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Biomedical subjects

D Ward

Publications and source records attributed to D Ward.

At least 91 records · Page 5Linked to original sources
PubMed1994

IRE-bubble PCR: a rapid method for efficient and representative amplification of human genomic DNA sequences from complex sources.

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and lambda phage and result in greater complexity and representation than standard inter-IRE PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.

Animals↗
PubMed1994

Large human YACs constructed in a rad52 strain show a reduced rate of chimerism.

Current YAC libraries are plagued by a high frequency of chimeras--that is, clones containing fragments from multiple genomic regions. Chimeras are thought to arise largely through recombination in the yeast host cell. If so, the use of recombination-deficient yeast strains, such as rad52 mutants, might be expected to alleviate the problem. Here, we report the construction of megabase-sized human YACs in the rad52 strain MHY5201 and the determination of their rate of chimerism by fluorescence in situ hybridization analysis. Examination of 48 YACs showed a rate of chimerism of at most 8%, whereas YACs constructed in the wildtype host AB1380 showed a rate of about 50%. These results show that it is possible to significantly decrease the rate of YAC chimerism through the use of appropriate yeast host strains.

Base Sequence↗
PubMed1994

Organization of the human keratin type II gene cluster at 12q13.

Keratin proteins constitute intermediate filaments and are the major differentiation products of mammalian epithelial cells. The epithelial keratins are classified into two groups, type I and type II, and one member of each group is expressed in a given epithelial cell differentiation stage. Mutations in type I and type II keratin genes have now been implicated in three different human genetic disorders, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, and epidermolytic palmoplantar keratoderma. Members of the type I keratins are mapped to human chromosome 17, and the type II keratin genes are mapped to chromosome 12. To understand the organization of the type II keratin genes on chromosome 12, we isolated several yeast artificial chromosomes carrying these keratin genes and examined them in detail. We show that eight already known type II keratin genes are located in a cluster at 12q13, and their relative organization reflects their evolutionary relationship. We also determined that a type I keratin gene, KRT18, is located next to its partner, KRT8, in this cluster. Careful examination of the cluster also revealed that there may be a number of additional keratin genes at this locus that have not been described previously.

Amino Acid Sequence↗
PubMed1994

Neutrophil dysfunction in end-stage renal failure: reduced response to priming by C5a.

We studied the effect of C5a pretreatment on phosphatidyl-inositol-4,5-bisphosphate (PIP2) hydrolysis and on the increase in peak and resting cytosolic calcium levels induced by C5a (0.1 and 10 nM) and/or N-formyl hexapeptide (FLPEP; 10 nM) in neutrophils isolated from patients with end-stage renal failure (ESRF) and those from healthy controls. We also investigated superoxide anion production under the same conditions using the fluorescent para-hydroxyphenylacetic acid assay. The hydrolysis of PIP2 induced by C5a or FLPEP alone was similar in neutrophils from patients with ESRF and in control cells. Likewise, pretreatment of patients' neutrophils with C5a prior to FLPEP did not affect hydrolysis or the increase in cytosolic calcium concentration as shown previously for control neutrophils. Resting calcium levels in both ESRF and control neutrophils, however, were significantly increased after priming with low C5a concentrations. After priming with low C5a, prior to FLPEP, there was also a significant increase in superoxide production. This increase was significantly lower in cells from uremic patients than in those from healthy controls. Our data suggest that priming-induced superoxide production in neutrophils is reduced in patients with ESRF.

Amino Acid Sequence↗
PubMed1994

Simultaneous determination of felbamate, primidone, phenobarbital, carbamazepine, two carbamazepine metabolites, phenytoin, and one phenytoin metabolite in human plasma by high-performance liquid chromatography.

An isocratic liquid-chromatographic method employing one extraction step has been developed for the quantitation of five drugs and three metabolites in human plasma. The method uses 0.100-ml aliquots of human plasma and two internal standards. Chromatographic conditions include a 4.6 mm x 150 mm Spherisorb ODS2, 3 microns a high-performance liquid chromatography, (HPLC) column, a phosphate buffer-acetonitrile-methanol (700:160:140) mobile phase, and ultraviolet (UV) absorbance detection at 210 nm. Analytes and linear quantitation ranges (microgram/ml) were felbamate (FBM) 0.391-200; primidone (PRIM), 0.098-100; phenobarbital (PHENO), 0.195-100; carbamazepine (CBZ), 0.195-100; phenytoin (PHT), 0.195-200. For CBZ-transdiol (CBZ-TR) CBZ-epoxide (CBZ-EP), and the PHT metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), the range was 0.049-25.0 micrograms/ml. Ethosuximide, methsuximide, 2-methyl-2-phenyl-succinimide (methsuximide metabolite), 2-ethyl-2-phenyl malonamide (PRIM metabolite, 5-ethyl-5-(4-hydroxyphenyl)-barbituric acid (PHENO metabolite), and mephenytoin do not interfere with quantitation of the above compounds.

Anticonvulsants↗
PubMed1994

Temporal influences on the prediction of postinfarction mortality by heart rate variability: a comparison with the left ventricular ejection fraction.

OBJECTIVE: To examine the influence of the duration of follow up on the values of heart rate variability (HRV) and the left ventricular ejection fraction (LVEF) for predicting mortality after infarction. BACKGROUND: HRV is an index of autonomic balance that identifies patients at a high risk of arrhythmic events. The index is most depressed during the first few weeks after myocardial infarction whereas left ventricular function tends to deteriorate with time. HYPOTHESIS: The value of depressed HRV measured before discharge from hospital for predicting mortality after infarction should decline with time. METHODS: The HRV and the LVEF were assessed in 433 survivors of a first acute myocardial infarction: HRV < 20 units and LVEF < 40% were taken as cut off points. Kaplan-Meier survival functions for total cardiac mortality and sudden cardiac death were calculated for the whole five year follow up period and for different intervening periods. RESULTS: During follow up of four weeks to five years there were 46 (10.6%) deaths and 15 (3.5%) patients died suddenly. Within the whole follow up period, HRV < 20 units and LVEF < 40% were both strongly associated with total cardiac mortality (p < 0.0001), but HRV was an independent predictor of total cardiac mortality only during the first six months of follow up. There were no deaths predicted by HRV < 20 units after the first year of follow up whereas LVEF < 40% had a sensitivity of 43% and a positive predictive accuracy of 9% for predicting death during this period. HRV < 20 units was better than LVEF < 40% in predicting sudden deaths during the first year of follow up but was an independent predictor only of those sudden deaths occurring within six months of infarction. CONCLUSIONS: The duration of follow up affects the prediction of sudden death and total cardiac mortality from HRV. Reduced HRV as measured before discharge from hospital does not seem to retain independent prognostic value after six months of follow up. These findings have potential implications for the serial evaluation of HRV and for the prevention of sudden death after myocardial infarction.

Death, Sudden, Cardiac↗