Biomedical subjects
R Mitchell
Publications and source records attributed to R Mitchell.
Binuclear centre structure of terminal protonmotive oxidases.
The recent proliferation of data obtained from mutant forms of cytochrome oxidase and analogous enzymes has necessitated a re-examination of existing structural models. A new model is proposed, consistent with these data, which brings several protonatable residues (Y244, D298, D300, T309, T316, K319, T326) into the vicinity of the binuclear centre, suggestive of a proton-transferring function. In addition, we also consider those residues which may participate in electron transport between CuA and haem a. We suggest several potential lines of investigation.
Functional expression of 5-HT1c receptor cDNA in COS 7 cells and its influence on protein kinase C.
Two subtypes of receptors for serotonin (5-hydroxytryptamine; 5-HT) are known to stimulate inositol (1,4,5)-trisphosphate production, the 5-HT1c and 5-HT2 receptors. In this study we investigated the ability of 5-HT1c receptors, transiently expressed in COS 7 cells, to functionally interact with protein kinase C-alpha, the indigenous (phorbol ester-responsive) isoform of the enzyme in those cells. Serotonin caused translocation of the [3H]phorbol 12,13-dibutyrate (PDBu) binding site of PKC-alpha from the cytosolic to the membrane fraction in a Ca(2+)-dependent manner which was prevented by the 5-HT1c receptor antagonist mianserin. The lipid activators of PKC, PDBu and 1,2-dioctanoyl-sn-glycerol (DOG) also caused translocation, but through a mechanism apparently quite independent of Ca2+.
The effects of neurokinin receptor antagonists on mustard oil-evoked activation of rat dorsal horn neurons.
Previous evidence indicated that brief nociceptive responses of neurons in laminae IV/V of both rat and cat dorsal horn are more readily inhibited by antagonists at NK2 rather than at NK1 neurokinin receptors. Further support for a role of spinal NK2 receptors in nociception has been provided from experiments assessing modulation of the nociceptive flexor reflex by tachykinins and activation of dorsal horn neurons by brief application of capsaicin to afferents. The present experiments were designed to compare the contribution of NK1 and NK2 receptors in dorsal horn to the sustained neuronal activity induced by peripheral application of the chemical algogen mustard oil (reported to be a selective activator of C afferents). In nearly all of the multireceptive laminae IV/V neurons tested, a selective NK2 receptor antagonist L 659,874 inhibited previously established mustard oil-induced activity. In contrast, two selective NK1 receptor antagonists L 668,169 and GR 82334 were only rarely effective. These results further underline the apparent importance of NK2 receptors in spinal nociceptive processing. NK1 receptors do not appear to play a major role in the present experimental protocol, but they may of course do so under different circumstances.
Inhibition by NK2 but not NK1 antagonists of carrageenan-induced preprodynorphin mRNA expression in rat dorsal horn lamina I neurons.
Previous evidence indicated that NK2 rather than NK1 receptors play a central role in mediating the electrophysiological responses of dorsal horn neurons to brief cutaneous stimuli such as noxious heat (but not noxious pinch) and moderately sustained stimuli such as mustard oil, topically applied over 10-20 min. The present experiments were designed to investigate, by in situ hybridisation histochemistry, a delayed genomic response in dorsal horn neurons (the expression of preprodynorphin mRNA induced by intraplantar carrageenan injection) and explore the role of NK1 and NK2 receptors in mediating this response. In anaesthetised rats with bilateral intraplantar injections of carrageenan, neurokinin receptor antagonists were administered unilaterally by prolonged ionophoresis into the superficial dorsal horn. The marked increase in preprodynorphin mRNA expression elicited by carrageenan was inhibited (both in terms of number of expressing cells and their level of expression) by NK2 but not NK1 antagonists.
The management of patients with carcinoma of the tongue.
Surgery, radiotherapy or the combination of these two modalities remains the accepted treatment for squamous carcinoma of the tongue. In the 10-year period between 1982 and 1992, 121 patients presented to the combined Maxillofacial/Radiation Oncology Clinic, Royal Infirmary, Edinburgh with carcinoma of the tongue. The majority of T1 tumours have been treated by surgical excision, almost equal numbers of T2 tumours were treated by surgery or radiotherapy, T3 and T4 tumours without evidence of nodal disease were treated by radiotherapy. Patients presenting with established nodal disease or involvement of bone were treated primarily by surgery. The patients presenting with T3 and T4 tumours were combined to produce a more realistic figure for statistical analysis. The actual 5-year survival rates for patients presenting from 1982 to 1987 for T1, T2 and T3/4 tumours are 83%, 63% and 0% respectively. Five year actuarial survival rates for patients from 1987 to 1992 are 89%, 69% and 47%. In the absence of nodal disease in the retrospective group, T1, T2 and T3/4 carcinomas of the tongue have actual 5-year survival rates of 93%, 87% and 0% respectively. Patients who present with or later develop nodal disease have a poorer prognosis with survival rates of 33%, 33% and 0% for T1, T2 and T3/4 tumours. The size of the primary tumour did not significantly affect the five year survival rate when nodal disease was present.
Three-dimensional colour Doppler imaging.
We have developed a system to acquire in vivo three-dimensional (3D) colour velocity images of peripheral vasculature. A clinical ultrasound system was modified by mounting the transducer on a motor-driven translation stage, allowing planar ultrasound images to be acquired along a 37 mm long stroke. A 3D velocity image is acquired by digitizing, in synchrony with the cardiac cycle, successive video images as the transducer is moved over the skin surface. 3D images require about 1 min to acquire and 10 min to reconstruct before being viewed interactively. Image acquisition at several points in the cardiac cycle permits a cine-type reconstructed image. Geometrical, temporal and velocity accuracy of the acquisition and reconstruction have been quantified and found not to degrade the image.
The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release.
We examined the role of voltage-activated, L-type, Ca2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K(+)-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K+ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K+, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K(+)-induced hormone release where staurosporine partially blocked K(+)-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K(+)-induced 45Ca2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.
Differential involvement of phospholipase A2 in phorbol ester-induced luteinizing hormone and growth hormone release from rat anterior pituitary tissue.
The protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu) induced the release of both luteinizing hormone (LH) and growth hormone (GH) from proestrous rat anterior pituitary pieces in vitro. Phorbol 12,13-dibutyrate-induced LH, but not GH release was readily inhibited by the phospholipase A2 (PLA2) inhibitors, quinacrine, aristolochic acid, ONO-RS-082 and chloracysine. Furthermore, PDBu induced release of [3H]arachidonic acid ([3H]AA) from pre-labelled anterior pituitary tissue that was prevented in the presence of quinacrine, aristolochic acid and ONO-RS-082 but not the diglyceride lipase inhibitor RHC 80267. The effect of PDBu was completely inhibited by staurosporine and the selective PKC inhibitor Ro 31-8220 but only partially by low concentrations of H7; consistent with the involvement of both H7-sensitive and H7-resistant forms of PKC in the activation of PLA2 by PDBu. The protein synthesis inhibitor cycloheximide inhibited the release of both [3H]AA and LH that had been induced by PDBu, whereas LH release induced by the PLA2 activator mellitin was cycloheximide-insensitive. These results suggest that PKC activators may induce LH but not GH release from anterior pituitary tissue by a mechanism involving activation of a PLA2, brought about by a process which is reliant on protein synthesis.
The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release.
We investigated the possibility that various protein kinase C (PKC) activators and inhibitors may differentially affect luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue, incubated in vitro. Activators of PKC induced LH release with the following order of potency: mezerein > phorbol 12,13-dibutyrate (PDBu). Mezerein and PDBu were equipotent on GH release. A range of PKC inhibitors (including compounds highly selective for PKC) potently and completely inhibited PKC activator-induced LH and GH release. Chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) were less potent inhibitors of PDBu-induced GH release than of LH release. A component of PDBu- and mezerein-induced LH release was inhibited by H7 with high potency, but a second H7-insensitive component was detected. Mezerein- and PDBu-induced GH release consisted of an H7-resistant component only. When the regulatory domain of PKCs from different sources was investigated by displacement of [3H]PDBu binding, the affinity for mezerein was 3-5-fold greater than that for PDBu at PKCs from cerebral cortex, lung and alpha and beta isoforms extensively purified from brain. Anterior pituitary PKCs were unusual in showing closely matched affinity for mezerein and PDBu, reminiscent of their equivalent potency on GH release. In order to investigate the potency of the catalytic domain inhibitor H7 on PKCs from different sources, enzyme activity assays were carried out on partially purified cytosolic PKCs from midbrain and anterior pituitary and on extensively purified PKC alpha and PKC beta. The Ca(2+)-independent component of PDBu-induced (phosphatidylserine-dependent) activity from anterior pituitary alone showed unusually low potency of inhibition by H7 but was potently inhibited by staurosporine and Ro 31-8220. In contrast, the Ca(2+)-dependent PKC activity in anterior pituitary was inhibited by H7, staurosporine and Ro-31-8220 with high potency as in all other preparations. These results are consistent with the presence and active role in secretion of pharmacologically distinct forms of PKC (or PKC-like kinases) in rat anterior pituitary cells.
Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.
Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro.
The absence of Harvey ras mutations during development and progression of squamous cell carcinomas of the head and neck.
We have examined the incidence of Harvey ras mutations in human squamous cell carcinomas (SCC) of the upper aerodigestive tract using the polymerase chain reaction (PCR) followed by direct sequencing. No mutations were detected at codons 12, 13, 59 or 61 of this gene in any of six papillomas, five erythroplakias, 56 squamous cell carcinomas, and 16 SCC cell lines. Some of the SCC were lymph node metastases (three) or tumours which had recurred following radiotherapy (seven). We conclude that Harvey ras mutations are not a common event in the pathogenesis or recurrence of SCCs from Caucasian subjects, in contrast to the situation with Indian populations (Saranath et al., 1991).
Activation of MAP kinase in alpha T3-1 cells by luteinising hormone-releasing hormone.
Explore the source record for details and available documents.
Biochemical characterisation of an apparently novel isoform of protein kinase C in pituitary.
Explore the source record for details and available documents.
Identifying training and technical assistance needs in community coalitions: a developmental approach.
Explore the source record for details and available documents.
The effects of indomethacin and naproxen on zidovudine pharmacokinetics.
The effects of indomethacin and naproxen on zidovudine (ZDV) pharmacokinetics were studied in six patients with the acquired immunodeficiency syndrome (AIDS), AIDS related complex (ARC) or asymptomatic HIV disease using a placebo-controlled crossover design. Indomethacin 25 mg twice daily or naproxen 250 mg twice daily did not alter ZDV pharmacokinetics compared with placebo. The mean AUC value for the glucuronidated metabolite, GZDV, was reduced from 26.6 +/- 11.7 mumol l-1 h in the presence of placebo to 20.9 +/- 8.3 mumol l-1 h (95% C.I. of the difference 1.39-9.98; P < 0.05) following treatment with naproxen 250 mg twice daily for 3 days. The small decrease in plasma GZDV in the naproxen phase reflects an increase in clearance of ZDV to other metabolites and/or a decrease in the formation clearance to GZDV and/or an increase in the clearance of GZDV. A decrease in formation clearance to GZDV would be consistent with the results of in vitro studies reported previously. No significant increase in ZDV concentration in the presence of naproxen may reflect a lower sensitivity of parent drug measurements to selective inhibition of parallel pathways of metabolism. The clinical significance of these findings is unknown but toxicity may be increased if a decreased formation of GZDV is accompanied by shunting of metabolism to 3'-amino-3'-deoxythymidine which is alleged to be cytotoxic.
Influence of demographic factors on antithrombin III activity in a healthy population.
Antithrombin III (AT III) activity has been measured in 9669 healthy blood donors (5525 male and 4144 female). The distribution of AT III is approximately 'normal' with mean 105.6 IU/dl and standard deviation 11.2; however, definite age and sex related variations are evident. Pre-menopausal females have lower mean AT III compared to their male contemporaries who have remarkably stable mean AT III until 45 years, after which there is a gradual decline. In contrast, post-menopausal females have higher mean AT III than both males of the same age and younger pre-menopausal females. Concurrent hormone replacement therapy inhibits this rise. The use of hormonal preparations is associated with a 4 IU/dl reduction of mean AT III in younger females but not in those over 30 years. Smoking may result in a mild increase in AT III of doubtful clinical significance. On-going genetic and family studies are expected to predict a prevalence rate of congenital AT III deficiency in excess of the previously reported figure of 0.02%. The authors consider these observed variations as minor and recommend the use of a single reference range for AT III activity, but that particular care be taken when interpreting results in pill-taking females and the elderly.
Elevated serum immunoreactive inhibin levels in peripubertal boys with chronic renal failure. Cooperative Study Group on Pubertal Development in Chronic Renal Failure (CSPCRF).
OBJECTIVE: Boys with chronic renal failure have delayed progress through puberty and have raised gonadotrophin and low testosterone levels indicative of disturbed hypothalamo-pituitary-testicular function. Most studies into the mechanisms underlying the dysfunction have concentrated on the LH-Leydig cell interaction. However, it is now possible to probe the FSH-Sertoli cell axis by measuring plasma immunoreactive inhibin, which is a marker of Sertoli cell function. This study investigated the FSH-Sertoli cell (immunoreactive inhibin) axis in boys with chronic renal failure on conservative and dialysis treatment as they progressed through puberty. The effect of renal transplantation in chronic renal failure was also investigated. DESIGN: Blood was drawn at 15-minute intervals between 2000 and 0700 h from 51 boys with chronic renal failure at various stages of puberty. The samples were divided into two pools, corresponding to the hormone secretion in the first and second part of the night. Single blood samples were drawn from a group of normal boys between 0800 and 1000 h. PATIENTS: A total of 37 normal boys and 51 boys with chronic renal failure were examined immediately before and during puberty. Of a total of 80 pulse profiles taken in chronic renal failure, 36 were from transplanted and 44 from non-transplanted uraemic subjects. MEASUREMENTS: Immunoreactive inhibin, FSH and testosterone were measured using standard radioimmunoassays. The subjects were pooled into pubertal stages I, II/III and IV/V for analysis of hormone data. RESULTS: Early morning levels of immunoreactive inhibin like molecules (i-Inh) rose steadily with pubertal progression for all subject groups, those for boys with chronic renal failure being significantly elevated over normal boys from pubertal stage II/III onwards. Uraemic boys had higher levels than those who had been transplanted at all pubertal stages (P < 0.05). Early morning levels of FSH were significantly higher in uraemic patients with pubertal stages IV/V compared to our normal boys. There were no differences in i-Inh levels in plasma pooled from the samples taken between 2000 and 0115 h and 0130 and 0700 h for either treatment group at any stage of puberty. Testosterone levels rose in the second part of the profile from pubertal stages II/III onwards for both treatment groups. The proportional increase of testosterone was lower by mid puberty in uraemic than in transplanted children (percentage increases of 92 +/- 29 and 569 +/- 190 respectively, mean +/- SEM). i-Inh failed to correlate with FSH at any Tanner stage or for any subject group. CONCLUSION: Peripubertal boys with chronic renal failure have highly elevated serum immunoreactive inhibin and FSH levels which are partially reduced by renal transplantation. There was no evidence of any relationship between i-Inh and FSH secretion in either normal boys or in uraemic or transplanted boys with the exception of a positive correlation in late pubertal patients after transplantation. Finally, despite problems associated with the current immunoassay for inhibin, this assay may still prove to be a useful marker of Sertoli cell function in testicular pathology.