribution↗
PubMed2002

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs.

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.

Bacterial Proteins↗
PubMed2001

Human cofilin forms oligomers exhibiting actin bundling activity.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys(39) and probably Cys(147) of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfide-linked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.

Actin Depolymerizing Factors↗
PubMed2001

Wortmannin-sensitive trafficking steps in the endocytic pathway in rat liver endothelial cells.

Liver endothelial cells (LECs) play an important homoeostatic role by removing potentially harmful macromolecules from blood. The extremely efficient endocytosis in LECs makes these cells an interesting model for the study of the involvement of phosphoinositides in the different steps of the endocytic process. In the present investigation we have studied the effect of wortmannin, an inhibitor of phosphatidylinositol kinases, on uptake, recycling and intracellular transport of (125)I-labelled ovalbumin, which is taken up in LECs via mannose-receptor-mediated endocytosis. Wortmannin was found to inhibit both uptake and degradation of ovalbumin. Further studies indicated that the reduced uptake via the mannose receptor was due both to a reduction of the number of surface receptors and a reduction in the rate of receptor-ligand internalization. Transport of ligand from endosomes to lysosomes was prevented, leading to increased recycling of internalized ligand. Wortmannin treatment released the Rab5 effector EEA1 from the endosomes and caused reduced size of early endosomes.

Androstadienes↗
PubMed2001

Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria.

The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium (M.) tuberculosis and Mycobacterium avium ssp. avium (M. avium), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis (M. ptb), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium, as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae. Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA-gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)-dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC-dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to approximately pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium. In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of approximately 5.2. After infection with M. smegmatis and M. gordonae, acidification was initially (1-5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.

Animals↗
PubMed2001

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium. Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

Actins↗
PubMed2001

Electron microscopy applications for quantitative cellular microbiology.

Recently, there has been a surge of interest in analysing intracellular trafficking of pathogenic microorganisms. This field relies heavily on a wide range of approaches developed by cell biologists. However, one well-established approach that seems to be underused in cellular microbiology is electron microscopy (EM). In this article, we emphasize the power of state-of-the-art EM approaches, especially when used in conjunction with the quantitative approaches provided by stereology. Together, this combination of techniques can be exploited to pursue kinetic analyses of the intracellular pathways followed by microorganisms. In addition, it will provide a more detailed description of the antigenic composition of relevant structures than is possible by light microscopy (LM). Here, we will explain how EM, applied in an integrated approach with LM, has distinct advantages and can provide unique insights into the cellular fate of microorganisms.

Cells↗
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

Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.

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

Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis.

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

Dithiothreitol↗
PubMed2001

Structure and assembly of intracellular mature vaccinia virus: thin-section analyses.

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.

Bacterial Proteins↗
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↗