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M J Smith

Publications and source records attributed to M J Smith.

At least 109 records · Page 6Linked to original sources
PubMed1998

Chemical cleavage of mismatch: a new look at an established method.

Chemical cleavage of mismatch (CCM), also known as chemical mismatch cleavage (CMC) or the HOT (hydroxylamine/osmium tetroxide) chemical method, has been used for detection of sequence variability with many systems since it was first described. Recently, adaptation to fluorescence-based detection systems has fundamentally changed both the execution and analysis of CCM. This review will outline major advances in the methodology of CCM, from the advent of PCR through fluorescent analysis, and includes applications and modifications of CCM.

DNA Mutational Analysis↗
PubMed1998

Critical parameters influencing hyperthermia-induced apoptosis in human lymphoid cell lines.

Brief mild hyperthermia is sufficient to induce apoptosis (programmed cell death) in many cell lines. Here we describe the effects of a number of factors modulating heat shock induced apoptosis outcomes. We report the effects of cell type, heat load, recovery times, cellular growth phase, and protein synthesis on the levels of apoptoses seen in heat stressed cell populations. We observe that a number of cell lines are competent to undergo heat stress induced apoptosis using both the comet assay and cellular and nuclear morphologies. Of the cell lines tested we saw a wide spectrum of sensitivities, ranging from resistant (less than 1% apoptotic after 12 h) to exquisitely sensitive (>95%). By incrementally increasing the heat load from 37-49 degrees C, we observed a gradual increase in apoptosis with a significant change from apoptotic to necrotic death at temperatures beyond 45 degrees C. The kinetics of the apoptotic response to heat shock were also examined. A time dependent increase in apoptotic cell death was seen after initial hyperthermic treatment with most cell types reaching a 'plateau' at 18 h. In addition to these parameters we report that growth phase has a strong influence on the number of apoptoses induced as a result of heat stress. Cultured cells, grown to a plateau, undergo apoptosis at a much higher level than similarly treated cells taken during an exponential phase of growth. Finally, we determined the necessity of protein synthesis for apoptotic competency.

Journal Article↗
PubMed1998

Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria.

A novel mitochondrial tRNA gene arrangement is described for two species of sea cucumber. The mitochondrial tRNA gene cluster common to sea stars, sea urchins, and the sea cucumber Parastichopus californicus has been significantly modified in the genus Cucumaria as a result of dispersal of the tRNA genes into two separate areas of the genome. The tRNA genes in the novel clusters are interspersed with short unassigned sequences (UASs). Alignment of the two separated novel clusters indicates that the rearrangement was most likely the result of a tandem duplication of approximately 7 kb, encompassing the putative control region, the tRNA cluster, NADH dehydrogenase subunits 1 and 2, the large ribosomal RNA (lrRNA), cytochrome oxidase subunit I, and tRNAArg. Subsequently, deletion of the duplicated lrRNA and protein-coding genes occurred. In addition, the degeneration of one of each of the duplicated tRNA gene pairs has resulted in the interspersed UAS segments observed in each cluster. In contrast, the second copy of the putative control region has been maintained with a very high degree of sequence conservation, suggesting either some functional constraint or concerted evolution for the duplicated element. Analysis of gene organization in other sea cucumber species may provide (1) important insights into the mechanism of mitochondrial gene rearrangements and (2) an informative character set for deep-level phylogenetic analysis of this echinoderm class.

Amino Acid Sequence↗
PubMed1998

Word recognition, discrimination accuracy, and decision bias in schizophrenia: association with positive symptomatology and depressive symptomatology.

The purpose of this experiment was to replicate and extend to a memory task Bentall and Slade's (1985) finding that hallucinations in schizophrenic patients were linked to a liberal decision bias. A word recognition task was administered to 40 schizophrenic patients and 40 normal controls that yielded two indices of performance: an index of discrimination accuracy (Pr) and one of decision bias (Br). Patients obtained a lower Pr than controls, whereas Br was similar in both groups. In patients, Br was selectively correlated with positive symptomatology: the more the positive symptoms, the more liberal the bias. In particular, there was a specific correlation between decision bias and hallucinations. Conversely, Pr was inversely correlated with severity of depression, but not with either positive or negative symptoms. Thus, positive symptomatology may be linked more to difficulties in distinguishing between representations of internal versus external events than to deficits in encoding external events.

Adult↗
PubMed1998

Sex ratio and absence of uniparental disomy in spontaneous abortions with a normal karyotype.

A series of spontaneous abortions collected in the South Wales region over a period of 18 months was karyotyped to identify those with a normal chromosome complement. Microsatellite polymorphisms distributed throughout all autosomes were typed by the polymerase chain reaction to determine the parental origin of each autosome pair in karyotypically normal spontaneous abortions. In 35 cases biparental inheritance of every autosome pair was demonstrated. The sex ratio of the normal spontaneous abortions of proven biparental origin was 0.77, but this was not significantly different from 1.00.

Abortion, Spontaneous↗
PubMed1998

Maintenance of baseline angiotensin II potentiates insulin hypertension in rats.

Chronic insulin infusion in rats increases mean arterial pressure (MAP) by a mechanism dependent on angiotensin II (Ang II). However, the fact that plasma renin activity (PRA) decreases with insulin infusion suggests that Ang II sensitivity is increased and that the parallel reduction in Ang II may partly counteract any hypertensive action of insulin. This study tested that hypothesis by clamping Ang II at baseline levels during chronic insulin infusion. Sprague-Dawley rats were instrumented with artery and vein catheters, and MAP was measured 24 hours per day. In seven angiotensin clamped rats (AC rats), renin-angiotensin II system activity was clamped at normal levels throughout the study by continuous intravenous infusion of the angiotensin-converting enzyme inhibitor benazepril at 5 mg/kg per day (which decreased MAP by 18+/-2 mm Hg) together with intravenous Ang II at 5 ng/kg per minute. Control MAP in AC rats after clamping averaged 99+/-1 mm Hg, which was not different from the 101+/-2 mm Hg measured before clamping Ang II levels. Control MAP in the 8 vehicle-infused rats averaged 105+/-2 mm Hg. A 7-day infusion of insulin (1.5 mU/kg per minute IV) plus glucose (20 mg/kg per minute IV) increased MAP in both groups of rats; however, the increase in MAP was significantly greater in AC rats (12+/-1 versus 5+/-1 mm Hg). This enhanced hypertensive response to insulin in AC rats was associated with a greater increase in renal vascular resistance (153+/-10% versus 119+/-6% of control) and a significant increase in renal formation of thromboxane (149+/-11% of control). Thus, decreased Ang II during insulin infusion limits the renal vasoconstrictor and hypertensive actions of insulin, and this may be caused, at least in part, by attenuation of renal thromboxane production.

Angiotensin II↗
PubMed1998

Effects of a split keyboard design and wrist rest on performance, posture, and comfort.

The purpose of this study was to examine differences in experienced typists' performance, posture, and musculoskeletal pain when using a split, adjustable (SA) computer keyboard and when using a standard flat keyboard. In addition, the use of a wrist rest was examined for performance, posture, and musculoskeletal pain effects. Eighteen participants were exposed to the SA keyboard and a flat keyboard in a laboratory study of text typing for four consecutive hours on five days. The results indicated that after just 2 h of orientation and practice, the participants could perform as well on the SA keyboard as on the flat keyboard. The SA keyboard provided advantages for reduced wrist/hand pronation. There was no difference between the keyboards in the level of musculoskeletal pain reported by participants after typing. However, they reported increased pain in the back, neck, shoulders, and wrists from the beginning to the end of each of the experimental periods for both keyboards.

Adolescent↗
PubMed1998

Familial Kallmann syndrome: a novel splice acceptor mutation in the KAL gene.

Kallmann syndrome is an inherited disease which is characterised by anosmia (inability to smell) and hypogonadotropic hypogonadism both of which are thought to occur as a result of a failure of correct neuronal migration. To date the only genetic lesions identified are mutations in the X-linked gene, KAL. We conducted a mutation screen of the KAL gene in a family with Kallmann syndrome. This identified a new mutation in the KAL gene which removed an acceptor site at the junction of exon 6/intron 5. Exon 6 of the KAL gene encodes the C-terminal portion of a fibronectin type III domain may be involved in axonal pathfinding. We presume that the described mutation would result in the removal of exon 6 resulting in a frame shift which terminates the protein prematurely. It has been proposed that both mental illness and vesico-ureteric reflux are associated with mutations in the KAL gene. However, results from the family presented here do not show an association between either trait and the KAL gene mutation.

Child↗
PubMed1997

The effect of blockade of kappa-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat.

The present study examined whether blockade of kappa-opioid receptors in the medial preoptic area (MPOA) prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory luteinizing hormone (LH) surge, and if so, whether norepinephrine (NE) is involved in mediating this response. In the first experiment, push-pull perfusion of the MPOA with nor-binaltorphimine (nor-BNI), a specific kappa-opioid receptor antagonist, was done in rats between 10.30 and 13.50 h on proestrus. To determine whether any resulting ovulation was due to a nor-BNI-induced increase in LH release, rats were injected with pentobarbital at 13.55 h to block the afternoon LH surge. In 7 of 10 rats, nor-BNI in the MPOA produced a large increase in LH release beginning between 12.30 and 13.30 h, and 5 of 7 ovulated. During MPOA perfusion with cerebrospinal fluid in our normal colony between 14.00 and 17.00 h, surges of LH release began in the majority of rats between 15.30 and 16.30 h. Thus blockade of MPOA kappa-opioid receptors advanced the LH surge by 3 h. The next experiment examined the effect of NE synthesis inhibition with bis(4-methyl-1-homopiperazinylthiocarbonyl) disulfide (FLA-63), or alpha-adrenergic receptor blockade with phenoxybenzamine (PBZ), on the nor-BNI-induced LH response. In 5 of 6 vehicle-treated rats, blockade of MPOA kappa-opioid receptors elicited a large increase in LH release and all 5 ovulated. In contrast, only 3 of 8 rats pretreated with FLA-63 had a large increase in LH release and ovulated, and PBZ prevented the nor-BNI-induced LH increase and ovulation in 4 of 4 rats. PBZ also prevented the afternoon LH surge and ovulation in 4 of 4 rats in our normal colony. Finally, HPLC measurement of NE levels in MPOA push-pull perfusate indicated no increase in NE release during the nor-BNI-induced or normal afternoon LH surges. These results indicate that antagonism of kappa-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA.

Animals↗
PubMed1997

Bioavailability and biodegradation of polycyclic aromatic hydrocarbons in soils.

Inoculation of soil with bacteria (a Gram-negative rod [PD2] and a 4-membered consortium [DC1]) accelerated mineralisation of phenanthrene and pyrene (but not naphthalene) added individually to a pristine sand and a pristine organic soil. The half-life of naphthalene was 3.5 days in both soils whether inoculated or non-inoculated. However, the half-life of phenanthrene decreased from 86 days in non-inoculated sand soil and 80 days in the non-inoculated organic soil to 3.6 days in the sand and 3.1 days in organic soil when inoculated with PD2, and to 6.6 days in the sand and 8.7 days in the organic soil when inoculated with DC1. Phenanthrene mineralisation ceased after 23 days in DC1-inoculated soil and was 71.3 +/- 3.6% (sand) and 63.3 +/- 2.8% (organic). This compared with 96.8 +/- 3.8% (sand) 102.8 +/- 2.5% (organic) after 8 days in PD2-inoculated soil. Inoculation with DC1 (but not PD2) also accelerated mineralisation of pyrene, where the half-life decreased from 155 days to 18 days in the sand soil, and from 216 days to 33 days in organic soil.

Biodegradation, Environmental↗
PubMed1997

Dobutamine pharmacokinetics during dobutamine stress echocardiography.

Many patients fail to achieve target heart rate during dobutamine stress echocardiography (DSE). We evaluated the pharmacokinetics of dobutamine during DSE to determine whether patients with an impaired chronotropic response have higher rates of dobutamine clearance and consequently relatively lower plasma dobutamine levels. Plasma dobutamine levels, heart rate, and left ventricular (LV) ejection fraction (EF) were measured in 13 male patients referred for DSE at baseline and at the end of stepped 3-minute dobutamine infusions of 5, 10, 20, and 30 microg/kg/min. Dobutamine levels increased with doses: 27 +/- 10, 111 +/- 17, 275 +/- 17, and 403 +/- 28 ng/ml (mean +/- SEM). There was no relation observed between the plasma dobutamine level achieved at the 30-microg infusion dose and the increase in heart rate from baseline (r = 0.066; p = 0.83). Baseline LVEF and a measure of chronotropic beta responsivity were identified as independent predictors of dobutamine clearance, together accounting for 73% of the variance in dobutamine clearance. In conclusion, (1) there is a dose-dependent increase in plasma dobutamine levels during DSE, (2) dobutamine clearance is positively related to baseline LVEF and is partially mediated by a beta-receptor mechanism, and (3) an impaired chronotropic response during DSE is not due to failure to achieve a sufficiently high dobutamine level. We conclude that in patients who lack an adequate heart rate response during the early stages of DSE (e.g., up to 20 microg/kg/min infusion), administration of atropine rather than progressively higher amounts of dobutamine may provide a more effective strategy to achieve target heart rate.

Adrenergic beta-Agonists↗
PubMed1997

Discrimination and response bias in memory: effects of depression severity and psychomotor retardation.

Although memory disorders have been well documented in depression, there is controversy concerning depressives' performance on recognition memory tasks; e.g. whether they have impaired discrimination and conservative or liberal response bias according to signal detection theory. In addition, symptomatic correlates of discrimination and response bias indices have been lacking. A word recognition memory task analyzed according to the two high threshold theory was administered to 26 depressives and 26 controls. Depressives obtained a lower index of discrimination (Pr) than controls. The index of response bias (Br) was not different between groups. In the depressed group, overall severity of depression was related to discrimination, whereas psychomotor retardation level was related to response bias. Cognitive performance of depressives could be advantageously analyzed in terms of these two dimensions of symptomatology.

Adult↗
PubMed1997

Bacterial protease Lon is a site-specific DNA-binding protein.

The product of the Escherichia coli lon gene is the ATP-dependent Lon protease. Lon contributes to the regulation of several important cellular functions, including radiation resistance, cell division, filamentation, capsular polysaccharide production, lysogeny of certain bacteriophages, and proteolytic degradation of certain regulatory and abnormal proteins. Lon homologues are also found in several widely divergent bacteria, as well as in the mitochondria of yeast and humans. E. coli Lon has long been known to bind to DNA, but this interaction has not been further characterized and has generally been assumed to be nonspecific. We now demonstrate that E. coli Lon can bind to a TG-rich DNA promoter element in a sequence-specific manner. This finding is based on the results of experiments employing SouthWestern blotting, protein purification, "shift-shift" electrophoretic mobility shift assays, electrophoretic mobility shift assays using in vitro transcribed and translated Lon, and DNase footprinting. Site-specific DNA binding is likely to be an additional important biochemical characteristic of the multifaceted Lon protease.

ATP-Dependent Proteases↗
PubMed1997

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro.

Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8+ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma.

CD4-Positive T-Lymphocytes↗