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Biomedical subjects

G Griffiths

Publications and source records attributed to G Griffiths.

At least 55 records · Page 3Linked to original sources
PubMed1999

Overall treatment time and the conventional arm of the CHART trial in the radiotherapy of head and neck cancer.

BACKGROUND AND PURPOSE: An analysis of the 366 patients treated with conventional radiotherapy in the CHART randomized trial was performed to determine whether prolongation of treatment time had any influence on tumour control or survival and to assess if this could have influenced the results of the randomized comparison of CHART against conventional radiotherapy. METHOD: After a preliminary analysis the cases were divided into two groups according to duration of treatment. RESULTS: Survival and primary tumour control showed small margins of deterioration when the 232 patients who had been treated up to 48 days were compared with 127 patients who had been treated in 49 or more days. These differences were not large with an estimated difference in 2-year survival of 8% and not conventionally statistically significant (P=0.25); furthermore, the survival difference was considerably reduced when the prognostic factors were taken into consideration in a Cox model. CONCLUSION: With a mean difference in treatment duration of 5.8 days, the marginal differences observed between patients treated for longer and shorter times are compatible with that which has been suggested in previous publications for the prolongation of treatment time in head and neck cancer. We conclude that the randomized comparison of CHART with conventional radiotherapy is unlikely to be affected by conventionally treated patients who took longer than 48 days to complete their treatment.

Carcinoma, Squamous Cell↗
PubMed1999

Continuous, hyperfractionated, accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small cell lung cancer: mature data from the randomised multicentre trial. CHART Steering committee.

BACKGROUND AND METHOD: A randomised controlled trial in locally advanced non-small cell lung cancer (NSCLC), compared CHART which employs 36 fractions of 1.5 Gy 3 times per day to give 54 Gy in 12 consecutive days with conventional radiotherapy-30 fractions of 2 Gy to a total dose of 60 Gy in 6 weeks. A total of 563 patients were entered between April 1990 and April 1995. This report is based upon the data updated to 1 April 1998. RESULTS: The analysis of the mature data shows that the benefits previously reported have been maintained. Overall there was a 22% reduction in the relative risk of death, which is equivalent to an absolute improvement in 2 year survival of 9% from 20 to 29% (P = 0.008) and a 21% reduction in the relative risk of local progression (P = 0.033). In the large subgroup of patients with squamous cell cancer which accounted for 81% of the cases, there was a 30% reduction in the relative risk of death, which is equivalent to an absolute improvement in 2 year survival of 13% from 20 to 33% (P = 0.0007) and a 27% reduction in the relative risk of local progression (P = 0.012). Furthermore, in squamous carcinoma there was a 25% reduction in the relative risk of local and/or distant progression (P = 0.025) and 24% reduction in the relative risk of metastasis (P = 0.043). There was no evidence that CHART gave more or less benefit in any other subgroup. CONCLUSION: This analysis of mature data confirms that CHART is superior to conventional radiotherapy in achieving local tumour control and survival in locally advanced NSCLC. This demonstrates the importance of cellular repopulation as a cause of failure in the radiotherapy of NSCLC. The reduction in the risk of metastasis confirms that improved local tumour control, even in lung cancer, can reduce the incidence of metastasis. This trial shows that control of local tumour can lead to an improvement in long term survival.

Adult↗
PubMed1999

Antisense-mediated depletion of potato leaf omega3 fatty acid desaturase lowers linolenic acid content and reduces gene activation in response to wounding.

Fatty acid omega3 desaturases act on membrane lipids to catalyse the formation of trienoic fatty acids, the most abundant in plant tissues being alpha-linolenic acid. This fatty acid is a precursor of jasmonic acid, a plant growth regulator involved in the control of wound-induced gene activation in plants and in the induction of tuberization in potato. We isolated a potato omega3 desaturase cDNA, possibly encoding a plastidial isoform, and used it to investigate its expression pattern throughout plant development and in response to wounding. Plastidial omega3 desaturase gene transcripts accumulate rapidly upon wounding, preceding the jasmonate-dependent induction of the wound-responsive proteinase inhibitor II gene. We generated transgenic potato plants constitutively expressing an antisense RNA to this plastidial omega3 desaturase. Selected transgenic lines in which the cognate omega3 desaturase mRNA is largely depleted show a marked reduction, of up to 60%, in trienoic acids in leaves and tubers. In these lines, a corresponding reduction in jasmonate content and proteinase inhibitor II expression is observed upon wounding. Our results indicate that a reduction in omega3 desaturase mRNA levels compromises the wound-induced activation of proteinase inhibitor II, suggesting that wound-induced synthesis of linolenic acid is required for jasmonic acid production. The antisense-mediated depletion of fatty acid omega3 desaturases is a viable alternative for reducing trienoic fatty acid content in plant species in which a mutant screening approach is not applicable.

Amino Acid Sequence↗
PubMed1999

GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6.

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

Amino Acid Sequence↗
PubMed1999

Two fatty acid delta9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutation.

Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases. LM9 and delta9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.

Fatty Acid Desaturases↗
PubMed1999

Localized adhesion molecule expression and circulating LFA-3 levels in adult and early onset forms of periodontitis.

Because of their importance in mediating cellular interactions in chronic inflammatory diseases, this study has examined the expression of a number of adhesion molecules in adult (n=11), generalized early onset (n=5) and localized early onset (n=2) forms of periodontitis. In comparison with immunostaining profiles of cryostat sections of healthy gingival tissue (n=7), the beta 1 integrins VLA-1, VLA-2 and VLA-4 were found to be up-regulated in periodontitis, with VLA-6 being markedly elevated. Although only small differences were observed in ICAM-1 and LFA-3 expression in the gingival epithelium, there was particularly notable up-regulation of these adhesion molecules within the inflammatory infiltrates of the diseased tissues. However, there were no statistically significant differences between the serum levels of a soluble form of LFA-3 in periodontitis patients (n=47) compared with healthy control subjects (n=40), although the generalized early onset and adult periodontitis groups exhibited wider ranges of circulating LFA-3. These findings show that there is localized modulation of adhesion molecule expression in the chronic inflammatory periodontal diseases studied, but that the levels of LFA-3 in the circulation nevertheless remain unaffected.

Adolescent↗
PubMed1999

Management of acute nontraumatic upper limb ischemia.

A retrospective review of all patients presenting to a tertiary referral center with acute nontraumatic upper limb ischemia between January 1992 and June 1997 was undertaken to examine the role of intraarterial thrombolysis in the management of such cases. Twenty-one patients were identified in the radiology and vascular surgery departments' registers. Twenty (95%) underwent angiography, demonstrating subclavian artery occlusion in four, axillary in two, brachial in 13, and one at the digital level. Intraarterial thrombolysis was attempted in 12 patients. There were three technical failures, all requiring embolectomy. Six had complete lysis and resolution of their symptoms. One patient had partial lysis but experienced no further rest pain. Thrombolysis was unsuccessful in two cases with one subsequently requiring embolectomy and the other surgical bypass. Three patients had surgical intervention as their primary procedure with two favorable outcomes and one ending in above-elbow amputation. Five patients were treated conservatively with heparin, resulting in three partial and two full recoveries. One patient experienced complete resolution of symptoms with an intravenous prostacyclin infusion. Both electrocardiograms (ECG) and echocardiograms (ECHO) were of limited diagnostic aid, and long-term warfarin anticoagulation was prescribed to all patients. There was no recurrence of upper limb ischemia at a median follow up of 18 months. Intraarterial thrombolysis is an effective first line treatment for acute nontraumatic upper limb ischemia in selected cases.

Acute Disease↗
PubMed1999

Conjugation of an anti-B-cell lymphoma monoclonal antibody, LL2, to long-circulating drug-carrier lipid emulsions.

Long-circulating submicron lipid emulsions, stabilized with poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE), are promising drug carriers with substantial capacity for solubilization of lipophilic anticancer agents. This study describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of lipid-emulsion globules by use of a novel poly(ethylene glycol)-based heterobifunctional coupling agent. The efficiency of coupling of LL2 to the lipid emulsion was 85% (approx.) and essentially independent of the LL2/emulsion particle ratio and amount of surface-bound PEG-PE. Results from sucrose-gradient centrifugation and Sepharose CL-4B gel filtration indicated stable binding of the antibody to the emulsion. The immunoreactivity of the emulsion-LL2 conjugates was tested with alkaline phosphatase-conjugated LL2 against a monoclonal anti-idiotype antibody, WN. The binding of the conjugates to WN increased with increasing surface density of LL2 up to 40 monoclonal antibodies/emulsion particle, and exceeded that for the free monoclonal antibody (approx. 20 molecules/particle). Results from competitive-binding ELISA were indicative of similar displacement curves for free LL2 and emulsion-LL2 conjugates. Direct cellular ELISA revealed similar binding of emulsion-LL2 complexes to three types of Burkitt's lymphoma cell lines, Raji, Ramos and Daudi. The results from this study indicate that emulsion-LL2 complexes might be a useful drug-carrier system for more specific delivery of anticancer drugs to B-cell malignancy.

Antibodies, Monoclonal↗
PubMed1998

Functional identification of a fatty acid delta5 desaturase gene from Caenorhabditis elegans.

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid delta5 desaturase. Saccharomyces cerevisiae expressing the full-length cDNA was able to convert di-homo-gamma-linolenic acid to arachidonic acid, thus confirming delta5 desaturation. The 1341 bp delta5 desaturase sequence contained an N-terminal cytochrome b5 domain and was located within a kilobase of the C. elegans delta6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a delta5 desaturase gene isolated from an animal.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages.

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.

Animals↗
PubMed1998

KIF2beta, a new kinesin superfamily protein in non-neuronal cells, is associated with lysosomes and may be implicated in their centrifugal translocation.

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2beta, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2beta is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2beta-transiently-transfected fibroblasts show KIF2 and KIF2beta primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2beta-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2beta does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2beta as a motor responsible for the peripheral translocation of lysosomes.

3T3 Cells↗
PubMed1998

Isolation of a Delta5-fatty acid desaturase gene from Mortierella alpina.

Arachidonic acid (C20:4 Delta5,8,11,14) is a polyunsaturated fatty acid synthesized by the Delta5-fatty acid desaturation of di-homo-gamma-linolenic acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Delta5-desaturase is the first example of a cloned Delta5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.

Amino Acid Sequence↗
PubMed1998

Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.

Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.

Amino Acid Sequence↗
PubMed1998

Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes.

In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.

Animals↗
PubMed1998

Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides.

The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained delta9, delta12 and delta6 desaturase activities. The delta9 desaturase exhibited characteristics similar to those of the animal and yeast delta9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the delta12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the delta12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine:acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. Delta6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and gamma-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.

Acyl Coenzyme A↗
PubMed1998

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant alpha-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.

Amino Acid Sequence↗