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D Ward

Publications and source records attributed to D Ward.

At least 37 records · Page 2Linked 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↗
PubMed1994

Labor and cost in AIDS family caregiving.

When the costs of care for persons with AIDS are calculated, the assumption is made that the care provided by the family or other social support network members is a social and not also an economic contribution. This article challenges the flawed assumption of "free" care, focuses on the labor and economic dimensions of family home care, and presents a calculation of the dollar value of this care for persons with AIDS (PWAs). The sample consisted of 53 persons, each of whom identified themselves as the primary caregiver for a PWA. Study findings revealed caregiver estimates of 5 hours a week of housework performed specifically for the PWA. Caregivers spent an average of 8.5 hours a day performing personal care tasks for the PWA. The three most common activities were providing companionship, running errands, and performing food/meal-related activities. Gender comparisons revealed that women performed more hours of housework than men but that both provided similar types of personal care for similar numbers of hours. Using a market valuation method, the value of a day's caregiving work was estimated to be $43.78. The annual value of unpaid care, including housework, for one PWA was calculated to be $25,857.88. These data document a significant economic contribution by families and friends caring for PWAs. This subsidy must be included as part of the policy and planning work essential to addressing the AIDS epidemic.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Genomic structure and chromosomal mapping of the human CD22 gene.

The human CD22 gene is expressed specifically in B lymphocytes and likely has an important function in cell-cell interactions. A nearly full length human CD22 cDNA clone was used to isolate genomic clones that span the CD22 gene. The CD22 gene is spread over 22 kb of DNA and is composed of 15 exons. The first exon contains the major transcriptional start sites. The translation initiation codon is located in exon 3, which also encodes a portion of the signal peptide. Exons 4 to 10 encode the seven Ig domains of CD22, exon 11 encodes the transmembrane domain, exons 12 to 15 encode the intracytoplasmic domain of CD22, and exon 15 also contains the 3' untranslated region. A minor form of CD22 mRNA likely results from splicing of exon 5 to exon 8, skipping exons 6 and 7. A 4.6-kb XbaI fragment of the CD22 gene was used to map the chromosomal location of CD22 by fluorescence in situ hybridization. The hybridization locus was identified by combining fluorescent images of the probe with the chromosomal banding pattern generated by an Alu probe. The results demonstrate that CD22 is located within the band region q13.1 of chromosome 19. Two closely clustered major transcription start sites and several minor start sites were mapped by primer extension. Similarly to many other lymphoid-specific genes, the CD22 promoter lacks an obvious TATA box. Approximately 4 kb of DNA 5' of the transcription start sites were sequenced and found to contain multiple Alu elements. Potential binding sites for the transcriptional factors NF-kappa B, AP-1, and Oct-2 are located within 300 bp 5' of the major transcription start sites. A 400-bp fragment (bp -339 through +71) of the CD22 promoter region was subcloned into a pGEM-chloramphenicol acetyltransferase vector and after transfection into B and T cells was found to be active in both B and T cells. Further studies of the CD22 gene should lead to a greater understanding of the expression of CD22 during B cell development and differentiation.

Antigens, CD↗
PubMed1993

Autonomic correlates of late infarct artery patency after first myocardial infarction.

Occlusion of the infarct-related artery has recently been associated with an increased risk of sudden death, particularly in patients with poor left ventricular function. Depressed heart rate variability (HRV) also identifies postinfarction patients at an increased risk of sudden death. The correlation between infarct artery patency, left ventricular function, and HRV was therefore examined in 186 survivors of a first myocardial infarction. Predischarge coronary angiography and Holter monitoring were carried out in 186 patients with a first acute myocardial infarction. Coronary angiography was performed because of abnormal predischarge exercise test findings. Mean age (56 +/- 9 years) and the proportions of type and site of infarction did not differ between patients with occluded or patent arteries or between patients who did or did not undergo coronary angiography. The mean left ventricular ejection fraction (EF) was 55 +/- 15% in patients with patent and 49 +/- 14% in those with occluded infarct arteries (p < 0.001), and the EF was < 40% in 17% and 28% of the respective groups (p < 0.05). HRV was < 20 U in 7 (18%) of the 39 patients with an EF < 40% but in only 7 (5%) of the 147 patients with an EF > 40% (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Laryngo-tracheo-oesophageal cleft: a plea for early diagnosis.

Congenital laryngeal clefts are rare. This paper reports on the management of 3 patients with Type 2 laryngo-tracheo-oesophageal cleft. One patient died following tracheopexy after successful cleft closure. Prognosis is related to the presence of other major anomalies, the early diagnosis of the lesion and institution of appropriate respiratory and nutritional care prior to correction of the defect. A greater awareness of the condition combined with aggressive diagnostic endoscopy should result in early diagnosis and improved survival.

Abnormalities, Multiple↗
PubMed1992

The physician and employee judgment system: reliability and validity of a hospital quality measurement method.

Hospitals engaged in quality improvement must measure and assess customer perceptions of hospital services. Hospital customers are either "external"--patients, their families, and payers--or "internal"--physicians and hospital employees. This article describes two measurement systems designed to gather information from internal customers about the quality of hospital services. One system targets physicians, the other hospital employees. This article describes how these systems were developed and pilot-tested, and how the results were analyzed. Analysis of results showed the systems to be reliable, valid, and representative. The article also addresses limitations of the research (focus on general acute-care hospitals), interesting findings and implications (the correlation between physicians' and employees' ratings of hospital quality), and uses of these measures in corporate-level and hospital-level quality improvement (measuring trends and identifying specific opportunities for improvement).

Attitude of Health Personnel↗