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J M Gilbert

Publications and source records attributed to J M Gilbert.

At least 73 records · Page 4Linked to original sources
PubMed1986

In vitro studies of the biosynthesis of brain tubulin on membranes.

Membrane elements in brain tissue contain relatively large amounts of alpha- and beta-tubulin (FIGURES 2 and 3). We have investigated the subcellular sites of tubulin biosynthesis in order to determine the origin of this membrane-associated tubulin. Free and membrane-bound polysomes from rat forebrain were separated by differential centrifugation, and the products of translation from these polysome populations were analyzed by 2DGE (FIGURES 4 and 6). Alpha- and beta-tubulin subunits were synthesized by the free polysome population (FIGURES 4 and 5A and B). The membrane-bound polysome fraction synthesized a protein with similar (but not identical) characteristics to alpha-tubulin (denoted as "MB" in FIGURE 6), including isoelectric point, molecular weight, peptide map, and copurification with microtubules after aggregation-disaggregation. Tubulin subunits synthesized in vitro by free polysomes could associate posttranslationally with a microsome fraction (FIGURE 7A). The association of the tubulin translation products with membranes was not disrupted by high salt; the associated tubulin, however, was susceptible to proteolytic digestion, with the exception of one of the beta-tubulin subunits (FIGURE 7B). There was an identical protease-resistant beta-tubulin subunit among the native proteins of the smooth microsome fractions. Our data is consistent with the conclusion that at least one beta subunit of membrane-associated tubulin is synthesized by free polysomes and becomes posttranslationally added to membrane structures. It is unlikely that a cotranslational mechanism is responsible, in which there is a signal-mediated insertion of a growing polypeptide chain to membrane. Our results, however, are consistent with a "membrane trigger" mechanism proposed by Wickner in which the membrane lipid bilayer triggers the folding of a polypeptide into a configuration that allows integral membrane insertion. The association of tubulin with membranes may also be secondary to the interaction of hydrophobic elements. The amino acid sequence of beta tubulin is known to contain several hydrophobic domains. Tubulin can be incorporated into phospholipid vesicles and various subcellular membrane elements. In our studies, in vitro synthesized tubulin from free polysome was found to be purified by hydrophobic affinity chromatography with ethane-sepharose (FIGURE 8). Thus, the hydrophobic characteristics of newly synthesized tubulin could be partially responsible for the posttranslational association of tubulin subunit with membranes. Native tubulin in a soluble fraction of CNS tissue was not purified by hydrophobic affinity chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Trials of adjuvant chemotherapy in colorectal cancer.

Chemotherapy has been extensively investigated in colorectal cancer. Evaluation in advanced disease has shown that only a limited number of drugs are active. This activity was assessed according to objective criteria for response of measurable lesions, but despite producing objective remissions, no agent has prolonged survival in advanced disease. Drugs showing activity in advanced disease have been evaluated subsequently as adjuvants to surgery. Single agents were evaluated first and 5-fluorouracil has been investigated most extensively. It has been administered by a variety of routes and in different regimens and in some trials it has produced results which border on statistical significance for both disease-free interval and survival. It is the drug of first choice in colorectal cancer, although in some trials no effect was seen. Razoxane (ICRF 159) has also been evaluated and has shown some promise, although also some toxicity. Trials of combinations of treatments have been performed and have used permutations of 5-fluorouracil, MeCCNU, BCG and radiotherapy. None of these trials has shown a definite advantage for chemotherapy and, in some, toxicity has been considerable. 5-Fluorouracil as a single agent has shown the best results to date, and is the treatment of first choice but no treatment has been shown to prolong survival unequivocally in colorectal cancer. Future trials should include a surgery-only control arm, against which any new treatment can be compared.

Antineoplastic Agents↗
PubMed1986

Detailed faecal bile acid profile: a diagnostic test for colorectal cancer?

Detailed profiles of bile acids in faeces were evaluated as a diagnostic test for colorectal cancer in rats. Twenty-seven bile acid peaks were measured using improved methods of extraction and separation followed by the sensitive and specific techniques of capillary column gas liquid chromatography and mass spectrometry. Colorectal cancer was induced in experimental animals (female Sprague-Dawley rats, n = 20) by subcutaneous injection of dimethylhydrazine (DMH) and faecal unconjugated bile acids compared with those in the control group (n = 20). The amount of total faecal unconjugated bile acids was lower in the animals administered DMH (255 mg/day vs 334 mg/day: (P = 0.04), and the excretion of seven individual bile acids was reduced when compared with those in the control group (P less than 0.01). In order to use the faecal bile acid profiles as a diagnostic test, linear discriminant analysis was performed. A discriminant score was derived which was applied to each profile, to determine to which group (control or DMH) each animal belonged retrospectively. All analyses were performed blind, and 90% of the animals were correctly assigned. In man, as in rats, the bile acid profile of faces is equally complex and the bile acid profile may be useful as a diagnostic test.

Animals↗
PubMed1985

Serum unconjugated bile acids: qualitative and quantitative profiles in ileal resection and bacterial overgrowth.

Qualitative and quantitative profiles of unconjugated bile acids in the serum obtained over a 24-h period from three patients with ileal resections and one with a bacterial overgrowth are described. Unconjugated serum bile acids were determined using the high sensitivity and resolution of capillary column gas liquid chromatography after their rapid extraction and isolation using reverse phase octadecylsilane bonded silica cartridges and the lipophilic gel Lipidex 1000. Unconjugated serum bile acid concentrations were elevated throughout the day in both ileum resected patients and in conditions involving bacterial overgrowth when compared to healthy subjects. Total conjugated cholic acid concentrations were expectedly low in both intestinal disorders and were without the postprandial increases generally observed in healthy subjects. Qualitative gas chromatographic profiles of serum unconjugated bile acids in bacterial overgrowth distinctly revealed a predominance of deoxycholic acid and other secondary bile acids in all samples, while, in conditions of an impaired enterohepatic circulation, deoxycholic acid was absent or present in only trace amounts. The potential significance of measuring serum unconjugated bile acids in intestinal disorders is discussed.

Adult↗
PubMed1985

Woodchuck hepatitis virus infection: serologic and histopathologic course and outcome.

Five out of seven American woodchucks inoculated with woodchuck hepatitis virus developed antigenemia after 2 to 13 weeks followed by an antibody response. One animal became a carrier, and another animal exhibited a primary antibody response. Clinical disease was not obvious and aminotransferase elevation could not be demonstrated. Liver biopsy showed mononuclear portal infiltration and little parenchymal cell necrosis.

Animals↗
PubMed1984

Sites of recurrent tumour after 'curative' colorectal surgery: implications for adjuvant therapy.

The pattern of recurrent tumour following 'curative' colorectal surgery was examined in a necropsy series and a prospective clinical series. In the necropsy series the commonest pattern of recurrence was disseminated disease (73 per cent) and recurrence in a single site was much less common (27 per cent). In the clinical series recurrence in a single site was commoner (55 per cent). The difference between the two series is statistically significant (P = 0.02, chi 2), and is probably due to under-diagnosis of disseminated recurrence in the clinical series. In both series local recurrence and hepatic metastases occurred almost equally but usually as part of disseminated disease. We conclude that after 'curative' surgery for colorectal cancer, recurrence is usually disseminated (73 per cent) and therefore therapy adjuvant to surgery should be active systemically. Adjuvant therapy directed at a single site (e.g. radiotherapy or intrahepatic chemotherapy) should be combined with a systemic therapy.

Adult↗
PubMed1984

Inhibition of experimental colorectal cancer by razoxane (ICRF-159).

Large bowel cancer induced in rats by dimethylhydrazine (DMH) closely resembles human disease both histologically and clinically. In this study it has been used to assess the antimitotic drug razoxane (ICRF-159). Dimethylhydrazine induced benign colorectal tumours after 20 weeks and adenocarcinomas from 30 weeks. Razoxane was given from week 25 until the end of experiment (week 35) to see if this drug could inhibit the development of tumours. Animals were randomly allocated to four groups: DMH + razoxane (26), DMH + control (26), control + razoxane (25), control + control (25). There were fewer malignant colorectal tumours in rats receiving razoxane than in the controls (10 vs. 24; P = 0.04). Some animals developed malignant tumours of the small intestine as well as the large bowel and the number of malignant tumours for the whole intestinal tract was also reduced in animals receiving razoxane (15 vs. 32; P = 0.025). This study demonstrates inhibition of the development of malignant tumours in rats by razoxane. This finding may have relevance to colorectal cancer in man where razoxane has been used in disseminated disease and as an adjuvant to surgery.

1,2-Dimethylhydrazine↗
PubMed1984

Cellular origin and biosynthesis of rat optic nerve proteins: a two-dimensional gel analysis.

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.

Animals↗
PubMed1984

Characterization and biosynthesis of cyclic-AMP-binding proteins in the rat central nervous system.

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Properties and function of brain carbonic anhydrase.

This chapter has described the characterization and biogenesis of soluble and membrane-bound CA in the central nervous system. The two forms of the enzyme appear to be quite similar in their molecular characteristics, however the data strongly indicate that they are synthesized on separate polysomal populations; the membrane-bound form resulting from synthesis on the RER. Our preliminary data suggest that the partitioning of mRNA for CA on the different polysomes results from the interaction of partial nascent chains with a specific receptor on the RER. We feel a function of membrane-associated synthesis is for the targeting of CA to sites in the cell where there are enzymes that can rapidly utilize the protons and bicarbonate produced by CA catalytic activity for ion exchange reactions. We have also presented arguments that CA may function as a bicarbonate source in the control of metabolism specifically in the acceleration of fatty acid synthesis in the oligodendrocyte.

Aging↗
PubMed1983

Fat and cancer.

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Animals↗
PubMed1983

Studies on the cell-free biosynthesis of CNS membrane proteins.

The biosynthesis of CNS membrane proteins was studied in cell-free systems containing membrane-bound polysomes (rough endoplasmic reticulum; RER) or free polysomes from rat forebrain. In previous studies of CNS membrane proteins using two-dimensional gel electrophoretic analysis, five proteins (mol. wt. -pI: 75K, 5.4, 68K 5.6, 61K 5.1, 58K 5.1, and 36K 5.6) were found in cell membrane fractions including preparations enriched in RER, smooth endoplasmic reticulum, and plasma membranes. One of these proteins, 68K 5.6, was also present in cytosol and comigrated with a microtubule-associated protein. In our present study, cell-free systems containing RER were found to synthesize the 75K, 5.4, 61K 5.1, and 58K 5.1 proteins. A protein, 34K 5.65, similar (but not identical) to the 36K 5.6 protein was also synthesized. After cell-free synthesis, 75K 5.4 and 58K 5.1 proteins could be purified by concanavalin A affinity chromatography. Of the five common membrane proteins previously identified, only 68K 5.6 protein was synthesized by the free polysome population. The free polysomes were also found to synthesize cyclic AMP binding proteins at 48K and 54K, known from previous studies to be present in both cytosol and plasma membrane fractions in mammalian brain tissue. In conclusion, RER synthesized proteins found exclusively in CNS membrane fractions, whereas free polysomes synthesized those proteins found in both soluble and membrane compartments.

Animals↗
PubMed1983

Characterization and biosynthesis of soluble and membrane-bound carbonic anhydrase in brain.

Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.

Animals↗
PubMed1983

Chemotherapy of chemically-induced colorectal tumours.

Benign and malignant tumours were induced in the large bowel of rats by the carcinogen 1,2-dimethylhydrazine (DMH). Benign tumours appeared from week 20 onwards and malignant tumours from week 30. 5-Fluorouracil was administered intraperitoneally in maximally tolerated doses from week 26 onwards and failed to influence significantly the development of either benign or malignant tumours. Tumours induced by DMH are a close model of human colorectal cancer and suitable for testing anticancer drugs. Agents with greater activity against colorectal cancer are required.

Animals↗
PubMed1982

Adjuvant oral razoxane (ICRF-159) in resectable colorectal cancer.

One hundred and seventy six patients (81 controls, 95 receiving treatment) have entered a prospective randomized trial of long-term oral adjuvant razoxane (ICRF-159) following removal of a colorectal cancer. The median follow-up is 34 months. The treated patients in Dukes' groups B and C have a significantly longer disease-free interval than the control patients (P = 0.01 'as randomized' and P = 0.004 'as treated'). The differences in survival for Dukes' groups B and C are not significant, although follow-up is short. In Dukes' groups B and C, however, 24 of 56 of the patients in the control group have died (43%), as against only 17 of 64 in the treatment group (27%). The treatment produces very few side-effects, is well tolerated by patients, and is taken orally.

Administration, Oral↗