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Biomedical subjects

P Watt

Publications and source records attributed to P Watt.

At least 19 recordsLinked to original sources
PubMed2001

A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevulinic acid-n-pentylester.

BACKGROUND: Topical 5-aminolaevulinic acid (ALA) is widely used in photodynamic therapy (PDT) to generate protoporphyrin IX (PpIX) in the skin. However, other prodrugs may be more effective. OBJECTIVES: The pharmacokinetics of ALA- and ALA-n-pentylester-induced PpIX, together with the phototoxicity after PDT, were compared in human skin in vivo, using iontophoresis as a quantitative drug delivery system. METHODS: A series of six increasing doses of equimolar prodrug solutions was iontophoresed into normal skin of the upper inner arms of 20 healthy subjects. The kinetics of PpIX metabolism in skin (n = 4) and the response to light exposure, performed at 4.5 h (n = 6) and 6 h (n = 10) after application, were assessed by skin surface PpIX fluorescence and postirradiation erythema. RESULTS: ALA and ALA-n-pentylester showed a linear correlation between logarithm of dose and PpIX fluorescence (P < 0.005), and logarithm of dose and skin phototoxicity with irradiation at 4.5 h (P < 0.001 and P < 0.005, respectively) and 6 h (P < 0.05 and P < 0.0001, respectively) after iontophoresis. Higher phototoxicity was observed with ALA-n-pentylester than with ALA when sites were irradiated at 6 h, as indicated by the significantly lower theoretical threshold dose for erythema (P < 0.05) and the shift of the PpIX fluorescence/phototoxicity curve towards greater skin erythema at equal PpIX fluorescence levels. Depth of PpIX fluorescence in skin, as determined by fluorescence microscopy, was similar for both prodrugs, but a more homogeneous distribution of PpIX was seen with the more lipophilic ALA-n-pentylester. CONCLUSIONS: The observed greater phototoxicity of ALA-n-pentylester relative to ALA may be attributable to a more favourable PpIX localization in tissue and/or greater intrinsic toxicity.

Adult↗
PubMed2001

Detection of cervical infections in colposcopy clinic patients.

The purpose of this study was to determine if Neisseria gonorrhoeae; Chlamydia trachomatis; herpes simplex virus; cytomegalovirus; Epstein-Barr virus; human herpesviruses 6, 7, and 8; or adeno-associated virus influenced the production of cervical intraepithelial neoplasia. Two hundred thirty-one cervical smear samples were tested for the presence of the organisms by PCR. In addition, human papillomavirus types in the samples were determined by PCR and classified into cancer risk types of high, moderate, and low. There was no link with cervical intraepithelial neoplasia status and detection of herpes simplex virus, cytomegalovirus, Epstein-Barr virus, human herpesviruses 6 and 8, gonorrhea, or chlamydia. However, high-grade cervical intraepithelial neoplasia was found more frequently with mixed infection by moderate-risk human papillomavirus types and human herpesvirus 7 than with these papillomavirus types alone. The presence of human herpesvirus 7 may increase the oncogenic potential of moderate-risk human papillomavirus types.

Adolescent↗
PubMed2001

HPV detection and measurement of HPV-16, telomerase, and survivin transcripts in colposcopy clinic patients.

AIMS: To determine whether the detection of high risk human papillomavirus (HPV) types is more predictive for high grade CIN than the current cervical smear test, and whether the production and measurement of HPV type 16 (HPV-16) and cellular survivin and telomerase transcripts can be used to discriminate between cervical HPV infections that self cure and those that induce high grade lesions. METHODS: Three hundred and fifty four cervical smear samples from women attending the colposcopy clinic were tested by the polymerase chain reaction (PCR) for the presence of HPV. Transcripts for HPV-16 E6, E6*I, E6*II, E7, and L1 as well as cellular survivin, telomerase RNA component, and telomerase reverse transcriptase were measured using fluorogenic probe (Taqman) assays. RESULTS: Referral smear grades of severe or moderate showed greater positive predictive values for CIN 2/3 than did the detection of high or moderate risk HPV types. HPV-16 transcripts from E6, E6*I, E6*II, and E7 showed high predictive values for CIN 2/3, but low sensitivity. The telomerase RNA component was detected in 53 of 57 samples and telomerase reverse transcriptase was only detected in one sample, whereas survivin transcripts were detected in 40% of samples. CONCLUSIONS: The detection of HPV-16 or cellular survivin or telomerase transcripts did not accurately predict the grade of CIN in the samples. The detection of HPV risk types correlated well with the grade of CIN; however, the referral grade smear was the most accurate predictor of the severity of the lesion. Of the 35 different HPV types detected, 18 are not included in the HPV hybrid capture II commercial test kit. The use of such kits would have missed HPV infection in 4.3% of clinic patients with CIN 2/3 lesions and 15.4% with CIN 0/1.

Adult↗
PubMed2000

Interaction trap experiment with CDC6.

CDC6 is an essential gene of yeast Saccharomyces cerevisiae. Although DNA sequence of the gene is available for a long time, biochemical function of Cdc6 protein in the cell cycle remains unclear. Using the interaction trap experiment we were looking for proteins interacting specifically with Cdc6. Four gene products interacting with Cdc6 were detected. By sequence analysis we found that ECM11 codes for the protein involved in the cell wall synthesis, YNL201 codes for the protein of unknown function, probably involved in the carbon metabolism, YOR279 codes for protein of completely unknown function with no significant similarity with any known protein, and the interaction with Ty1 retrotransposition element was also found. The strongest interaction with Cdc6 bait measured as beta-galactosidase activity was observed with ECM11 and YNL201; YOR279 interacts slightly weaker. The weakest beta-galactosidase activity was obtained by Ty1A element. The strongest suppression of cdc6-1 mutation was observed by Ty1A element, the slight one with ECM11 and YNL201 but no suppression of thermosensitive mutation was detected for YOR279.

Cell Cycle Proteins↗
PubMed1999

Detection and typing of human papillomavirus DNA in paired urine and cervical scrapes.

The prevalence of human papillomavirus (HPV) in paired cervical scrape and urine specimens from 144 women attending a clinic for genitourinary medicine was determined by polymerase chain reaction (PCR) and nested PCR, using degenerate and general primer pairs localized within the L1 region. HPV typing was by restriction fragment length polymorphism (RFLP), type-specific PCR (HPV 6, 11, 16, 18, 33), and partial DNA sequencing of PCR products. HPV DNA was detected in 114 (84%) women. HPV DNA was detected in the specimens of 58 patients after amplification with MY09/MY11 primers and in a further 54 patients after nested PCR with the GP5+/GP6+ primers. A total of 106/136 (78%) of women had HPV DNA positive cervical scrapes and 89 (65%) had HPV DNA positive urine specimens. Both the urine and cervical specimens of 81 women were positive. In 25 women HPV DNA was detected in the cervical specimen only, and in 8 women HPV DNA was detected in the urine specimens only. A total of 108 specimens from 75 patients were typed. For 33 patients HPV typing was achieved in both the cervical and the urine specimens and 19 women had identical types in paired specimens. Multiple HPV infections could be detected in 15 (20%) of 75 women where either the cervical and urine specimen or both of the specimens could be typed. More then one HPV type was found in 8 specimens and from multiple sites (cervix and urinary tract) in the same patients on 7 occasions. The results of this study indicate that the detection of HPVs in the urogenital tract can be maximised through the testing of both cervical scrapes and urine specimens in conjunction with the use of a nested PCR to increase the sensitivity of HPV DNA detection. Also, urine cannot be a direct substitute for a cervical scrape as different HPV types are often detected in the urine compared with those detected in the cervix.

Adolescent↗
PubMed1999

Monocular and binocular depth discrimination thresholds.

BACKGROUND: Measurement of stereoacuity at varying distances, by real or simulated depth stereoacuity tests, is helpful in the evaluation of patients with binocular imbalance or strabismus. Although the cue of binocular disparity underpins stereoacuity tests, there may be variable amounts of other binocular and monocular cues inherent in a stereoacuity test. In such circumstances, a combined monocular and binocular threshold of depth discrimination may be measured--stereoacuity conventionally referring to the situation where binocular disparity giving rise to retinal disparity is the only cue present. A child-friendly variable distance stereoacuity test (VDS) was developed, with a method for determining the binocular depth threshold from the combined monocular and binocular threshold of depth of discrimination (CT). METHODS: Subjects with normal binocular function, reduced binocular function, and apparently absent binocularity were included. To measure the threshold of depth discrimination, subjects were required by means of a hand control to align two electronically controlled spheres at viewing distances of 1, 3, and 6m. Stereoacuity was also measured using the TNO, Frisby, and Titmus stereoacuity tests. BTs were calculated according to the function BT= arctan (1/tan alphaC - 1/tan alphaM)(-1), where alphaC and alphaM are the angles subtended at the nodal points by objects situated at the monocular threshold (alphaM) and the combined monocular-binocular threshold (alphaC) of discrimination. RESULTS: In subjects with good binocularity, BTs were similar to their combined thresholds, whereas subjects with reduced and apparently absent binocularity had binocular thresholds 4 and 10 times higher than their combined thresholds (CT). The VDS binocular thresholds showed significantly higher correlation and agreement with the TNO test and the binocular thresholds of the Frisby and Titmus tests, than the corresponding combined thresholds (p = 0.0019). CONCLUSION: The VDS was found to be an easy to use real depth (distance) stereoacuity test. The method described for calculating the BT provides one simple nonlinear solution for determining the respective contributions of binocular and monocular (MT) depth discrimination to the combined depth threshold.

Adolescent↗
PubMed1999

Could it be whooping cough?

BACKGROUND: Pertussis is an uncommon but serious illness, mainly affecting children under the age of 5 years. OBJECTIVE: Affected children usually contract the disease from older children or adults in whom the disease presents in an atypical way, their coughing being attributed to other aliments. This article discusses the epidemiology, clinical course and management of pertussis. Prophylaxis is also discussed. DISCUSSION: Pertussis presents as a hacking, mostly nocturnal cough, before becoming constant and occurring in paroxysms. Even though children appear sick, they classically are afebrile. Erythromycin is the treatment of choice for affected children and their immediate family. Achieving a 90% vaccination coverage would eradicate pertussis, but booster vaccinations later in life have been proposed since the protection of vaccination wanes rapidly over a period of 6-10 years.

Adult↗
PubMed1999

Acute gastroenteritis in children.

BACKGROUND: Acute gastroenteritis is a common childhood illness. Most cases are caused by rotavirus infection. OBJECTIVE: This article discusses the classic presentation of gastroenteritis in children, which includes a short history of vomiting followed by diarrhoea. DISCUSSION: Dehydration is the main problem in children with gastroenteritis, and severe dehydration is a medical emergency. Mild to moderately dehydrated children can be safely treated at home with oral rehydration therapy, however parents require careful explanation of the disease, its course and potential complications. Early reintroduction of usual foods (breast milk, formula or solids) has been shown to shorten the illness. Babies may need to be reviewed within 8 hours and older children within a day.

Acute Disease↗
PubMed1998

Sif2p interacts with Sir4p amino-terminal domain and antagonizes telomeric silencing in yeast.

Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, we have cloned SIF2, which encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improved telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression altered the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains were hypersensitive to a range of stress conditions, but did not have decreased viability and did not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci.

Binding, Competitive↗
PubMed1995

The effect of temperature on spontaneous action potential discharge of the isolated sinus venosus from winter and summer plaice (Pleuronectes platessa)

The spontaneous cardiac pacemaker activity and conformation were recorded in vitro, using intracellular recording methods, from heart tissue of summer- and winter-caught plaice. The effects of changing temperature on the pacemaker rate, duration of action potential and diastolic depolarization were investigated by altering the temperature of the superfusing medium. The resting intrinsic rate of discharge was significantly greater in pacemaker cells from winter plaice (P=0.05), but there was no significant difference between winter and summer fish in the apparent Arrhenius activation energies for this process. However, there was a significant difference in the estimated intercept, indicating a thermal shift in the processes underlying the spontaneous pacemaker rhythm. There was no significant difference in the diastolic depolarization duration recorded from winter and summer fish over the temperature range 4&shy;22 &deg;C. The major effect of previous environmental temperature was on the duration of the action potential (P<0.02), indicating that the observed changes in pacemaker discharge rate were not influenced by the processes that determine the duration of the pacemaker diastolic depolarisation but were modulated by the channel events that give rise to the action potential.

Journal Article↗
PubMed1994

Regulatory role of intraislet somatostatin on insulin secretion in the isolated perfused human pancreas.

This study was undertaken to determine whether intraislet somatostatin inhibits insulin secretion in the human islet. A high-affinity monoclonal somatostatin antibody was used to immunoneutralize somatostatin in the isolated, perfused human pancreas. Single pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs medium with either 3.9 or 12.9 mM glucose. Sequential test periods separated by basal periods were performed with either somatostatin-14 (SS-14), somatostatin monoclonal antibody (CURE.S6), or a combined infusion. Infusion of SS-14 resulted in inhibition of insulin secretion under both low glucose (delta X = -712 +/- 212 pM) (p < 0.05) and high glucose (delta X = -21,913 +/- 10,003 pM) (p = 0.06) conditions. Immunoneutralization of intraislet somatostatin with CURE.S6 resulted in a significant increase in insulin secretion under both low glucose (454 +/- 162 pM) (p < 0.05) and high glucose (2,177 +/- 829 pM) (p < 0.05) conditions. Combined infusion of SS-14 and CURE.S6 resulted in a reversal of the inhibitory effect of exogenous SS-14. The data suggest that intraislet somatostatin has an inhibitory role in the regulation of insulin secretion in the human islet.

Adolescent↗
PubMed1994

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Adenosine Triphosphatases↗
PubMed1994

DETERMINATION OF PROTEIN SYNTHESIS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, USING A STABLE ISOTOPE

It has been suggested (Houlihan, 1991) that the consumption of 1 g of protein in a variety of species of fish stimulates the synthesis of, approximately, an equal amount of protein. Although synthesis of protein may account for as much as 40 % of the whole-animal oxygen consumption (Lyndon et al. 1992), only about 30 % of the synthesized proteins are retained as growth (Houlihan et al. 1988; Carter et al. 1993a,b). Thus, one focus of attention is the potential advantage gained by fish in allocating a considerable proportion of assimilated energy to protein turnover in contrast to relatively low-cost, low-turnover protein growth (Houlihan et al. 1993). Rates of protein synthesis in several species of fish have been measured using radioactively labelled amino acids, frequently given as a flooding dose (reviewed by Fauconneau, 1985; Houlihan, 1991). These measurements cannot be made for longer than a few hours because of the decline in specific radioactivity in the amino acid free pool. However, as protein synthesis rates vary during the course of a day as a result of the post-prandial stimulation, and since radiolabelled amino acid methodology is invasive, short-term and terminal, it has been difficult to be certain of the relationship between protein growth measured in the long term and protein synthesis rates measured in the short term. This paper addresses these problems by developing a method using 15N in orally administered protein to measure protein synthesis rates in fish over relatively long periods, the aim being to use procedures that are as non-invasive and repeatable as possible. The use of stable isotopes to measure protein metabolism is well established in terrestrial mammals (see Rennie et al. 1991; Wolfe, 1992), but to our knowledge the only published data for aquatic ectotherms are on the blue mussel (Mytilus edulis L.) (Hawkins, 1985). In the present study, rates of protein synthesis of individual rainbow trout [Oncorhynchus mykiss (Walbaum)] were calculated from the enrichment of excreted ammonia with 15N over the 48 h following the feeding of a single meal (dose) containing protein uniformly labelled with 15N by use of an end-point stochastic model (Waterlow et al. 1978; Wolfe, 1992). Application of this type of modelling would appear to be ideal for measuring ammonotelic fish nitrogen metabolism since, unlike the situation in mammals, the catabolic flux of amino acids through urea is very small. Further, ammonia is excreted directly into the surrounding water via the gills and is not stored for any length of time, in contrast to the situation in mammals, so the rate of tracer appearance is easily measurable.

Journal Article↗
PubMed1993

Distinct negative regulation of the human embryonic globin genes zeta and epsilon.

The human embryonic globin genes zeta and epsilon are expressed when erythropoiesis is initiated at about the third week of development but are subsequently repressed as expression of the fetal globins, alpha and gamma, begins. We have examined the promoter region of the human zeta-globin and epsilon-globin genes in order to identify regulatory sequences that may be involved in this process. Stable transfection of the human erythroid cell line K562 with either a truncated form of the zeta-globin gene, containing 112 base pairs (bp) of 5'-flanking sequences, or a larger zeta-globin construct, containing several hundred bp of 5'-flanking sequence, revealed that the zeta-globin gene is subject to negative regulation by its 5'-flanking region. We have defined the sequences responsible for this negative regulation to a 22 bp region immediately upstream of the proximal promoter sequence of the zeta-globin gene. A 22 bp oligonucleotide including this negative element was found to inhibit both the zeta-globin and HSV TK promoters. We have also analyzed the promoter of the human epsilon-globin gene, since it is coordinately expressed with zeta-globin. We show that it is likewise subject to negative regulation, though in this case from a distal silencer element. Gel retardation and methylation interference assays have provided evidence of a factor which binds specifically to the epsilon-globin silencer. However, no obvious sequence homology exists between the zeta and epsilon negative elements, and at least some of the factors that recognize these elements are distinct. We postulate that the negative transcriptional control elements in the human embryonic globin gene promoters contribute to the observed reduction in zeta- and epsilon-globin gene expression that occurs during development.

Base Sequence↗
PubMed1992

Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells.

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

Adenine Phosphoribosyltransferase↗