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Biomedical subjects

A T Jones

Publications and source records attributed to A T Jones.

At least 19 recordsLinked to original sources
PubMed2001

Pulmonary perfusion in supine and prone positions: an electron-beam computed tomography study.

Acute respiratory distress syndrome is characterized by alterations in the ventilation-perfusion ratio. Present techniques for studying regional pulmonary perfusion are difficult to apply in the critically ill. Electron-beam computed tomography was used to study the effects of prone positioning on regional pulmonary perfusion in six healthy subjects. Contrast-enhanced sections were obtained sequentially in the supine, prone, and (original) supine positions at full inspiration. Regions of interest were placed along the nondependent to dependent axis and relative perfusion calculated. When corrected for the redistribution of lung parenchyma, a gravitational gradient of pulmonary perfusion existed in both supine and prone positions. The distribution of perfusion between the supine or prone positions did not differ, but data analysis using smaller regions of interest demonstrated marked heterogeneity of perfusion between anatomically adjacent regions of lung. The distribution of lung parenchyma was more uniform in the prone position. Gravity was estimated to be responsible for 22-34% of perfusion heterogeneity in the supine and 27-41% in the prone positions. These data support the hypothesis that factors other than gravity may be at least as important in determining the distribution of pulmonary perfusion in humans. The influence of nongravitational factors may not be detectable if techniques that sample large tissue volumes are employed.

Adult↗
PubMed2000

Differences between the coat protein amino acid sequences of English and Scottish serotypes of Raspberry ringspot virus exposed on the surface of virus particles.

The region of the RNA 2 coding for the putative helper/movement protein and the coat protein (CP) of each of six isolates of Raspberry ringspot virus was sequenced and these sequences were compared with the published sequence of the Scottish type isolate. Minimal differences were detected among the putative translations of the helper/movement proteins, however, multiple alignment and phylogenetic analysis of the putative CPs separated the English and Scottish serotypes into two distinct clades. Superimposing the amino acid sequences of the CPs of these two serotypes on the 3D model for the CP of a comovirus/nepovirus, showed that eight of the differences identified between the two serotypes occurred on the surface of the protein. Inspection of the recently reported structure of the capsid protein of Tobacco ringspot virus, the type member of the genus Nepovirus, indicated identical locations for these differences. The change of H (Scottish isolates) to R (English isolates) at position 219 in the amino acid sequences of the viruses occurred on an exposed, erect surface loop. The potential role of this change, and other unique differences between the amino acid sequences of the two serotypes, in the specificity of nematode transmission of the virus is discussed.

Amino Acid Sequence↗
PubMed2000

Black currant reversion disease--the probable causal agent, eriophyid mite vectors, epidemiology and prospects for control.

Black currant reversion disease and the vector of its causal agent, the black currant gall mite Cecidophyopsis ribis, have been recognised for at least 100 years and are the two most damaging organisms of black currant crops world-wide. However, the molecular characterisation of these two organisms has begun to be determined in only the last few years. The probable causal agent of reversion disease, Black currant reversion associated virus (BRAV), belongs to the genus Nepovirus, has isometric particles c. 28 nm in diameter that contain a single major polypeptide of c. 55 KDa and two polyadenylated ssRNA species of 7700 nt and 6400 nt. Some particle preparations also contain a satellite ssRNA species of 1432 nt. Using immuno-capture RT-PCR and primers based on the genomic RNA of BRAV, this virus was shown to be closely associated with reversion disease. Analysis of Cecidophyopsis mite rDNA, identified rapidly and unambiguously the three known species on Ribes and distinguished four new ones. Resistance to the reversion agent and to the gall mite vector has been introduced into black currant and has given effective control of these respective organisms in the field. These findings and their significance for the ecology, epidemiology and control of variants of these two organisms are reviewed and discussed.

Animals↗
PubMed1999

Identification of cecidophyopsis mites (Acari: Eriophyidae) based on variable simple sequence repeats of ribosomal DNA internal transcribed spacer-1 sequences via multiplex PCR.

A PCR multiplex technique was developed for identifying Cecidophyopsis mites using species-specific differences in rDNA ITS-1 sequences. Four PCR primers derived from ITS-1 were used for the simultaneous amplification (multiplex PCR) of interspecifically variable simple sequence repeats (vSSRs). Mites were identified by electrophoresing PCR products alongside those obtained from plasmids containing ITS copies of known mite species. The multiplex PCR assay was rapid, reproducible and had a sensitivity comparable to sequencing. It was used to identify mite specimens on Ribes from around the world. It also identified a profile from mites on R. rubrum that had no equivalent amongst the known Cecidophyopsis species. Sequence and ecological analysis of this mite suggest that it is a new species of nongall-forming Cecidophyopsis mite.

Animals↗
PubMed1999

Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.

ADP-Ribosylation Factors↗
PubMed1998

Inhibition of in vitro endosomal vesicle fusion activity by aminoglycoside antibiotics.

The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC50 values of approximately 45 microM and approximately 1 mM, respectively. Because the IC50 for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP2), these data suggest that negatively charged lipids are required for endosome fusion. A role for negatively charged lipids in membrane traffic has been postulated to involve the activity of a PIP2-dependent phospholipase D (PLD) stimulated by the GTP-binding protein ADP-ribosylation factor (ARF). Although neomycin blocks endosome fusion at a stage of the in vitro reaction that is temporally related to steps inhibited by cytosolic ARFs when they bind guanosine-5'-gamma-thiophosphate (GTPgammaS), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP2-independent PLD does not suppress neomycin inhibition of endosome fusion; moreover, in vitro fusion activity is not affected by the pleckstrin homology domain of phosphoinositide-specific phospholipase C delta1, which binds to acidic phospholipids, particularly PIP2, with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTPgammaS-bound ARF, these effects are unrelated to inhibition of the PIP2-dependent PLD activity stimulated by this GTP-binding protein. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway.

ADP-Ribosylation Factors↗
PubMed1998

Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes.

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098-1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.

Animals↗
PubMed1998

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Alkyl and Aryl Transferases↗
PubMed1997

Inhibition of calcium-independent mannose 6-phosphate receptor incorporation into trans-Golgi network-derived clathrin-coated vesicles by wortmannin.

The transport of pro-cathepsin D from the trans-Golgi network (TGN) to the endosomal pathway is dependent on binding to the calcium-independent mannose 6-phosphate receptor (ci-M6PR), which is incorporated into TGN-derived clathrin-coated transport vesicles (CCVs). Inhibition of this transport step by wortmannin has led to the proposal that it is dependent upon a phosphoinositide 3-kinase activity necessary for ci-M6PR recruitment into TGN-derived CCVs or in the formation of those vesicles (Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H., and Balch, W. E. (1995) J. Cell Biol. 130, 781-796; Davidson, H. W. (1995) J. Cell Biol. 130, 797-806). In this study we have addressed the effect of wortmannin on the TGN step of the ci-M6PR cycle. CCVs from K562 cells, pretreated or not with 250 nM wortmannin, were purified on equilibrium density gradients. Quantification of TGN-derived CCVs, assessed by gamma-adaptin content in purified vesicle fractions, showed that the formation of the vesicles was only marginally decreased after 20 min of treatment with the drug, while for the same wortmannin treatment, the amount of ci-M6PR recruited into those vesicles was decreased by 70% compared with control. At a later time point (2 h), a reduction in the amount of gamma-adaptin in CCV fractions was also observed. These findings demonstrate that inhibition of ci-M6PR recruitment into CCVs but not of vesicle formation is the primary reason for the observed defect in cathepsin D transport following wortmannin treatment.

Androstadienes↗
PubMed1997

Regulation of early endosome fusion by phospholipase D activity.

In vitro or cell-free assays of homotypic fusion between early endosomes have provided several insights into the means by which an intracellular membrane fusion event can be regulated. In this report we show that homotypic fusion between early endosomes from baby-hamster kidney cells is partially blocked by 340 mM ethanol (2% v/v) and by 100 mM butan-1-ol, but not by the secondary alcohol butan-2-ol. We ascribe the effect of primary alcohols to their participation in a well-characterised transphosphatidylation reaction catalysed by phospholipase D activity, which results in the production of phasphatidylalcohol at the expense of phosphatidic acid. In accordance with this interpretation, we find that addition of exogenous phospholipase D results in stimulation of early endosome fusion.

1-Butanol↗
PubMed1996

Electrospray mass spectrometric evidence for the occurrence of two major variants in native pig pepsin A.

Native pig pepsin was analysed by negative ion electrospray mass spectrometry in order to rationalize anomalies between the published sequences. Outstanding variations in otherwise identical sequences indicate that amino acid residue 242 is either Asp or Tyr, and in some determinations an additional Ile is inserted at position 230. Mass spectrometric evidence is consistent with the presence, in the native enzyme, of two variants in comparable abundance, with either Asp or Tyr at residue 242. There is no evidence for the additional Ile at position 230.

Animals↗
PubMed1996

The effects of Helicobacter pylori colonization on gastric function and the incidence of portal hypertensive gastropathy in patients with cirrhosis of the liver.

OBJECTIVES: To investigate the prevalence of Helicobacter pylori infection in 50 cirrhotic patients with biopsy-proven cirrhosis with and without portal hypertensive gastropathy and to study whether or not the effects of H. pylori colonization of the stomach on gastric acid and pepsin secretion, serum gastrin and pepsinogen I levels, gastric mucus, and gastric emptying contributed to the development of portal hypertensive gastropathy in cirrhotics. METHODS: All patients underwent an upper GI endoscopy followed by determination of basal and pentagastrin and insulin-stimulated gastric acid and pepsin secretion and serum gastrin and pepsinogen I levels. The gastric biopsies were stained to detect H. pylori infection, portal hypertensive gastropathy, and gastritis. The amount of gastric mucus was estimated by a microanalytical technique. The rate of gastric emptying was assessed by the radionuclide method using a semi-solid meal. RESULTS: Thirty-three (66%) patients had endoscopic evidence of portal hypertensive gastropathy, 10 with the severe (20%) and 23 with mild form (46%). Twenty (40%) patients had histological evidence of H. pylori infection. Eleven out of 33 (33%) patients with endoscopic portal hypertensive gastropathy had microscopic evidence of H. pylori infection. Eighteen out of 20 (90%) patients with chronic active gastritis had concomitant H. pylori colonization. In contrast, the gastric mucosa was histologically normal in 21 of the 30 patients (70%) not infected with H. pylori. Marked hypochlorhydria and reduced pepsin secretion associated with a tendency to hypergastrinemia were observed in cirrhotic patients colonized with H. pylori compared with those without. However, there was no significant difference in serum pepsinogen I concentrations, the ratio of polymeric to degraded gastric mucus, or the rate of gastric emptying between cirrhotics with and without H. pylori colonization of the stomach. Furthermore, these parameters were not significantly different in patients with portal hypertensive gastropathy with and without H. pylori infection. CONCLUSION: These observations suggest that H. pylori infection is unlikely to be involved in the pathogenesis of portal hypertensive gastropathy.

Adolescent↗
PubMed1995

Phosphatidylinositol 3-kinase activity is required for early endosome fusion.

The homotypic fusion between early endosomes from baby-hamster kidney cells is blocked by addition of the fungal metabolite wortmannin with an IC50 of approx. 15 nM. Over this concentration range, wortmannin has been regarded as a specific inhibitor of phosphatidylinositol (PI) 3-kinase. Further confirmation of the participation of a PI 3-kinase in the fusion reaction has been obtained by demonstrating a sensitivity to an additional, structurally unrelated, PI 3-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. Assays constructed such that only the membranous component has been incubated with wortmannin show in vitro fusion to be sensitive to treatment with the drug. Assays in which only the cytosolic component has been treated with wortmannin also showed inhibition of in vitro fusion, but to a lesser extent. PI 3-kinase action almost certainly involves direct regulation of membrane fusion, as no vesicular intermediate has been identified, despite previous extensive morphological examination of in vitro endosome fusions.

Androstadienes↗
PubMed1995

Phosphatidylinositol 3-kinase regulation of fluid phase endocytosis.

Endocytosis of the fluid phase marker, horse radish peroxidase, into baby hamster kidney cells is inhibited by treatment of cells with the fungal metabolite wortmannin. The IC50 of approximately 5 nM is consistent with the well-described action of wortmannin upon phosphatidylinositol (PI) 3-kinase. Analysis of the kinetics of uptake indicates a > 50% decrease in the initial rate of marker internalisation, a concomitant decrease in the volume of the early endosome and an increased efficiency of recycling of that marker which is internalised. As PI 3-kinase binds to activated growth factor receptors our data suggest that receptor activation can be coupled to receptor internalisation (down regulation) by localising PI 3-kinase stimulation of endocytosis.

Androstadienes↗
PubMed1995

Species identification of Cecidophyopsis mites (Acari: Eriophyidae) from different Ribes species and countries using molecular genetics.

Cecidophyopsis mites were studied by PCR amplification of parts of their ribosomal DNA, followed by restriction enzyme analysis. Mite specimens on Ribes nigrum (black currant) from six countries gave the same digestion pattern, which was distinct from the pattern for mites found on R. rubrum from Poland and Finland and for R. grossularia from the USA. This suggests that each Ribes species is host to a different mite species: C. ribis, C. selachodon and C. grossulariae, respectively. Two other mite samples from R. alpinum and R. aureum were identical but were distinct from each of the other species.

Animals↗