Biomedical subjects
G Thomas
Publications and source records attributed to G Thomas.
MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways.
Activation of protein synthesis is required for quiescent cells to transit the cell cycle, and seems to be mediated in part by phosphorylation of the 40S ribosomal protein, S6. A mitogen-activated S6 kinase of relative molecular mass 70,000 (70K) has been isolated from mouse fibroblasts as well as from avian, rat and rabbit tissues. Comparison of complementary DNA sequences shows that this enzyme is distinct from S6 kinase II (92K) found in Xenopus eggs and fibroblasts. Both kinases are activated by serine/threonine phosphorylation, suggesting that at least one serine/threonine kinase links receptor tyrosine kinases with S6 kinases. A candidate for this link is MAP2 kinase, which is rapidly activated by tyrosine/threonine phosphorylation following mitogenic stimulation. Incubation of MAP2 kinase from insulin-treated 3T3-L1 adipocytes with phosphatase-inactivated S6 kinase II from Xenopus leads to partial reactivation and phosphorylation of the enzyme. These and other findings have led to the suggestion that MAP2 kinase also activates the 70K S6 kinase. Here we refute this idea by showing that the two kinases lie on distinct signalling pathways.
Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population.
We describe a simple method for characterizing a frequent polymorphism (that substitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer.
Genotypic differences in hemangiopericytic meningioma.
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Micro- and macropurification methods for protein kinases.
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Affinity purification of protein kinases using adenosine 5'-triphosphate, amino acid, and peptide analogs.
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Purification and properties of mitogen-activated S6 kinase from rat liver and 3T3 cells.
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The prognostic importance of site and type of radiation-induced bowel injury in patients requiring surgical management.
A multivariate analysis was performed to determine the outcome, and factors prognostic for outcome, in 57 patients requiring surgical intervention for radiation bowel injury. The actuarial 2- and 5-year cause-specific survival (CSS) was 76 and 74%, respectively, with a median follow-up of 62 months for the survivors. The median time from surgery to death from complications was 4 months. Identified sites of injury were both large and small bowel. The types of injury were defined as stricture, perforation, inflammation, and fistula. At surgery 9 patients had more than one site, and 15 patients had more than one type of injury. Cox proportional hazards regression models relating survival to individual patient characteristics were constructed using surgical procedure, radiation-surgery interval, age, stage, radiotherapy technique and dose, and the individual sites and types of injuries. Only the site of injury was found to be of prognostic significance for CSS (P less than 0.03). However, when the site and type of injury were recoded as single or multiple, Cox regression analysis found both the site (P = 0.008) and the type (P = 0.02) of injury to be statistically significant for CSS (favoring single sites and types). Stepwise multivariate regression analysis found type of injury to be insignificant when site of injury was already in the model. Ileal damage was associated with the lowest CSS of any single site of injury (56%) and also appeared to be responsible for the poor CSS of those with multiple sites of injury (46%).
Intracellular messengers and the control of protein synthesis.
The molecular events responsible for controlling cell growth and development, as well as their coordinate interaction is only beginning to be revealed. At the basis of these controlling events are hormones, growth factors and mitogens which, through transmembrane signalling trigger an array of cellular responses, initiated by receptor-associated tyrosine kinases, which in turn either directly or indirectly mediate their effects through serine/threonine protein kinases. Utilizing the obligatory response of activation of protein synthesis in cell growth and development, we describe efforts to work backwards along the regulatory pathway to the receptor, identifying those molecular components involved in modulating the rate of translation. We begin by describing the components and steps of protein synthesis and then discuss in detail the regulatory pathways involved in the mitogenic response of eukaryotic cells and during meiotic maturation of oocytes. Finally we discuss possible future work which will further our understanding of these systems.
Photodynamic therapy for chest wall recurrence in breast cancer.
Photodynamic therapy is the use of a sensitizer (dihematoporphyrin ethers) which is preferentially retained in tumor cells and activated by subsequent light delivery resulting in a selective tumoricidal effect. Between 1986 and 1989, we treated 20 patients with photodynamic therapy for chest wall recurrence of breast cancer. Responses were seen (20% complete response, 45% partial response, 35% no response), but the duration of response was short (average 2.5 months). Complications, in decreasing frequency, included pain, ecchymoses, blistering, ulceration and necrosis in the area of tumor involvement on the chest wall. One patient required skin flap reconstruction for full thickness necrosis. A limitation to this mode of therapy is that the sensitizer currently used is activated by light at a wavelength of 630 nm. This light can penetrate to a tissue depth of only 0.5 to 1.0 cm; thus, deeper disease cannot be treated. Future research must focus on the development of a clinically useful photosensitizer that can be activated by light at longer wavelengths and thereby achieve deeper tissue penetration. This would greatly expand the patient population for which this therapy is useful.
Mapping of human chromosome 22 with a panel of somatic cell hybrids.
The adenylosuccinate lyase (ADSL) which is essential for generating adenylate, maps to the long arm of chromosome 22. By using a Chinese hamster ovary cell line deficient in ADSL activity, we have constructed a set of 17 somatic cell hybrids containing defined regions of human chromosome 22. This panel was extended with six additional hybrids, obtained in other laboratories using various methods of selection. Southern analysis of the hybrids with 38 chromosome 22 probes defined 14 different subregions which could be linearly organized on the long arm of chromosome 22. The order of the probes thus deduced is fully compatible with their previous localization and with the genetic map. The ADSL gene was further sublocalized between the MB and D22S22. This panel, which enables the rapid assignment of chromosome 22 single copy probes to small subregions, will be an important tool in the construction of a detailed physical map of this part of the genome.
Organization and expression of the lambda-like genes that contribute to the mu-psi light chain complex in human pre-B cells.
Lambda-like genes encode a polypeptide chain that associates with VpreB and mu chain in the so-called mu-psi light chain complex specifically expressed in pre-B cells. In humans, the lambda-like gene cluster contains three genes, termed 14.1, 16.1, and F lambda 1. The 14.1 gene contains three exons and was previously sequenced. The 16.1 and F lambda 1 have been isolated from cosmid libraries. They both contain only the exons 2 and 3 that appear highly homologous to their 14.1 counterparts. The lambda-like gene cluster has been mapped on chromosome 22, close and distal to the BCR gene, in the order (14.1; F lambda 1) and 16.1. Hybridization with the 14.1 exon 1 probe did not detect an equivalent on 16.1 and F lambda 1 genes, but instead, only revealed the X6 gene, that was previously identified 5 kb upstream of the IGLC locus. It appears, therefore, that the gene organization of the lambda and lambda-like loci is strikingly similar, with a three-exon-containing gene 5' of a series of J lambda-C lambda or J lambda-C lambda-like tandemly organized genes. Analysis of the transcripts in a 8 week old human fetal liver clearly indicated that 14.1 was the only functional gene of the lambda-like cluster. Various forms of transcripts resulting from alternate splicing have been characterized. The major component consisted of a full-length (exons 1-2-3) mRNA, whereas a minor (1-3) transcript was identified. Only the full-length transcript could encode a functional polypeptide chain corresponding to the 22 kDa light chain surrogate.
Effects of gonadotrophin releasing hormone antagonist and agonist on the pulsatile release of gonadotrophins and alpha-subunit in postmenopausal women.
OBJECTIVE: The present study was designed to further assess the mechanism of action of GnRH and GnRH analogues. DESIGN AND PATIENTS: Both the Nal-Glu GnRH antagonist and the D-Trp6 GnRH agonist were administered sequentially to nine normal, post-menopausal women. MEASUREMENTS: A baseline study of pulsatile LH, FSH and free alpha-subunit secretion was performed, with sampling every 10 min for 8 h, and then repeated 8 h after a single subcutaneous injection of Nal-Glu GnRH antagonist (5 mg). Sampling was repeated 21 days after the intramuscular injection of a depot preparation of D-Trp6 GnRH (3.75 mg) in the same women. RESULTS: The baseline sampling period showed synchronous pulses of LH and free alpha-subunit. The antagonist Nal-Glu decreased plasma LH (71%) and free alpha-subunit (43%). However, with the single dose of 5 mg, pulsatile LH and free alpha-subunit release were not completely suppressed and remained temporally correlated. The GnRH agonist had a potent inhibitory action on plasma immunoreactive LH (IRMA) (93%). In contrast, it increased the mean plasma levels of free alpha-subunit from 1.66 +/- 0.01 to 5.06 +/- 0.02 micrograms/l (205%). The pulsatile secretory patterns of both LH and free alpha-subunit were abolished by the agonist. Immunoreactive FSH levels were decreased by the antagonist (24%) and suppressed by the agonist (93%). CONCLUSIONS: The pulsatile study confirms the different mechanism of action of GnRH analogues. Following antagonist administration, low amplitude free alpha-subunit pulses persist and are synchronous with residual LH pulses. In contrast, LH and free alpha-subunit are not maintained under agonist treatment. These data provide evidence for the differential regulation of LH and free alpha-subunit by GnRH.
Steady-state population pharmacokinetics of sustained release theophylline in adult asthmatic patients.
The steady-state population pharmacokinetics of theophylline were studied in 52 asthmatic adult patients who received sustained-release theophylline as armophylline or euphylline. A total of 92 steady-state plasma theophylline concentration-dosage pairs were analyzed using a nonlinear mixed effects model. The pharmacokinetic model used was a one-compartment open model with single path Michaelis-Menten elimination. Dosage was adjusted to body weight. The effects of age, gender, alcohol consumption, cigarette smoking, dosage form, concurrent treatment with beta-agonists or steroids, outpatient dosing, and plasma caffeine concentration on maximum elimination rate (Vm) and Michaelis constant for theophylline metabolism (Km) were investigated. Hypothesis testing produced a final model in which Km = 0.42 (mg/l), and Vm (mg/kg per day) was based on cigarette smoking and dosage form, with Vm = 7.54 + 2.01 (smoking) + 1.08 (euphylline). Estimated coefficients of variation for interindividual variability in Km and Vm were 162.6% and 48.1%, respectively. Residual variability in dosage rates was estimated as 0.90 mg/kg per day. The identification of factors influencing theophylline disposition should prove useful for the a priori design of theophylline dosage regimens and monitoring of drug levels during therapy.
Influence of age on the responsiveness of the gonadotrophs to luteinizing hormone-releasing hormone in males.
The mechanisms that most likely underly the attenuated response of plasma LH to antiopioids or antiestrogens and the reduced frequency of large amplitude LH pulses in elderly men are decreased LHRH release and/or decreased sensitivity of the gonadotrophs to LHRH. To test the latter hypothesis, the LH response, measured both as bioactivity (B) and immunoactivity (IR), to increasing doses of LHRH ranging from 0.625-5 micrograms, administered as an iv bolus, was evaluated in 10 young (20-45 yr old) and 10 elderly (64-78 yr old) monks. Both basal B- and IR-LH levels were significantly higher in elderly men. The maximal response to each dose was similar in young and elderly men when measured as IR-LH, but the B-LH response was higher in elderly men. The B/IR LH ratio increased with increasing LH levels. The mean half-life of plasma IR-LH in the elderly was significantly longer than that in the group of young men and significantly longer than the mean half-life of B-LH. It is concluded that the sensitivity of the gonadotrophs to LHRH is not decreased in elderly men. This suggests that the attenuated response of LH levels to antiopioids and antiestrogens, respectively, as well as the reduced number of spontaneous high amplitude LH pulses in elderly men may, instead, be the consequence of the release of a decreased mass of LHRH at each pulse.
Effect of substituted arginine compounds on superoxide production in the rabbit aorta.
Superoxide anion (O2-) blocks the vascular effect of endothelium-derived relaxing factor (EDRF). Previous reports implicate L-arginine or N alpha-substituted arginine compounds as precursors of EDRF. Employing a microassay for the measurement of O2- production by rabbit aortic rings, which is based on the established method of reduction of cytochrome c by O2-, we studied the interaction between O2- and EDRF using L-arginine, an N-substituted arginine compound, namely, N alpha-benzoyl-L-arginine ethyl ester (BAEE), sodium nitroprusside and NG-monomethyl-L-arginine (NMMA), a putative specific inhibitor of EDRF synthesis. Measurements of O2- production were made under basal conditions and upon stimulation with alloxan, the diabetogenic, O(2-)-generating compound. Both BAEE, an EDRF-generating agent (0.3 and 3.0 mM) and sodium nitroprusside, an NO-generating compound (1.7 and 3.4 microM), significantly reduced alloxan-stimulated O2- production, but L-arginine (0.6-3.0 mM) paradoxically increased O2- generation. NMMA (1.0 mM) blocked the inhibitory effect of BAEE on O2- production in an endothelium-dependent manner. Because NMMA is an inhibitor of EDRF, these results suggest that BAEE, but not L-arginine, reduces O2- produced by the endothelium of the rabbit aorta through a mechanism involving EDRF generation.
Protein-DNA interactions in vivo--examining genes in Saccharomyces cerevisiae and Drosophila melanogaster by chromatin footprinting.
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Effects of guanidino compounds on the endothelium-derived relaxing factor inhibitor NG-monomethyl L-arginine.
The vasoconstrictor effect of NG-monomethyl L-arginine (L-NMMA) is believed to be due to the inhibition of the synthesis of endothelium-derived relaxing factor (EDRF) from L-arginine. Here, we tested a series of guanidino compounds other than L-arginine on the rat aorta preparation with and without endothelium present. None of the compounds promoted vascular relaxation like N alpha-benzoyl L-arginine ethyl ester or elicited vasoconstriction like L-NMMA. We discovered that the two guanidino compounds, L-homoarginine (L-HA) and L-amino-tau-guanidino butyric acid (L-AGBA), behave like L-arginine and reversed the vasoconstrictor effect of L-NMMA. This effect was stereospecific and concentration-dependent. The order of potency to overcome the effects of L-NMMA was L-HA, followed by L-AGBA. This was the same order of potency for overcoming the inhibitory effects of L-NMMA on cyclic GMP formation. Two related compounds, L-amino guanidino propionic acid and guanidine, were ineffective. Furthermore, high performance liquid chromatography analysis showed that rat aortic vessels contain the same amount of L-arginine in the presence or absence of endothelium, and no detectable amount of L-citruline is formed during endothelium-dependent relaxation. We conclude that the reversal of the effects of L-NMMA by L-HA and L-AGBA is due to their structural similarities to L-NMMA and not to synthesis of an EDRF-like material from these guanidines. We suggest that some of the inhibition of L-NMMA by L-arginine may have a similar basis of structural antagonism.