Search PubMed⌕ Search

Biomedical subjects

S Richards

Publications and source records attributed to S Richards.

At least 91 records · Page 5Linked to original sources
PubMed1996

32P-incorporation PCR for the detection of rearrangements at the TCR-gamma locus.

We have adapted and developed a PCR (polymerase chain reaction)-based technique for the T-cell receptor (TCR)-gamma chain gene, which has subsequently been used for routine diagnosis. Variable-region oligonucleotide primers were chosen from subgroups I and II, and the joining region primer was from the J2 segment. The primers were used to perform a 32P-incorporation PCR, and the products were then separated on an 8% denaturing polyacrylamide gel. In our hands, this technique is more reliable than cold methods, when separation is performed on either agarose or nondenaturing polyacrylamide. The radioactive technique was used to look at 102 T-cell proliferations, of which eight of eight T-acute lymphoblastic leukemia (ALL), 24 of 34 T-non-Hodgkin's leukemia (NHL), and 35 of 60 large granular lymphocyte (LGL) expansions were clonal. Of 122 B-cell proliferations investigated, including 72 cases of B-cell lineage ALL, 36 demonstrated a T-cell rearrangement (33 ALLs and three myelomas). Samples from nonlymphoid tumors were tested and produced a normal distribution ladder of PCR products after autoradiography, a pattern also observed with antenatal and preoperative patients. The radiolabel-incorporation method detected an abnormal pattern of a ladder with prominent dark bands in 29 of 122 B-cell and 27 of 102 T-cell cases and in 0 of 49 of the nonlymphoid and normal samples. The abnormal banding patterns obtained in a proportion of the B- and T-cell cases was not readily discernible by nondenaturing-acrylamide or agarose-separation methods.

Blotting, Southern↗
PubMed1996

Essential characteristics of self-determined behavior of individuals with mental retardation.

Despite increased emphasis on self-determination for individuals with mental retardation, only a few theoretical models have been formulated that specify measurable characteristics for the promotion and evaluation of this outcome. We propose that self-determination refers to acting as the primary causal agent in one's life and making choices and decisions regarding one's quality of life free from undue external influence or interference. Self-determined behavior is autonomous, self-regulated, based on psychological empowerment, and self-realizing. We evaluated this definition by asking participants with mental retardation to complete various instruments that measured self-determined behavior and these essential characteristics. Discriminant function analysis indicated that measures of essential characteristics predicted differences between groups based on exhibition of self-determined behavior.

Activities of Daily Living↗
PubMed1995

Functional activity of the human prolactin receptor and its ligands.

Prolactin receptors (PRLR) have been identified in a number of human tissues and cell lines, although little is known about the human receptor protein. The cloning of the human PRLR cDNA has enabled further characterization of the receptor protein in transfected cells. Since the human cDNA is expressed at lower levels than the rat cDNA, we have constructed a hybrid cDNA (pECE r5'hPRLR) containing nucleotides of the 5' untranslated region and signal peptide of the rat PRLR and the protein coding and 3' untranslated portion of the human receptor. Expression of the hybrid receptor was increased more than two-fold compared to the human receptor as detected by specific binding of 125I-human growth hormone (GH) to transfected COS-7 cells. The relative molecular mass of the receptor was 93,000 Da, as determined by chemical cross-linking studies. Transcriptional assays were used to show the human PRLR was able to activate two milk protein genes; ovine beta-lactoglobulin and rat beta-casein. Transfected cells expressing the human PRLR receptor, treated with human GH or prolactin (PRL), induced a dose-dependent increase in transcriptional activation of the beta-casein/luciferase fusion gene. Glycosylated, and non-glycosylated human PRL, and ovine PRL were equally effective in activating the beta-casein promoter. Human placental lactogen and bovine PRL could also induce a greater than 10-fold induction, whereas insulin did not significantly stimulate the beta-casein promoter. The results show that the human PRLR can activate both beta-lactoglobulin and beta-casein milk gene promoters and that these reporter genes can be used to evaluate the functional activity of agonists and antagonists of the human PRLR.

Animals↗
PubMed1995

Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology.

The purpose of this study is to demonstrate the value of a multiplex amplification and readout system. The validation was done using as a model system the detection of deletions in nine possible dystrophin exons: 4, 8, 12, 17, 19, 44, 45, 48, and 51. The amplification system was gap ligase chain reaction, adapted to amplify selected regions of multiple exons simultaneously. The amplified products were read out with an immunochromatographic methodology, adapted from that used in the Abbott product line commercialized under the name Test Pack Plus. In each amplification, the beta-globin gene was incorporated and served as a procedural control. The complete process takes < 3 hr from DNA sample to result. The procedure is therefore rapid and simple, as well as being potentially very cost effective. The combination of these two technologies is shown to be a useful tool for the determination of deletions in the nine exons of the dystrophin gene. The results of a 100-patient sample study showed concordance with cDNA and PCR in current use. Equivalent performance at two sites was shown.

Base Sequence↗
PubMed1995

Competing HMOs collaborate to improve preventive services.

BACKGROUND: In July 1993, an unusual collaboration developed between competing managed care plans and with competing primary care clinics as part of a federally funded research grant (IMPROVE from the Agency for Health Care Policy and Research). The goal of this collaboration is to scientifically test the ability of an health maintenance organization (HMO) to improve the delivery of eight adult preventive services by training and facilitating the use of continuous quality improvement and prevention systems by contracted private primary clinics. METHODOLOGY: In order to conduct this effectiveness study, it was necessary for two HMOs to come to a structural and functional understanding of how to operate jointly. Investigators recruited 44 private clinics for a randomized controlled trial in which 22 are being assisted in improving the process used to deliver these preventive services and 22 are being left alone as comparison clinics. The intervention is a train-the-trainer and consultation approach focused on clinics as collaborating customers. The comparison will be based on repeated surveys of patients and clinic personnel as well as chart audits to measure changes in systems and prevention rates. SUMMARY: Although this project was made possible by a number of unusual favorable factors, it can serve as a model for support of the clinician leadership that is essential to true health care delivery reform.

Adult↗
PubMed1995

High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE).

Genotyping (typing of genetic variation) typically involves PCR followed by an allele-specific oligonucleotide-binding assay, restriction enzyme digest or direct check of the outcome of a PCR designed to distinguish genotype. Electrophoresis can resolve "bound" from "free" oligonucleotide, as well as resolve PCR fragments and digests, but it is traditionally regarded as cumbersome and laborious in comparison with solution assays. Here we describe simple horizontal polyacrylamide gels which can receive a 96-well array of samples directly, which can be stacked in tanks and which are bound to a robust support of glass. The line of electrophoresis is on a 71.6 degree diagonal relative to the columns of the array (microplate array diagonal gel electrophoresis [MADGE]). Several thousand reactions can conveniently be analyzed in a shoebox-sized apparatus in a couple of hours. High resolution is achieved in the range of 20-1000 bp, information processing is simplified and automation is possible.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

Base Sequence↗
PubMed1994

Epidermal growth factor receptor expression by human squamous cell carcinomas of the head and neck, cell lines and xenografts.

Epidermal growth factor receptor (EGFR) overexpression has been associated frequently with squamous cell carcinomas (SCC) and SCC cell lines. In most cases the level of EGFR on the tumours from which the cell lines were derived has not been determined, nor have EGFR levels been determined for xenograft tumours from the cell lines. In this study we determined EGFR expression on a new series of head and neck SCC (SCCHN)-derived cell lines, which were obtained from tumours representing a spectrum of malignant progression, and two cell strains derived from erythroplakia premalignant lesions. The level of EGFR on cell lines was determined by [125I]EGF competitive binding assays. EGFR levels on some of the original tumours and xenografts of the cell lines were determined on cryosections by a competitive binding assay based on [125I]EGFR1, an EGFR-specific monoclonal antibody. EGFR expression on the tumour cryosections was compared with expression on cryosections of skin and buccal mucosa. Eight of the ten tumour cell lines had elevated EGFR. Two of the tumour-derived cell lines and the two erythroplakia-derived cell strains expressed EGFR at levels similar to that detected on normal keratinocytes in tissue culture. Only two of the tumours overexpressed EGFR when compared with normal tissue. The other tumours had levels similar to that detected on the basal layers of skin or buccal mucosa. The xenografts expressed EGFR, as did the original tumours, even though they were derived from cell lines that displayed significant overexpression of EGFR. This study suggests that most tumours have a latent potential to overexpress EGFR which is realised in tissue culture.

Animals↗
PubMed1994

Severe cytopenias associated with the sequential use of busulphan and interferon-alpha in chronic myeloid leukaemia.

We describe four patients who developed severe thrombocytopenia which progressed to aplasia after the use of alpha-interferon in maintenance therapy of chronic phase CML after busulphan induction. On reviewing over 400 patients in the MRC CML III trial we found that there is a risk of cytopenia developing after busulphan therapy and a lesser risk of cytopenias developing after alpha-interferon therapy. If the therapies are given in a sequential fashion the risk of cytopenia developing appears to be additive, may be pronounced, and may lead to clinically significant problems. Hydroxyurea alone does not lead to sustained cytopenia. Care should be taken to ensure that counts are stable after the use of busulphan before starting alpha-interferon as maintenance therapy.

Adult↗
PubMed1994

Dystrophin and DNA diagnosis in a large pediatric muscle clinic.

Eighty-seven unrelated patients from a large muscle clinic setting were analyzed by DNA for deletions in the dystrophin gene for diagnosis of Duchenne/Becker muscular dystrophy. The clinical phenotype of the patient population included 72% Duchenne, 13% Becker, and 15% outlier patients. Dystrophin gene deletions were detected in 61% of these patients, and disease phenotype was predicted by DNA with an accuracy of 95%. While DNA did not confirm diagnosis in all patients, dystrophin analysis of muscle biopsies, when available, predicted a disease phenotype. In the 66 patients in which muscle biopsies were available for analysis, the results of the dystrophin analysis agreed with actual clinical phenotype with 86% accuracy. Less agreement between dystrophin and clinical phenotype predictions were found in the Becker patient population. We suggest that, in at least 61% of Duchenne/Becker patients, DNA analysis provides a rapid and accurate diagnosis. DNA is less invasive and less expensive than biopsy and may allow family risk assessment. Therefore, DNA analysis may become the first recommended laboratory procedure for Duchenne/Becker diagnosis, and muscle biopsy with dystrophin analysis may become necessary only for those patients with undetectable gene mutations.

Adolescent↗
PubMed1993

Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.

The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.

Adult↗
PubMed1993

Binding and specificity of major immunoglobulin classes of preformed human anti-pig heart antibodies.

Preformed human anti-pig antibodies isolated from perfused pig hearts were used to analyze the binding of various immunoglobulin classes to cultured pig kidney cells. All anti-pig immunoglobulins (i.e., IgG, IgA, and IgM) were localized on the cell surface by the use of an indirect immunofluorescence technique. Anti-pig immunoglobulins also competed for the pig cell surface epitopes with Griffonia simplicifolia lectin (GS-I-B4), which is specific for alpha-galactosyl residues. This study provides further evidence that preformed human antibodies recognizing alpha-galactosyl-containing epitopes (anti-gal antibodies) could be an important factor in hyperacute rejection of pig organs.

Animals↗
PubMed1993

Fine structure of the human FMR1 gene.

The fragile X syndrome is due to a CGG triplet expansion in the first exon of FMR1, resulting in hypermethylation and extinction of gene expression. To further our understanding of the gene's involvement in the syndrome, we report the physical structure of this locus. A high resolution restriction map of the FRAX(A) locus has been prepared encompassing approximately 50 kb. Using exon-exon PCR and restriction analysis, the FMR1 gene has been determined to consist of 17 exons spanning 38 kb of Xq27.3. Each intron-exon boundary has been sequenced. In general, the splice donors and acceptors located in the 5' portion of the gene demonstrate greater adherence to consensus than those in the 3' end, providing a possible explanation for the finding of alternative splicing in FMR1. The elucidation of the exon composition of the FMR1 gene and its flanking region will enhance detection of coding sequence mutations possible in fragile X phenocopy individuals.

Alternative Splicing↗