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Biomedical subjects

L Clarke

Publications and source records attributed to L Clarke.

At least 19 recordsLinked to original sources
PubMed1992

Qualitative research: meaning and language.

Qualitative methodologies, for example those determined by ethnography, are being reported by a growing number of British nurses. It is this author's contention that these studies are seriously deficient in areas such as sampling, reliability and, particularly, the attribution of meaning to statements given by interviewees. Whilst it is acknowledged that this kind of research is necessary for analyses of inter-personal relations, it is asserted that such analyses have inadequately dealt with psychological factors of perception and interpretation. Although some of these problems may be partially resolved, there remains a requirement that such ethnographies acknowledge the tentative quality of their conclusions given the subjectivity of the methods used.

Bias↗
PubMed1992

Adenosine receptors on human airway epithelia and their relationship to chloride secretion.

1. We have characterized an adenosine receptor subtype present in human airway epithelial cells by measuring the changes in the intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and the rate of transepithelial Cl- secretion. 2. Primary cultures of human nasal epithelium obtained from excised surgical airway epithelial tissues and the cell lines BEAS39 and CF/T43 derived from human airway epithelium were grown on plastic dishes and labelled with [3H]-adenine for measurement of intracellular cyclic AMP accumulation. Primary cultures were loaded with the calcium indicator fura-2 to measure [Ca2+]i and studied as polarized, ion transporting epithelia on collagen matrix supports for measurement of Cl- secretion. 3. Adenosine analogues stimulated cyclic AMP accumulation with a rank order of potency characteristic of an A2-receptor: 5-N-ethyl-carboxamidoadenosine (NECA) greater than adenosine greater than R-phenylisopropyladenosine (R-PIA), 6-N-cyclopentyladenosine (CPA) greater than S-PIA. NECA increased cyclic AMP accumulation in normal and cystic fibrosis (CF) primary cells as well as in the CF/T43 and BEAS39 cell lines with K0.5 values ranging from 0.3 to 3 microM. Preincubation with NECA resulted in the homologous desensitization of airway epithelial cells. The effect of NECA was specifically inhibited by the adenosine receptor antagonist, aminophylline, in a competitive manner. 4. The A1-adenosine receptor agonists CPA and R-PIA did not inhibit isoprenaline-stimulated cyclic AMP accumulation in CF/T43 cells, and potentiating effects of the adenosine analogues were observed on forskolin-stimulated cyclic AMP accumulation. Adenosine analogues did not cause significant changes in intracellular Ca2+ ([Ca2+]i) in airway epithelium.5. Adenosine analogues, applied to either the serosal or mucosal side of the polarized amiloride pretreated primary cultures, induced changes in I,, with a rank order of potency of agonists similar to that observed for stimulation of cyclic AMP accumulation. Intracellular microelectrode studies indicated that the locus of action was the apical membrane Cl- conductance. Adenosine failed to stimulate C1- secretion in CF airway epithelium.6. These results provide evidence for the existence of an A2-adenosine receptor that modulates intracellular levels of cyclic AMP in human airway epithelium. Activation of this receptor might lead to stimulation of Cl- secretion in amiloride pretreated normal but not CF cells.

Adenosine↗
PubMed1992

Effect of maternal cold exposure on brown adipose tissue and thermogenesis in the neonatal lamb.

1. This study examines the effect of chronic cold exposure during pregnancy, induced by winter shearing twin-bearing ewes 4 weeks before predicted lambing date, on O2 consumption and CO2 production during non-rapid-eye-movement (REM) sleep in lambs maintained for at least 1 h at warm (28-18 degrees C) and cold (14-5 degrees C) ambient temperatures at 1, 4, 14 and 30 days of age. This was combined with measurement of the thermogenic activity (GDP binding to uncoupling protein in mitochondrial preparations) of perirenal adipose tissue from lambs immediately after birth and at 33 days of age. 2. Lambs born from shorn (cold-exposed) ewes were 15% heavier (P < 0.01) and possessed 21% (P < 0.01) more perirenal adipose tissue that contained 40% more protein and mitochondrial protein than unshorn (P < 0.05) controls. Total GDP binding in perirenal adipose tissue was 40% greater (P < 0.05) in lambs born from shorn ewes but there was no difference in lipid content of this tissue between the two groups. 3. At 1 day of age, lambs born from shorn ewes exhibited a 16% higher (P < 0.05) rate of O2 consumption (per kilogram bodyweight) at the warm temperature and a 40% greater metabolic response to the cold ambient temperature. All lambs born from shorn ewes responded to cold exposure without shivering (i.e. via non-shivering thermogenesis) whilst shivering was measured in four out of seven lambs in the unshorn group. These differences had disappeared by 4 days of age as a result of a 25% increased (P < 0.01) rate of O2 consumption in the warm in lambs born from unshorn ewes and a 20% decrease (P < 0.05) in the response to the cold in lambs from shorn ewes. Shivering during cold exposure was measured in six out of nine lambs born from shorn ewes indicating a rapid alteration in thermoregulatory responses to cold during the first few days of life. 4. The levels of GDP binding and mitochondrial protein in perirenal adipose tissue fell by one-third in both groups of lambs during the first 33 days of life whereas lipid content either increased or was unchanged. This indicated that brown adipose tissue (BAT) was developing the characteristics of white adipose tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1992

Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy.

A generalized deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is the inborn error in gyrate atrophy (GA), an autosomal recessive degenerative disease of the retina and choroid of the eye. Mutations in the OAT gene show a high degree of molecular heterogeneity in GA, reflecting the genetic heterogeneity in this disease. Using the combined techniques of PCR, denaturing gradient gel electrophoresis, and direct sequencing, we have identified three nonsense-codon mutations and one nonsense codon-generating mutation of the OAT gene in GA pedigrees. Three of them are single-base substitutions, and one is a 2-bp deletion resulting in a reading frameshift. A nonsense codon created at position 79 (TGA) by a frameshift and nonsense mutations at codons 209 (TAT----TAA) and 299 (TAC----TAG) result in abnormally low levels of OAT mRNA in the patient's skin fibroblasts. A nonsense mutation at codon 426 (CGA----TGA) in the last exon, however, has little effect on the mRNA level. Thus, the mRNA level can be reduced by nonsense-codon mutations, but the position of the mutation may be important, with earlier premature-translation termination having a greater effect than a later mutation.

Adult↗
PubMed1991

Clustered tRNA genes in Schizosaccharomyces pombe centromeric DNA sequence repeats.

The centromere-associated B' and B DNA sequence repeats of Schizosaccharomyces pombe chromosomes I and II have been found to contain clusters of tRNA genes. The centromere II region (cen2) includes at least 22 tRNA genes distributed among five copies of the B sequence repeat containing genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Val). Individual B repeats are variously associated with other tRNA genes, including those specifying tRNA(Lys), tRNA(Arg), and tRNA(Glu2). The centromere I region (cen1) contains at least six tRNA genes in two copies of the B' repeated element, including genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Glu3). Multiple tandemly arranged clusters of tRNA genes are presumably conserved due to restricted recombination frequencies in the centromere regions.

Base Sequence↗
PubMed1991

Human immunodeficiency virus-1 infection in an infertile population.

OBJECTIVE: The aim of our study was to determine the prevalence of human immunodeficiency virus-1 (HIV-1) in an infertile population. DESIGN: The study design included a retrospective anonymous survey of clinical data and screening for HIV-1 antibody by enzyme-linked immunoabsorbent assay (ELISA) in stored sera. Samples that were ELISA-positive were further tested by Western blot assay. Frequency distributions were analyzed by Fisher's exact test. SETTING: University tertiary care center. PARTICIPANTS: Based on availability of stored frozen sera, the total study population included 182 of 304 consecutively registered infertile couples. RESULTS: Seventy-five percent of the study population were found to have one or more risk factors for HIV infection. Of the 252 sera tested, 10 were repeatedly reactive by ELISA, and Western blot testing confirmed HIV-1 infection in one woman and two men. CONCLUSIONS: This relatively high HIV-1 seroprevalence (male: 2.6%; female: 0.6%) in a low-middle class infertile population emphasizes the urgent need to implement on-site HIV-infection counseling aimed at preventing the spread of disease to the healthy partner and fetus and to discuss therapeutic and reproductive options.

Adolescent↗
PubMed1991

The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function.

We have examined the chromatin structure of centromere regions from the fission yeast Schizosaccharomyces pombe. The large and complex centromere regions of the S. pombe chromosomes encompass many kilobase pairs of DNA and contain several classes of tandemly repeated DNA sequences. The repeated sequences are further organized into a large inverted repeat flanking a central core, a conserved structural feature among all three centromeres in S. pombe. The nucleosomal configuration of the centromere regions is nonuniform and highly varied. Most of the centromere-specific repeated DNA sequences are packaged into nucleosomes typical of bulk chromatin. However, the central core and core-associated repeated sequences from the centromere regions of chromosomes I (cen1) and II (cen2), when present in S. pombe, show an altered chromatin structure, with little or no evidence of regular nucleosomal packaging. The atypical chromatin organization of the cen2 central core is not due to transcription, as no transcripts from this region were detected. These same DNA sequences, however, are packaged into nucleosomes typical of bulk chromatin when present in a nonfunctional environment on a minichromosome in the budding yeast Saccharomyces cerevisiae. Because the cen2 central core sequences themselves do not preclude regular nucleosomal packaging, we speculate that in S. pombe they constitute a specialized site of kinetochore protein assembly. The atypical nucleosomal pattern of the cen2 central core remains constant during the cell cycle, with only minor differences observed for some sequences. We propose that the unusual chromatin organization of the core region forms the basis of a higher order structural differentiation that distinguishes the centromere from the chromosome arms and specifies the essential structure for centromere function.

Blotting, Northern↗
PubMed1991

Attitudes and interests of students and applicants from two branches of the British nursing profession.

The attitudes and interests of psychiatric students and applicants are compared with general nursing students and applicants using the Attitudes to Treatment Questionnaire (ATQ), the Wilson-Patterson Attitude Inventory (WPAI) and the Direction of Interest Questionnaire (DIQ). The student groups are also examined in respect of changes in attitude (ATQ) and interests (DIQ) during their training. Results indicate significant differences between nurse applicants for attitudes to treatment and direction of interest, the psychiatric applicants being more liberal in their attitudes to treatment and less practical in their interests. Similarly, psychiatric students demonstrated more liberalism in their attitudes to treatment as well as less conservatism than general students. No differences emerged for students in terms of direction of interest. Differences within groups occurring during training were complex. The implications of these findings are discussed and some suggestions made for further research.

Adolescent↗
PubMed1991

Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I.

We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding a 4- to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the cen1 region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.

Centromere↗
PubMed1990

Centromeres of budding and fission yeasts.

Centromeres of the budding yeast Saccharomyces cerevisiae are structurally relatively simple, are specified by only about 125 base pairs of DNA, and contain no repeated DNA sequences. The centromere regions in the fission yeast Schizosaccharomyces pombe span many kilobase pairs of DNA and contain repeated DNA sequences that appear to be necessary for full centromere function. A portion of the repeated sequences is organized into a large inverted repeated structure in the centromere region of each S. pombe chromosome. Fission yeast provides an excellent model system for studying the role of repeated DNA sequences in centromere function.

Base Sequence↗
PubMed1990

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.

Base Sequence↗
PubMed1990

Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin.

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3-9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values = 142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Centromere structure and function in budding and fission yeasts.

Functional centromeric DNAs have now been isolated and characterized from both budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts. Artificial chromosomes containing these centromere DNA sequences segregate faithfully in both mitotic and meiotic cell divisions, but only in the parent organism. Structure-function analyses have revealed surprising fundamental differences between these two centromere classes. In the budding yeast centromeres, a 125-bp consensus DNA sequence contains all the information needed in cis to provide proper chromosome segregation. In contrast, the fission yeast centromeres each contain a long run (40 to 100 kb) of untranscribed repetitive DNA sequences arranged into a large inverted repeat, most of which is required for full centromere function. The fission yeast centromere-kinetochore appears to be a highly relevant experimental model for analysis of the mechanism of chromosome segregation in higher eukaryotes, in which the centromere regions often contain megabases of transcriptionally silent repetitive DNA sequences of unknown function.

Base Sequence↗
PubMed1989

Evidence for enzymatic activation and oxygen involvement in cytotoxicity and antitumor activity of N,N',N''-triethylenethiophosphoramide.

The cytotoxicity of N,N',N''-triethylenethiophosphoramide (thiotepa) was studied in vitro in the MCF-7 human breast carcinoma cell line and in vivo using the EMT6 mouse mammary tumor model, under various conditions of oxygenation and in the presence and absence of Aroclor 1254-induced liver preparations. The cytotoxicity of thiotepa toward exponentially growing MCF-7 cells was markedly dependent on the presence of oxygen during the period of drug exposure, with 3 log greater cell kill at 500 microM thiotepa being observed when the cells were normally oxygenated compared with hypoxic cells. Incubation of thiotepa with an Aroclor 1254-induced rat liver S-9 homogenate, in the presence of a NADPH-regenerating system, resulted in an 8-fold increase in cytotoxicity towards the MCF-7 cells over a wide range of drug concentrations. Thiotepa was shown to be metabolized under these conditions in a NADPH- and O2-dependent reaction that was catalyzed by one or more microsomal cytochrome P-450 enzymes that were present in the S-9 fraction. The thiotepa metabolite triethylene phosphoramide, which hydrolyzes significantly faster than thiotepa, was significantly less cytotoxic toward the MCF-7 cells than was thiotepa itself, suggesting that it is unlikely to be the S-9 metabolite responsible for the observed increase in drug cytotoxicity. Moreover, triethylene phosphoramide cytotoxicity was only partially O2 dependent and was largely unaffected by incubation in the presence of the S-9 preparation, indicating a mechanism of action distinct from that of thiotepa. Tumor cell survival experiments with the EMT6 mouse mammary carcinoma system revealed that a 3.6-fold increase in thiotepa cytotoxicity was obtained by prior administration of the liver inducer Aroclor 1254 to the tumor-bearing animals, 5 days before drug treatment. Finally, the therapeutic effectiveness of thiotepa was significantly enhanced (3- to 5.8-fold increase in tumor growth delay) when an increase in oxygenation was achieved, by carbogen breathing, in animals given the perfluorochemical emulsion Fluosol-DA. These findings establish that the cytotoxic effects of thiotepa are oxygen dependent and may involve, at least in part, metabolic processes catalyzed by cytochrome P-450 enzymes.

Animals↗
PubMed1989

Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation.

Cytochrome P-450-catalyzed activation of cyclophosphamide to alkylating metabolites was studied in isolated rat liver microsomes and purified, reconstituted P-450 enzyme systems in order to identify the major enzymatic catalysts of drug activation in both uninduced and drug-induced liver tissue. P-450 form PB-4 (P-450 gene IIB1) activated cyclophosphamide with high efficiency [Vmax (app) = 18.2 nmol metabolite/min/nmol P-450; Km (app) = 0.16 mM] via the formation of 4-hydroxycyclophosphamide, which was quantitatively trapped as a bisulfite adduct then characterized following its conversion to cyano derivatives. Antibodies to P-450 PB-4 inhibited cyclophosphamide activation catalyzed by phenobarbital-induced adult male rat liver microsomes (specific activity, 5.4 nmol metabolite/min/mg liver microsomes) in a selective and near quantitative (greater than 80%) fashion; little or no inhibition was obtained using antibodies inhibitory towards six other rat hepatic P-450 forms. Cyclophosphamide activation catalyzed by uninduced adult male rat liver microsomes (specific activity, 0.68 nmol/min/mg), although not inhibited by anti-P-450 PB-4 antibodies, was partially inhibited (approximately 60%) by antibodies to P-450 PB-1 (gene IIC6) and more completely inhibited (greater than 95%) by antibodies reactive with both P-450 PB-1 and P-450 2c (gene IIC11). Consistent with these observations, P-450 PB-1 and P-450 2c both activated cyclophosphamide at moderate rates in reconstituted systems (turnover, 1.6-2.7 nmol metabolite/min/nmol P-450), while seven other purified hepatic P-450 forms exhibited significantly lower activities (turnover less than or equal to 0.5 nmol metabolite/min/nmol P-450). Further studies revealed that the changes in liver microsomal cyclophosphamide activation rates with age and sex and in response to in vivo administration of cisplatin primarily reflect changes in the levels of P-450 forms PB-1 and 2c. These studies establish that P-450 forms PB-1, 2c, and PB-4 are the major catalysts of cyclophosphamide activation in rat hepatic tissue and that the modulation of microsomal cyclophosphamide activation with development and in response to drug exposure largely reflects alterations in the levels of these three hepatic P-450 enzymes.

Animals↗