Improved method for direct PCR amplification from whole blood.
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Base Sequence↗
Biomedical subjects
T Smith
Publications and source records attributed to T Smith.
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Communication↗
Stimulation of the thrombin receptor of human glomerular mesangial cells by Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe peptide.
We have studied the effects of thrombin (alpha-thrombin) and Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SFLL), a peptide agonist of the platelet thrombin receptor in cultured human mesangial cells, and find that SFLL can reproduce the biochemical and morphological effects of thrombin. Treatment of mesangial cells with cAMP-elevating agents causes fragmentation of stress fibers, loss of the vitronectin receptor from sites of focal adhesion, and produces a change in shape from a flat to a more arborized configuration. These effects are prevented by both thrombin and SFLL. Thrombin and SFLL also initiate biochemical signaling events in mesangial cells by stimulating the metabolism of phospholipids. Both thrombin and SFLL stimulate release of inositol phosphates from [3H]inositol-labeled cells, elevation of cytosolic calcium, the formation of [3H]myristic acid-labeled diacylglycerol, an increase in the mass of diacylglycerol, 32P incorporation into phospholipids, and release of unesterified [3H]arachidonic acid from cells prelabeled with [3H]arachidonic acid. When present together, the effects of SFLL and thrombin on diacylglycerol formation, arachidonic acid production, and inositol phosphate production were not additive. This suggested that SFLL and thrombin were acting on the same receptor. This was further supported by our observations that cells pretreated with SFLL and subsequently exposed to thrombin (or vice versa) did not show elevated cytosolic calcium. We also show that phospholipase D is activated by demonstrating production of radiolabeled phosphatidylethanol when cells are treated with SFLL in the presence of ethanol. These findings indicate that SFLL can be used to study the receptor-mediated effects of thrombin in mesangial cells, thereby avoiding thrombin's proteolytic actions.
Actins↗
Prediction of gene structure.
We have developed a hierarchical rule base system for identifying genes in DNA sequences. Atomic sites (such as initiation codons, stop codons, acceptor sites and donor sites) are identified by a number of different methods and evaluated by a set of filters and rules chosen to maximize sensitivity; these are combined into higher-order gene elements (such as exons), evaluated, filtered and combined as equivalence classes into probable genes, which are evaluated and ranked. The system has been tested on an extensive collection of vertebrate genes smaller than 15,000 bases. Results obtained show that, on average, 88% of the predicted coding region for a transcription unit is actually coding, and 80% of the actual coding is correctly predicted. This will, in most applications, be sufficient for a search against protein sequence databases for the identification of probable gene function. In addition, the system provides a general test platform for both gene atomic site identification and the rules for their evaluation and assembly.
Algorithms↗
Identification of USF as the ubiquitous murine factor that binds to and stimulates transcription from the immunoglobulin lambda 2-chain promoter.
To study the specificity and identity of NF-lambda 2, a ubiquitous murine nuclear factor that interacts specifically with the promoter of the lambda 2-chain gene and stimulates its transcription, competition experiments were carried out using DNA fragments from various immunoglobulin regulatory elements. The results showed that a fragment containing the H-chain enhancer competed efficiently for the binding of NF-lambda 2. Dissection of the H-chain enhancer revealed that the microE3 motif contributed the competing ability. Additionally, a regulatory region found in the adenovirus major late promoter, which interacts with the human general transcription factor USF, competed very efficiently for binding to NF-lambda 2. This region contains a sequence, CACGTGAC, which is identical to a region within the NF-lambda 2 motif. The pattern of complexes formation using oligonucleotide probes corresponding to the NF-lambda 2 and USF motifs were identical, and they both differed from that displayed by the E3 probes. Antisera against different domains of USF also react specifically with NF-lambda 2 showing that this factor is antigenically related, if not identical, to USF. Furthermore, the activity of the lambda 2 promoter in an in vitro transcription assay was significantly reduced when the nuclear extract used was USF-depleted. Addition of exogenous USF to this extract restored the transcription activity. Therefore, we conclude that NF-lambda 2 is the murine homologue of USF.
Animals↗
Sustained-release ganciclovir therapy for treatment of cytomegalovirus retinitis. Use of an intravitreal device.
A surgically implantable device for sustained intravitreal release of ganciclovir has been developed. The device delivers ganciclovir intraocularly over approximately 4 to 5 months. Eight patients with acquired immunodeficiency syndrome (AIDS) and associated cytomegalovirus (CMV) retinitis were recruited as part of a phase 1 study. Thirteen eyes with active CMV retinitis underwent surgical implantation of the ganciclovir device. All eyes showed resolution of the CMV retinitis; none showed progression. Visual acuity remained unchanged in three eyes, improved in six eyes, and decreased in four eyes. Surgical complications included mild vitreous hemorrhage, astigmatism, and suprachoroidal placement of the device. Retinal detachment occurred in three eyes as the retinitis resolved. This new intraocular drug delivery system offers many advantages compared with intravenous therapy or repeated intravitreal ganciclovir injections for the management of CMV retinitis in patients with AIDS.
Acquired Immunodeficiency Syndrome↗
Ampicillin-sulbactam vs clindamycin in head and neck oncologic surgery. The need for gram-negative coverage.
This study was undertaken to assess whether gram-negative antimicrobial coverage is required in patients undergoing head and neck oncologic surgery. Ampicillin sodium-sulbactam sodium and clindamycin phosphate were compared in a prospective, randomized, parallel, double-blind trial of 212 patients undergoing head and neck procedures involving clean-contaminated wounds. Both antibiotics were given up to 1 hour before surgery and continued at 6-hour intervals after surgery for an additional eight doses. Fourteen infections occurred in the ampicillin-sulbactam-treated group (13.3%) and 29 infections in the clindamycin-treated group (27.1%). From patients receiving clindamycin, 29 gram-negative organisms were isolated, compared with six from those patients receiving ampicillin-sulbactam. This finding supports the need for gram-negative coverage in patients undergoing clean-contaminated head and neck oncologic surgery.
Adult↗
Gut protection by cyclophosphamide "priming" in patients receiving high-dose melphalan--effect of drug scheduling.
A "priming" injection of cyclophosphamide (400 mg/m2 given i.v. on day -7) has been shown to reduce intestinal permeability and thus gut toxicity in patients receiving high-dose melphalan. To determine the optimal timing for this injection, patients receiving 200 mg/m2 melphalan with an autologous bone marrow transplant were randomly assigned to receive cyclophosphamide at 5, 7 or 9 days before the melphalan. The median percentage of [51Cr]-ethylenediaminetetraacetic acid excretion was similar (9.1% vs 7.1% vs 7.7%, respectively), with equivalent duration of WHO grade 2-4 mucositis and diarrhoea being recorded for each group. Thus, the timing of the cyclophosphamide prime is not critical, and the priming injection may be given between 5 and 9 days prior to high-dose melphalan.
Bone Marrow Transplantation↗
Prognostic significance of elevated serum beta 2-microglobulin levels in adult acute lymphocytic leukemia.
PURPOSE: Elevated serum beta-2 microglobulin (beta 2M) levels are associated with poor prognosis in several lymphoproliferative disorders including multiple myeloma and lymphoma. Their prognostic relevance in acute lymphocytic leukemia (ALL) is unknown. We analyzed the associations of serum beta 2M levels at diagnosis with pretreatment characteristics and with prognosis in adult ALL. PATIENTS AND METHODS: One hundred fifty-nine adults with newly diagnosed ALL were investigated. Serum beta 2M levels were determined at diagnosis, on fresh peripheral blood samples, using a radioimmunoassay, the Pharmacia beta 2 Micro RIA (Pharmacia Diagnostics, Uppsala, Sweden). Statistical correlations were assessed by standard methods, and further independent prognostic value of serum beta 2M was determined by multivariate analysis. RESULTS: Patients with beta 2M levels of 4.0 mg/L or above had a lower complete response rate (61% versus 80%; p = 0.02), a significantly worse survival (p < 0.01), and a significantly higher association with development of central nervous system (CNS) leukemia (p < 0.01). High beta 2M levels were more common among patients with older age, with elevated creatinine, bilirubin, and alkaline phosphatase levels, with low albumin levels, and with B-cell disease. Multivariate analysis for survival indicated the beta 2M level to be an independent prognostic variable (after adjusting for pretreatment creatinine level and age). The evaluation of beta 2M levels within low- and high-risk groups for CNS disease suggested an association of elevated beta 2M levels with a worse incidence of CNS disease in the high-risk patients. CONCLUSION: Monitoring serum beta 2M levels may provide significant prognostic information in adults with ALL and should be included in their pretreatment evaluation. Its importance in childhood ALL requires investigation.
Adult↗
Lead inhibits secretion of osteonectin/SPARC without significantly altering collagen or Hsp47 production in osteoblast-like ROS 17/2.8 cells.
In an effort to better understand the consequences of lead (Pb2+) on skeletal growth, the effects of Pb2+ were investigated using ROS 17/2.8 bone-like cells in vitro. These studies revealed that Pb2+ (4.5 x 10(-6) M -4.5 x 10(-7) M) has little or no effect on cell shape except when added immediately following seeding of the cells. However, proliferation of ROS cells was inhibited, in the absence of serum, at concentrations of 4.5 x 10(-6) M Pb2+. Protein production was generally increased, however, the major structural protein of bone, type I collagen, production was only slightly altered. Following treatment of ROS cells with Pb2+, intracellular levels of the calcium-binding protein osteonectin/SPARC were increased. Osteonectin/SPARC secretion into the media was delayed or inhibited. Coincident with retention of osteonectin/SPARC there was a decrease in the levels of osteonectin/SPARC mRNA as determined by Northern analysis. These studies suggest that processes associated with osteonectin/SPARC translation and secretion are sensitive to Pb2+.
Animals↗
Bone remodelling does not decline after menopause in vertebral fracture osteoporosis.
There is considerable current interest in whether activators of bone remodelling, such as IL-1 and other cytokines, are involved in the pathogenesis of osteoporosis. We have therefore studied indices relating to remodelling activation in 50 patients with postmenopausal vertebral osteoporosis and 12 with hip fracture osteoporosis in comparison with 25 age- and sex-matched controls. Because of uncertainty regarding the accuracy of current biochemical markers of bone formation with respect to the estimation of whole body rates of bone formation, a 85Sr-based radioisotopic method was used. This method was previously validated by comparison with data obtained after double in vivo labelling of transiliac biopsies taken nearly simultaneously. Bone resorption was estimated from urinary hydroxyproline data. Controls selected for their continued good health showed a progressive and statistically highly significant decline in indices of bone formation with time after menopause. No such decline was seen in the vertebral fracture patients (P less than 0.005). There were no hip fracture patients within 10 years of menopause so this statistical test could not be applied appropriately to them. The hydroxyproline data were consistent with the suggestion arising from the bone formation data that remodelling declines progressively after menopause in the controls but not in the vertebral fracture patients. The data also suggested that these two fracture groups were in more negative calcium balance than the controls, this being particularly marked in the hip fracture cases. Plasma osteocalcin data correlated moderately well with the kinetic measurements of bone formation. It is concluded that vertebral fracture osteoporosis is associated with prolongation of menopausal levels of bone remodelling which is inappropriate by comparison with healthy controls.
Age Factors↗
Effects of a beta 2-adrenergic agonist, cimaterol and corticosterone on growth and carcass composition of male rats.
1. Growth rate and carcass composition were measured in rats treated with cimaterol or vehicle only for 21 days, with corticosterone administered during the last 7 days of cimaterol or vehicle only treatment. 2. Cimaterol significantly stimulated the amount of protein in the carcass as well as gastrocnemius and plantaris muscles by 9-15%, and decreased carcass fat by 24%. 3. Corticosterone treatment significantly decreased protein in the carcass and soleus, plantaris and gastrocnemius muscles by 13-33%. 4. The catabolic effects of corticosterone on carcass and muscle protein were reduced by pretreatment with cimaterol, suggesting that beta-adrenergic agonists may have some potential use in preventing muscle wasting during stressed states.
Adrenergic beta-Agonists↗
Transfer of staff training from workshops to group homes: a failure to generalize across settings.
Two experiments were conducted to assess acquisition and generalization of skills acquired in a workshop by trainees who were primary caregivers on the staffs of group homes for developmentally disabled clients. In Study 1, 31 staff trainees received an intensive, 1-week workshop in behavioral theory and treatment techniques. When assessed at the workshop site, these staff trainees showed increased treatment skills, relative to 18 staff trainees who did not participate in the workshop. In Study 2, pre- and postworkshop observations were taken on 53 developmentally disabled clients in group homes where the staff trainees worked. These observations provided no evidence that the workshop had any effect on group home client functioning. Future training programs for caregivers may be more successful if they occur in the group home, involve clients in the home, and enlist the support of supervisory staff, rather than focusing only on primary caregivers.
Activities of Daily Living↗
Persistent iron overload 4 years after inadvertent transplantation of a haemochromatotic liver in a patient with primary biliary cirrhosis.
An iron-loaded liver from a 20-year-old man with occult haemochromatosis was inadvertently transplanted into a 64-year-old lady with primary biliary cirrhosis. Increased hepatic iron storage was observed in biopsy specimens undertaken at 18, 30 and 36 months after transplantation. Serum iron levels and transferrin iron saturation increased and remained elevated 4 years after surgery. Ferritin levels were also consistently increased. Iron absorption test, performed at 4 years after transplant, was increased at 38%. Our data suggest that an intrahepatic defect in haemochromatosis cannot be excluded, and casts some doubt on the assertion that a precirrhotic haemochromatotic liver can be transplanted without sequelae.
Adult↗
Whole-body and site-specific bone remodelling in patients with previous femoral fractures: relationships between reduced physical activity, reduced bone mass and increased bone resorption.
1. A new tracer method is described for the non-invasive measurement of bone formation in the proximal femur. The method is based on our previously described whole-body method using 85Sr as the tracer (Reeve, J., Hesp, R. & Wootton, R. Calcif. Tissue Res. 1976; 22, 191-206). It allows correction to be made for long-term exchange processes within the skeleton. 2. The method has been applied in a study of regional and whole-body bone formation in 12 rehabilitated patients who had previously suffered a fracture of the proximal femur. Twelve healthy control subjects were studied, who were selected for their good health and continued physical activity. The aim was to explore the relationship between bone formation and physical activity. 3. Bone formation was similar in the two groups, both regionally and in the whole body. Based on analyses of four cadaver specimens, bone formation in the proximal femur was about one and two-thirds times that in the whole skeleton when related to mass of calcium in the region of interest. 4. Whole-body bone resorption, estimated from five measurements per subject of hydroxyproline excretion in relation to creatinine excretion, was significantly higher in the fracture patients (P < 0.01, Wilcoxon's test). 5. Estimates of current physical activity (and immediate pre-fracture physical activity) were made with a newly devised questionnaire. Historical levels of physical activity (at ages 15-45 years) were determined with Astrom's questionnaire. No bone formation index correlated with any index of physical activity. Urinary hydroxyproline excretion correlated inversely both with current physical activity and historical physical activity (for both regression coefficients P < 0.01). 6. The results are discussed in the light of our current understanding of the control of bone remodelling by the discrete basic multicellular units of bone. The opportunity to study regional bone resorption by the additional use of serial dual X-ray absorptiometry of the same region will in future allow the direct monitoring of the effects of therapeutic interventions which have been designed to prevent contralateral hip fracture.
Aged↗
Comparison of two methods of quantitation in human studies of biodistribution and radiation dosimetry.
A simple method of quantitating organ radioactivity content for dosimetry purposes, based on relationships between organ count rate and the initial whole body count rate, has been compared with a more rigorous method of absolute quantitation using a transmission scanning technique. Comparisons were on the basis of organ uptake (% administered activity) and resultant organ radiation doses (mGy MBq-1) in 6 normal male volunteers given a 99Tcm-labelled myocardial perfusion imaging agent intravenously at rest and following exercise. In these studies, estimates of individual organ uptakes by the simple method were in error by between +24 and -16% compared with the more accurate method. However, errors on organ dose values were somewhat less and the effective dose was correct to within 3%. These radiation dose effects were largely influenced by radioactivity in the remaining body (i.e. excluding the organs specifically analysed) which accounted for the major proportion of the 99Tcm residence time. Effects due to different biodistributions between rest and exercise studies were also observed and it is concluded that although the simple method was acceptable for the present dosimetry study it should only be used with caution, and a more accurate method is recommended.
Adult↗
Validation of a method of treating menorrhagia by endometrial ablation.
Menstrual blood loss was measured by intravenous injection of radioactive iron (59Fe) and whole-body counting in 19 women complaining of menorrhagia due to dysfunctional bleeding. Fifteen patients were then treated by radiofrequency endometrial ablation, after which blood loss was re-measured. The majority of the patients were bleeding excessively before treatment (mean 281 (SD 156) ml per cycle averaged over two cycles, range 49-665 ml, n = 19). In the 15 treated patients, average blood loss was reduced to 52 (SD 39) ml per cycle and the mean reduction in blood loss was highly significant (P < 0.001). These quantitative data correlate well with the patients' subjective reports.
Adult↗
The relative effects of sex and deprivation on the risk of early death.
Socio-economic factors and sex are both known to have a pronounced bearing on the risk of early death. To determine the relative effects of these factors in our Health Board, an analysis was carried out of all deaths under the age of 70 registered in Tayside during 1989 and 1990, by age, years of life lost, diagnosis and postcode sector/deprivation category. A yearly average of 14,520 years of life up to the age of 70 were lost by males compared with 8248 by females. When cancers of the breast and sex organs were excluded, just over a quarter (28 per cent) of the difference was due to the higher death rate in males resulting from injuries and poisoning and another quarter to ischaemic heart disease (23 per cent), with infant deaths in third place (17 per cent). Deprivation category analysis showed that deaths and years of lost life from ischaemic heart disease and injuries and poisoning in males in the most deprived areas were approximately one-third higher than in the most affluent. At the same time, the death rate in males from these causes in each area, from the most affluent to the most deprived, was approximately two and a half to three times as high as in females, as were lost years of life.(ABSTRACT TRUNCATED AT 250 WORDS)
Cause of Death↗