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Biomedical subjects

D Holt

Publications and source records attributed to D Holt.

At least 37 records · Page 2Linked to original sources
PubMed1999

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.

Animals↗
PubMed1999

New PCR primers for the sensitive detection and specific identification of group B beta-hemolytic streptococci in cerebrospinal fluid.

Group B beta-hemolytic streptococci (GBS) are a major cause of sepsis and meningitis in newborn babies. Although penicillin remains an effective treatment, there has been no decline in mortality. The rapid identification of GBS in cerebrospinal fluid (CSF) would improve the diagnosis of meningitis, but data from several previous studies indicate that the sensitivity of polymerase chain reaction (PCR) is not better than culture. Following extraction and precipitation of DNA, we have used semi-nested PCR to amplify a 450 base-pair and a 265 base-pair product of the 16S rRNA gene. This method has a lower detection limit of 50 fg of DNA and six CFU of GBS per millilitre. Specificity was confirmed by analysing a range of Gram-positive and Gram-negative organisms and pediatric isolates. Polymerase chain reaction analysis of 56 cerebrospinal fluid specimens from infants under 1 year of age was performed and compared with culture. False-negative results were only encountered when processed CSF supernatant was analysed. False-positive results were obtained when DNA from Streptococcus porcinus was amplified, however this is a rare organism which has yet to be isolated as a cause of neonatal meningitis. The data indicate that semi-nested PCR is a rapid tool which might be used to confirm a diagnosis of GBS meningitis in infants and newborn babies.

Animals↗
PubMed1999

PET measures of amphetamine-induced dopamine release in ventral versus dorsal striatum.

Regional differences in dextroamphetamine (AMPH)-induced dopamine (DA) release in the baboon striatum were assessed using positron emission tomographic (PET) measures of [11C]raclopride specific binding to DA D2/D3 receptors acquired before and after AMPH administration. The magnitude of the reduction in [11C]raclopride binding, following AMPH administration, was two-fold greater in the anteroventral striatum (comprised of ventral caudate, anteroventral putamen, and nucleus accumbens) than the dorsal striatum (dorsal caudate). A simulation study demonstrated that any potential biases due to resolution (partial volume) and alignment effects were significantly smaller than the magnitude of the observed results. These regional differences in the sensitivity of AMPH are compatible with microdialysis evidence in rats indicating that the magnitude of DA release in response to AMPH concentrations in the range tested is greater in ventral than dorsal striatal regions. Post hoc tests involving measures in other striatal regions showed that the baseline DA D2/D3 binding was highest and the correlation between AMPH dose and change in [11C]raclopride binding most significant in the putamen.

Amphetamine↗
PubMed1999

The experimental evaluation of an oral cancer information leaflet.

The aims of our series of studies were: (1) to prepare an information leaflet about oral cancer for the general public; (2) to design a questionnaire to assess the level of knowledge about oral cancer; and (3) to evaluate the newly prepared leaflet. A study was conducted for each aim. For study 1, multiple drafts of oral cancer leaflets were distributed for comment and editing. For study 2, a large number of statements (100) about oral cancer were compiled and judged. Redundant items were withdrawn. The final version was administered to criterion groups (students, dentists and the public) to assess reliability and validity. For study 3, a multigroup pre- and post-test randomised design (comprising an experimental and two control groups) was adopted to evaluate knowledge improvement from exposure to the oral cancer leaflet. The participants in each study were as follows: in study 1, questions were judged by the authors, colleagues (n=2) and a non-patient group (n=10); in study 2, undergraduate psychology students (n=41), medical students (n=60), dental students (n=82), members of the public (n=54) and general dental practitioners (n=22) were respondents for the reliability/validity assessment; and in study 3, the dental students and members of the public from the previous study participated. The measure was a true/false 36-item questionnaire with additional demographic questions. In study 1 the Flesch readability index for the leaflet equalled approximately 80, i.e. 'fairly easy'. In study 2 the Kuder-Richardson 20 reliability coefficient of the questionnaire equalled 0.76. Criterion validity was confirmed with general dental practitioners scoring greater knowledge than members of the public (p<0.05). In study 3 participants who had access to the leaflet showed a significant increase in knowledge at post-test in comparison to pre-test. Control group results were supportive of a positive effect from leaflet exposure. In conclusion, the design of a health information leaflet, the assessment of reliability and validity of an associated knowledge questionnaire and evaluation of the influence of such a leaflet should be conducted in a series of planned steps. This approach can be used for other health-related issues which require dissemination of information. Three studies have demonstrated the successful application of this design and evaluation model to the area of oral cancer education.

Alcohol Drinking↗
PubMed1999

Interindividual variation in mitotic recombination.

Mitotic recombination (MR) between homologous chromosomes is a mutational event that results in loss of heterozygosity in half of the segregants at mitosis. Loss of heterozygosity may have important biological consequences. The purpose of this study was to describe human variation in the spontaneous frequency of MR. Using an immunoselection technique for isolating the somatic mutations that result in loss of expression of one of the codominant alleles at the HLA-A locus, we have measured the frequency and molecular basis of somatic mutations in lymphocytes from a population of young adults. Mutations were classified as being the result of intragenic changes, major deletions, or MR. Here we show that the MR mutation frequency in females was significantly greater than that in males but that intragenic mutation frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed. Furthermore, the observed number of individuals from whom no mutants resulting from MR were obtained was significantly greater than was expected. The endogenous level of MR may be under genetic control. Given the association of loss of heterozygosity with cancer initiation and progression, low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply.

Adult↗
PubMed1999

Pathogenesis, diagnosis, and therapy of feline idiopathic megacolon.

Many cats have one or two episodes of constipation without further recurrence, although others progress to complete colonic failure. Middle-aged male cats are particularly at risk for the clinical continuum of constipation, obstipation, and dilated megacolon. Pelvic canal stenosis and nerve injury are minor causes in the development of this syndrome. In most affected cats, the underlying pathogenesis appears to involve colonic smooth muscle dysfunction. In this group of cats, it is not yet clear whether this disorder represents a primary or secondary (resulting from long-standing constipation and colonic distension) abnormality. Many cats with mild to moderate constipation respond to conservative medical management (e.g., dietary fiber supplementation, emollient or hyperosmotic laxatives, colonic prokinetic agents). Indeed, early use of colonic prokinetic agents is likely to prevent the progression of constipation of obstipation and dilated megacolon in many cats. Some cats may become refractory to these therapies, however, as they progress through moderate or recurrent constipation to obstipation and dilated megacolon. These cats eventually require colectomy. Cats have a generally favorable prognosis for recovery following colectomy, although mild to moderate diarrhea may persist for 4 to 6 weeks postoperatively in some cases.

Animals↗
PubMed1998

Test-retest variability of serotonin 5-HT2A receptor binding measured with positron emission tomography and [18F]altanserin in the human brain.

The role of serotonin in CNS function and in many neuropsychiatric diseases (e.g., schizophrenia, affective disorders, degenerative dementias) support the development of a reliable measure of serotonin receptor binding in vivo in human subjects. To this end, the regional distribution and intrasubject test-retest variability of the binding of [18F]altanserin were measured as important steps in the further development of [18F]altanserin as a radiotracer for positron emission tomography (PET) studies of the serotonin 5-HT2A receptor. Two high specific activity [18F]altanserin PET studies were performed in normal control subjects (n = 8) on two separate days (2-16 days apart). Regional specific binding was assessed by distribution volume (DV), estimates that were derived using a conventional four compartment (4C) model, and the Logan graphical analysis method. For both analysis methods, levels of [18F]altanserin binding were highest in cortical areas, lower in the striatum and thalamus, and lowest in the cerebellum. Similar average differences of 13% or less were observed for the 4C model DV determined in regions with high receptor concentrations with greater variability in regions with low concentrations (16-20%). For all regions, the absolute value of the test-retest differences in the Logan DV values averaged 12% or less. The test-retest differences in the DV ratios (regional DV values normalized to the cerebellar DV) determined by both data analysis methods averaged less than 10%. The regional [18F]altanserin DV values using both of these methods were significantly correlated with literature-based values of the regional concentrations of 5-HT2A receptors determined by postmortem autoradiographic studies (r2 = 0.95, P < 0.001 for the 4C model and r2 = 0.96, P < 0.001 for the Logan method). Brain uptake studies in rats demonstrated that two different radiolabeled metabolites of [18F]altanserin (present at levels of 3-25% of the total radioactivity in human plasma 10-120 min postinjection) were able to penetrate the blood-brain barrier. However, neither of these radiolabeled metabolites bound specifically to the 5-HT2A receptor and did not interfere with the interpretation of regional [18F]altanserin-specific binding parameters obtained using either a conventional 4C model or the Logan graphical analysis method. In summary, these results demonstrate that the test-retest variability of [18F]altanserin-specific binding is comparable to that of other PET radiotracers and that the regional specific binding of [18F]altanserin in human brain was correlated with the known regional distribution of 5-HT2A receptors. These findings support the usefulness of [18F]altanserin as a radioligand for PET studies of 5-HT2A receptors.

Adult↗
PubMed1998

Serum sialic acid concentration is not associated with the extent or severity of coronary artery disease in patients with stable angina pectoris.

BACKGROUND: Total serum sialic acid concentration has been reported to predict death from cardiovascular disease. This study was performed to assess the relation between serum sialic acid concentration and the angiographic extent and severity of coronary atheroma in patients with stable angina. METHODS: Quantitative coronary angiography was performed in 40 patients with stable angina with either triple-vessel disease (23 patients) or normal/nearly normal coronary arteries (17 patients). A colorimetric assay for the enzymatic determination of serum sialic acid was used. RESULTS: Serum sialic acid concentration was not significantly different in patients with normal or nearly normal coronary angiograms compared with those with triple-vessel disease (68+/-10 mg/100 mL and 68+/-11 mg/100 mL, respectively). Neither was there any association between the extent or severity of coronary disease and serum sialic acid levels. CONCLUSIONS: Serum sialic acid concentration does not appear to be associated with the extent or severity of coronary artery disease in patients with stable angina pectoris. Thus the previously described association between serum sialic acid and cardiovascular death may reflect the role of mechanisms other than the severity of coronary artery narrowings.

Aged↗
PubMed1998

Differential plasma endothelin levels in subgroups of patients with angina and angiographically normal coronary arteries. Coronary Artery Disease Research Group.

BACKGROUND: Raised plasma endothelin concentrations have previously been reported in patients with cardiac syndrome X, but it is not known whether these levels vary between clinically distinct subgroups in this heterogeneous condition. METHODS AND RESULTS: We compared plasma immunoreactive endothelin levels in 54 patients with angina pectoris and normal coronary angiograms and 21 healthy control subjects. The patient group was divided into 4 clinically distinct subgroups: 7 with left bundle branch block (group A); 7 with previous myocardial infarction (group B); 24 with positive exercise electrocardiography (group C); and 16 with negative exercise electrocardiography (group D). The plasma endothelin concentration was significantly higher in patients compared with control subjects (3.7 [2.9 to 4.3] vs 2.96 [2.4 to 3.4] pg/mL, respectively, median [interquartile range]; P=0.002). Endothelin concentrations were most significantly elevated in group A and group B (4.5 [3.6 to 5.2] pg/mL; P=0.005 and 4.1 [3.9 to 4.5] pg/mL; P=0.001, respectively). Plasma endothelin concentrations were also significantly elevated in group C (3.7 [2.8 to 4.1] pg/mL; P=0.02) but not in group D (3.0 [2.5 to 3.8] pg/mL; P=0.3). CONCLUSIONS: Plasma endothelin concentration is elevated in patients with angina pectoris and angiographically normal coronary arteries, particularly those with left bundle branch block or previous myocardial infarction.

Aged↗
PubMed1998

Raised endothelin 1 levels in patients with colorectal liver metastases.

BACKGROUND: Endothelin 1 (ET-1), a vasoconstrictor peptide, has been implicated as a tumour growth stimulator and an angiogenesis factor. METHODS: To assess the involvement of ET-1 in colorectal cancer, immunoelectron microscopy for ET-1 was performed in colorectal liver metastases and normal liver (n = 6). ET-1 plasma levels were measured by radioimmunoassay in patients with colorectal cancer, with (n = 18) and without (n = 12) liver metastases, and in controls (n = 22). RESULTS: In normal liver, ET-1 was present in endothelial cells; in tumour, it was observed in endothelial cells, tumour cells and myofibroblasts. Mean(s.d.) plasma ET-1 levels were 2.75 (1.37) pg/ml in controls, 4.53(1.61) pg/ml in patients with colorectal liver metastases (P = 0.001) and 3.92(1.32) pg/ml in patients without metastases (P = 0.02). CONCLUSION: ET-1 was present in various cell types within colorectal liver metastases and raised levels were found in the plasma of patients with colorectal cancer. ET-1 may not only modulate tumour vascular tone but also act on tumour growth and angiogenesis, both locally and systemically.

Aged↗
PubMed1998

General practitioner screening for melanoma: sensitivity, specificity, and effect of training.

OBJECTIVE: To measure the performance of trained and untrained general practitioners (GPs) in screening men and women aged 50 or more for melanomas. METHODS: GPs trained in melanoma diagnosis, untrained GPs, and skin cancer specialists examined groups of volunteers, each of which included a small number of subjects with prediagnosed suspicious pigmented lesions (SPLs) that were subsequently excised for histopathological examination. RESULTS: Trained and untrained GPs achieved mean sensitivities of 0.73 and 0.71, and mean predictive values of 0.40 and 0.37, respectively, for the detection of prediagnosed SPLs. When the SPLs had been excised and examined histopathologically, reanalysis showed mean sensitivities of 0.98 and 0.95, mean specificities of 0.52 and 0.49, and mean positive predictive values of 0.24 and 0.22 for the detection of subjects with melanomas by trained and untrained GPs respectively. Trained GPs were significantly better than untrained GPs at diagnosing as melanomas SPLs that subsequently proved to be melanomas (p = 0.04). CONCLUSIONS: GPs in this study achieved high sensitivities in screening older Australian men and women for melanomas, but at the cost of low specificities and positive predictive values. Training in melanoma diagnosis had no significant effect on sensitivity, specificity, and positive predictive value for screening. Data from the study were tested in a model of population screening for melanomas, and costs per life year saved for men aged 50-70 ranged from $A11,852 to $A40,259 depending upon the screening interval and whether the GPs excised the SPLs diagnosed, or referred all patients to skin cancer specialists; this would be as cost effective as cervical cancer screening.

Female↗
PubMed1998

Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons.

Parasitic nematodes of the genus Strongyloides are remarkable for their ability to switch between alternative free-living developmental pathways in response to changing internal environmental conditions. After exiting the host, soil-dwelling larval stages may develop either to infectivity via 2 microbiverous stages (homogonic development) or to free-living adulthood via 4 microbiverous larval stages (heterogonic development). The progeny of these adults then give rise to the infective stage. In the latter case, free-living existence is extended in time and the number of infective larvae is greatly amplified. Anterior chemosensory neurons (amphidial neurons) are thought to respond to environmental cues and via signal transduction pathways control the direction of larval development. We now demonstrate by laser microbeam ablation that 2 classes of amphidial neurons (ASF and ASI), acting together, control the direction of free-living larval development. Larvae in which the neurons were killed developed to infectivity via the homogonic route rather than to adulthood via the otherwise predominant heterogonic route. These neurons are probable homologues of neurons ADF (=ASF) and ASI in Caenorhabditis elegans, suggesting the control of development at the cellular level is conserved among divergent taxa of nematodes. These observations also have important implications for the evolution of nematode parasitism and the design of new prophylactic measures against parasitic nematodes of medical and veterinary medical importance.

Animals↗
PubMed1997

Application of composite estimation in studies of animal population production with two-stage repeated sample designs.

This paper reports results from two example data sets of a two-stage sampling design where sampling (in panels) both farms and animals within selected farms increases the efficiency of parameter estimation from measurements recorded over time. With such a design, not only are farms replaced from time-to-time but also animals subsampled within retained farms are subject to replacement. Three general categories of parameters estimated for the population (the set of animals belonging to the universe of farms of interest) were (1) the total at each measurement occasion; (2) the difference between means or totals on successive measurement occasions; (3) the total over a sequence of successive measurement periods. Whereas several responses at the farm level were highly correlated over time (rho 1), the corresponding animal responses were less correlated over time (rho 2)-leading to only moderate gains in relative efficiency. Intraclass correlation values were too low in most cases to counteract the overall negative impact of rho 2. In general, sizeable gains in relative efficiency were observed for estimating change-confirming a previous result which showed this to be true provided that rho 1 was high (irrespective of rho 2).

Animal Husbandry↗
PubMed1997

Barren female Strongyloides stercoralis from occult chronic infections are rejuvenated by transfer to parasite-naive recipient hosts and give rise to an autoinfective burst.

It is widely assumed that barren Strongyloides stercoralis occurring in chronically infected carriers can become fecund when immunity wanes. Evidence for this involves corticosteroid treatment of hosts harboring occult infections that subsequently return to patency. However, nematodes have ecdysteroid receptors, and it has been suggested that corticosteroids act directly on the parasite, inducing autoinfective development, rather than indirectly by suppressing host immunity. To test these competing concepts, barren females were recovered from donor dogs when the dogs' fecal examinations turned negative. Groups of 100 active barren worms were surgically transplanted into the small intestines of each of 6 naive canine recipients. Three were examined at necropsy at 4-5 days postinfection (PI), before autoinfection could amplify the number of successfully transferred parasites. The remaining recipients were examined 21-22 days PI when, if autoinfection had occurred, the worm populations should have increased. At 4-5 days, gravid worms occurred in each of the recipients (19 +/- 6 worms/dog). By 21-22 days, a remarkable population increase had occurred (522.6 +/- 296 worms/dog). Worms from chronically infected donors were stunted, and electron microscopy revealed damage to the intestine and ovaries. Successfully transplanted worms recovered at days 4-5 PI were ovigerous and less stunted and showed repair of intestinal and ovarian tissues.

Animals↗