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Biomedical subjects

E Lyon

Publications and source records attributed to E Lyon.

At least 19 recordsLinked to original sources
PubMed2001

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

DNA↗
PubMed2001

Hereditary hemochromatosis since discovery of the HFE gene.

BACKGROUND: Hereditary hemochromatosis is an inherited disorder of iron metabolism that is characterized by excessive iron deposition in major organs of the body. Chronic increased iron absorption leads to multiorgan dysfunction. Since the discovery of the gene responsible for the majority of cases, research has progressed rapidly to identify the gene product, the effects of mutations, and the implications for different populations. The protein product of the HFE gene is a transmembrane glycoprotein, termed HFE, that modulates iron uptake. Mutations in the HFE protein compromise its function and produce disease symptoms. Two mutations, C282Y and H63D, have been linked to the majority of disease cases. APPROACH: We reviewed the recent literature for the molecular basis of hereditary hemochromatosis. Genotypic information was combined with biochemical and clinical phenotypic information to achieve a better understanding of the disease mechanism. CONTENT: This review provides a comprehensive discussion of known mutations in the HFE gene and their phenotypic expression. Diagnostic criteria using molecular genetic techniques in conjunction with traditional biochemical tests are provided. Current methods and limitations of molecular testing are examined in detail. A strategy for population screening and an algorithm for diagnosis that incorporates molecular testing are presented. Treatment by therapeutic phlebotomy and the use of blood obtained from hemochromatosis patients are discussed. SUMMARY: Although the disease mechanism has not been completely elucidated, phenotypic and penetrance data are becoming available. Controversy still exists concerning the role of genetic testing in diagnosis and population screening.

Genetic Testing↗
PubMed2000

Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes.

BACKGROUND: Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). METHODS AND RESULTS: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. CONCLUSIONS: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.

DNA↗
PubMed2000

Comparison of automated short tandem repeat and manual variable number of tandem repeat analysis of chimerism in bone marrow transplant patients.

Hematopoietic chimerism can be monitored in bone marrow transplant patients at DNA polymorphic sites. In this study, allele detection and quantification by ethidium bromide-stained agarose gels were compared with automated fluorescent sizing on an artificially mixed system and on chimeric post-transplant whole blood and sorted cell populations. A panel of five variable number of tandem repeats (VNTRs) were amplified and quantified visually on an ethidium bromide-stained gel. The ten short tandem repeats (STRs) were amplified as a multiplex polymerase chain reaction (PCR) and fluorescently detected on a DNA sequencer. Fluorescent band intensities were converted to fluorescent peak areas for allele quantification. Using mixed DNA of different proportions, both STRs and VNTRs showed linearity and appeared equally sensitive. However, case studies showed STRs to be more sensitive (<5%) than VNTRs (<10%). The STRs more accurately quantified the minor DNA component at low concentrations.

Alleles↗
PubMed1999

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

Artificial Gene Fusion↗
PubMed1998

Implications of a novel cryptic splice site in the BRCA1 gene.

This study was designed to determine the significance of a single intronic base change (IVS5-12 G-->A) found in a family with a history of breast cancer. This change is predicted to form a cryptic splice site resulting in the addition of 11 nucleotides to the BRCA1 transcript. The BRCA1 gene of the relatives and control individuals was sequenced and analyzed using RT-PCR, ASO hybridization, and size fractionation. All patients showed an 11 nucleotide insert at the intron 5/exon 6 boundary. This variant is likely to form a short protein product incapable of the hypothesized tumor suppressor functions of the BRCA1 gene. This information is important for providing counseling for families with this cryptic splice site and a family history of breast cancer.

Base Sequence↗
PubMed1994

Monoclonal antibodies to glioma-derived growth factor(s).

Several monoclonal antibodies (mAb) reactive against a high-molecular-weight growth factor from human glioblastoma cell lines have been produced by immunizing mice with partially purified preparations from conditioned media. Antibody-secreting colonies were selected by their capacity to bind 35S-labeled glioma cell protein and by reactivity in indirect enzyme-linked immunoadsorbent assay (ELISA), using high-molecular-weight gel filtration fractions and preparative isoelectric focusing fractions containing growth factor activities. Two of the select mAbs (20F3 and 12A12) depleted mitogenic activity (> 50% inhibition, p < 0.05) from gel filtration fractions by immunoprecipitation, but could not neutralize mitogenic activity directly. Mitogenic activity recovered from affinity columns prepared with mAb 20F3 eluted at 48% and 52% acetonitrile from HPLC C4 reversed-phase columns. Immunoprecipitation of 35S-labeled cell lysates with 20F3 followed by resolution with SDS-PAGE autoradiography revealed one unique protein of 170 kD. Established glioma cell line D-54 MG showed perinuclear and cytoplasmic staining with mAb 20F3. mAb 20F3 should prove useful in purification and characterization of these glioma-derived growth factor(s).

3T3 Cells↗
PubMed1993

Partial characterization of glioma-derived growth factor 2: a novel mitogenic activity from human cell line D-54 MG.

We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111:B4, 1 microgram/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells. This glioma-derived growth factor (GDGF-2) acts like a 'competence' factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serum-free conditioned culture medium. GDGF-2 is resistant to heat (100 degrees C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGF alpha, TGF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 x 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF beta antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha 2-macroglobulin (alpha 2M), which is known to bind TGF beta; however, immunoprecipitation of alpha 2M did not deplete TGF beta or GDGF-2 activity. Further, neither GDGF-2 or TGF beta can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.

Biological Assay↗
PubMed1993

Hospital staff reactions to accounts by survivors of childhood abuse.

Staff reactions to hospitalized patients' accounts of their abuse were found to resemble some symptoms of post-traumatic stress disorder (PTSD). These reactions are described, their implications for a better understanding of the interpersonal effects of trauma are discussed, and interventions to maintain the effectiveness of the therapeutic milieu are suggested.

Adaptation, Psychological↗
PubMed1992

A comparison of the currency of secondary information sources in the biochemical literature. II. MEDLINE online and on CD-ROM.

The currency of selected versions of the MEDLINE database on CD-ROM and in online format was studied. The arrival of issues from 72 monthly journals and nine weekly titles during a 3-month period was monitored and their appearance in the various MEDLINE formats were recorded. Availability data for MEDLINE published by the National Library of Medicine were used as a baseline and subsequent distribution delays for the different MEDLINE formats were calculated. The study suggests a delay of approximately 2 weeks for indexing and mounting the MEDLINE file on to host computers for online access. A delay of 6-8 weeks was calculated for the MEDLINE data to be encoded on to compact disk, shipped and posted to the library customer. The currency of the MEDLINE database formats was compared with a weekly current-awareness service, CURRENT CONTENTS ON DISKETTE LIFE SCIENCES (CCOD). The majority of papers were indexed and distributed in CCOD within 10 weeks of publication date. Most papers appeared within 15 weeks in the online MEDLINE format and within 20 weeks in CD-ROM MEDLINE. The primary journal arrived at the library site within 5 weeks in most cases.

CD-ROM↗
PubMed1991

A comparison of the currency of secondary information sources in the biomedical literature. I. Weekly current awareness services.

Three biomedical current awareness products which are distributed on floppy disc, were studied over a period of 3 months. They were monitored for the speed with which primary journal issues were indexed, and for the coverage of the journals which were selected for study. Data on issues of 72 monthly journals (38 North American and 34 European) and nine weekly titles (four from North America and five from Europe) were recorded. The time to elapse between publication date and arrival of hard copy in the British libraries and appearance in secondary sources varied enormously. The findings suggest that monthly journal issues appear in the secondary sources on average 6-7 weeks after publication date and weekly journals appear after approximately 4 weeks. Indexing of North American monthly journals is 1 week ahead of European titles in the two products from the US, but the European service indexed European publications sooner. One product, Current Contents on Diskette, contained all issues included in the 3-month period covered by the study, but Reference Update omitted 9.1% of the issues and Medical Science Weekly 1.6%, including leading biomedical journals.

Abstracting and Indexing↗
PubMed1990

The clinical 'end user'--a neglected resource in patient care?

In both medicine and science it is difficult to keep up to date with the large and rapidly increasing literature. Whilst a medical academic is likely to use electronic bibliographic databases for his research, he is less likely to use them in clinical practice. Although the potential advantages of an online search in a clinical situation have been appreciated for some time, in the UK it remains an under-utilised resource. In a pilot end user training project, 9 preclinical and 4 clinical academic staff were taught Data-Star command language together with methods of searching the Medline database. In the 12 weeks follow-up period only 1 of the 4 clinical staff had used online searches to help solve clinical problems. To maximise the use of bibliographic database searches in clinical practice, future end user training projects should aim to overcome the problems of the inaccessibility of computers and their command languages.

Adult↗
PubMed1989

The questionnaire--a quality control method for online searching?

The quality of the online search service in a university medical school/large hospital library was investigated by user survey. The use of a questionnaire as a tool to measure quality was assessed. The survey indicated that most users were satisfied with the quality of the service. User ratings for all search performance criteria were high and most readers found online searches to be good value for money. Information about user groups, frequency of searching, search applications, pre-search work and user expectations was obtained. The most important aspect of a computer search to the reader was 'savings in your time'. Three types of user were described based on search output analyses and reading levels. Some areas where the existing service could be improved were identified. The introduction of a standard questionnaire for monitoring the quality of computer searches and other library services was proposed.

Consumer Behavior↗
PubMed1989

MEDLINE-for-Medics: an outline searching course for health-care professionals.

An online searching course for health professionals was initiated at a university medical school. A group of end users were taught to search the MEDLINE database on DATA-STAR and their subsequent online progress was monitored. The preliminary survey by questionnaire, pretraining interviews, the tutorial programme, course evaluation and end-user feedback were described. Logsheets showed that most end-user searches were for research purposes, although some were for patient care. The effects of CD-ROM on online end-user searching were discussed and it was suggested that direct online access to information might be a more appropriate medium for busy clinical and academic staff.

Academic Medical Centers↗
PubMed1985

Endoscopic ureteropyelostomy: opening the obliterated ureteropelvic junction with nephroscopy and flexible ureteropyeloscopy.

Endoscopic re-establishment of a totally obstructed ureteropelvic junction was accomplished in 2 patients. The combined use of rigid percutaneous nephroscopy and flexible ureteropyeloscopy provided full visualization of the obliterated segment, and allowed accurate electroincision into the ureter and placement of a wire and catheter into the lumen. This technique demonstrates the potential therapeutic applications of flexible ureteropyeloscopy and should be considered for the initial treatment of the obliterated ureteropelvic junction.

Aged↗
PubMed1981

Biologic properties of iscador: a Viscum album preparation I. Hyperplasia of the thymic cortex and accelerated regeneration of hematopoietic cells following X-irradiation.

We demonstrated that a preparation of Viscum album (Iscador) has unusual effects on the immune system. In CD-1 mice, the mean weight of the thymus increased from 45 to 85 mg. following six daily intraperitoneal injections of 2.5 mg. of Iscador. The thymic trophic influence was maintained over a long period of time. Sprague-Dawley rats treated with Iscador for 16 weeks showed an increase of 78% in thymic weight over controls. The increased weight of the thymus was due to increased proliferation of cortical thymocytes. The number of blast cells in the outer cortex of the thymus increased within 2 days of the first injection and remained at high levels. Thymocytes of Iscador-treated animals were 29 times more responsive to Concanavalin A than those of untreated controls. Iscador increased the amount of antibody produced by animals injected with sheep red blood cells if it was injected after the antigen. Iscador also had effects on hematopoietic cells. It accelerated the recovery of hematopoietic tissue in bone marrow and spleens of irradiated animals. We believe that V. album preparations may be valuable reagents for perturbing thymus function.

Animals↗