Biomedical subjects

D C Ward

Publications and source records attributed to D C Ward.

At least 199 records · Page 11Linked to original sources

PubMed1988

Inhibition of lipopolysaccharide activation of 7OZ/3 cells by anti-lipopolysaccharide antibodies.

We have investigated the ability of mAb against LPS to inhibit LPS-induced activation of 7OZ/3 pre-B cells. The fine specificity and relative affinity of these mAb for lipid A and LPS were also determined. We found that antibodies inhibited only the activity of glycolipids which they bound with relatively high affinity. However, two high affinity antibodies binding to non-lipid A epitopes did not block cellular activation. Some, but not all, relatively high affinity antibodies binding to the lipid A region of the LPS molecule inhibited biologic activity. The inhibitory antibodies bound to at least two distinct epitopes within the lipid A region. These data suggest that LPS interacts with 7OZ/3 cells in a highly specific fashion.

Animals↗

PubMed1988

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzyme-labeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laser-scanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

Chromosomes, Human↗

PubMed1988

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes.

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

Biotin↗

PubMed1988

Replication of rat virus in neonatal calvaria in culture.

Rat Virus, a parvovirus of rodents that produces a variety of developmental disturbances of the head and face in neonatal animals, was examined for its ability to replicate in neonatal calvariae in vitro. The bones were isolated and infected with RV within 1 d of birth and cultured for up to 7 d. Virus from the bones and supernatant was titered, and the cellular location of replication determined using in situ hybridization. The virus readily replicated in the isolated bony tissues, reaching titers of nearly 10(7) plaque-forming units/ml. Using viral and complementary strand-specific probes, replication sites were located in the sutures and calvarial bones, as well as in cartilages thought to be part of the neurocranium. Results suggest that the virus localizes and replicates in cells necessary for the normal growth and development of the skull.

Animals↗

PubMed1988

Rapid detection of human chromosome 21 aberrations by in situ hybridization.

Plasmid clones containing up to 94 kilobases of single-copy DNA from band q22.3 of chromosome 21 and a complete pool of insert DNA from a chromosome 21 recombinant library have been used to rapidly detect numerical and structural aberrations of chromosome 21 by in situ hybridization in both metaphase and interphase cells. A trisomic karyotype, diagnostic of Down syndrome, is readily detected in nonmitotic cells because the majority of their nuclei exhibit three discrete foci of hybridization, in contrast to normal diploid cells, which show two foci. Chromosomal translocations involving chromosome 21 sequences were also detected with these probes, and the intranuclear location of 21q22.3 DNA sequences in "normal" human brain neurons was established with the plasmid DNA probe set. These results suggest that chromosome 21-specific probes may have utility in clinical diagnostics, especially by facilitating the direct analysis of interphase cells.

Brain Chemistry↗

PubMed1988

Monoclonal antibodies to salmonella lipopolysaccharide: functional analysis of anti-lipid A antibodies.

We have produced monoclonal antibodies (MoAb) to the Rb core and lipid A regions of Salmonella lipopolysaccharide (LPS) and have assessed their ability to inhibit LPS-mediated mitogenic responses in vitro, and to protect against LPS toxicity and lethal Salmonella infection in vivo. Monoclonal antibodies RC-8 and RC-16 were specific for LPS Rb core determinants, and MoAb LA-1, LA-2, LA-3, LA-4 and LA-5 were specific for lipid A. Anti-lipid A MoAb LA-2, LA-3 and LA-5 were found to abrogate mitogenic responses of C3H/HeN spleen cells to smooth S. typhimurium LPS (S LPS) and to rough S. minnesota R595 LPS (Re LPS). Monoclonal antibody LA-5 was effective in extending the median length of survival of C3H/HeN mice challenged with a lethal dose of either S LPS or Re LPS. Antibody LA-2 could extend the median length of survival of C3H/HeJ mice challenged with Re LPS but not with S LPS, and failed to extend significantly the length of survival of S LPS-challenged C3H/HeN and DBA/2 mice. Neither 20 micrograms of anti-Rb core or anti-lipid A MoAb nor 200 micrograms of anti-lipid A MoAb were able to protect C3H/HeN or BALB/c mice, respectively, against lethal infection with S. typhimurium SR-11. These results suggest that the importance of anti-lipid A antibodies in host defence may lie more in their ability to neutralize pathological effects of LPS, than in their ability to protect against bacterial infection.

Animals↗

PubMed1987

Highly recurring sequence elements identified in eukaryotic DNAs by computer analysis are often homologous to regulatory sequences or protein binding sites.

We have used computer assisted dot matrix and oligonucleotide frequency analyses to identify highly recurring sequence elements of 7-11 base pairs in eukaryotic genes and viral DNAs. Such elements are found much more frequently than expected, often with an average spacing of a few hundred base pairs. Furthermore, the most abundant repetitive elements observed in the ovalbumin locus, the beta-globin gene cluster, the metallothionein gene and the viral genomes of SV40, polyoma, Herpes simplex-1 and Mouse Mammary Tumor Virus were sequences shown previously to be protein binding sites or sequences important for regulating gene expression. These sequences were present in both exons and introns as well as promoter regions. These observations suggest that such sequences are often highly overrepresented within the specific gene segments with which they are associated. Computer analysis of other genetic units, including viral genomes and oncogenes, has identified a number of highly recurring sequence elements that could serve similar regulatory or protein-binding functions. A model for the role of such reiterated sequence elements in DNA organization and function is presented.

Animals↗

PubMed1987

Gastric emptying and clinical outcome after Roux-en-Y diversion.

The results of 48 Roux-en-Y (RY) diversion procedures are reported: 41 were performed as secondary procedures and 7 as part of a primary operation for peptic ulcer. There was no operative mortality, but four patients developed temporary fistulae in the postoperative period and three patients required reoperation. Good clinical results were found when RY diversion was performed as a primary procedure or when the indication for operation was peptic ulceration. The overall results, however, were poor: 24 patients (50 per cent) felt that they had not benefited and 32 patients (67 per cent) remained in Visick grades III or IV. The main cause of failure was gastric stasis, especially of solid food. Gastric emptying studies were carried out after RY diversion in 22 patients, most of whom had symptoms of stasis. Emptying of liquids was found to be normal in most patients, but emptying of solids was delayed, the median t 1/2 for solids being 160 (75-370) min compared with 67 (50-85) min in DU patients. Bilious vomiting improved significantly after RY diversion, but 18 patients (38 per cent) complained of vomiting food and 32 patients (67 per cent) experienced postprandial distress or pain. Loss of the antral mill, vagotomy of the gastric remnant and, perhaps, resistance to gastric emptying by the Roux loop itself may together explain the delay in gastric emptying of solids after RY diversion.

Adult↗

PubMed1987

Detection of guinea pig cytomegalovirus nucleic acids in cultured cells with biotin-labelled hybridization probes.

Biotin labelled hybridization probes prepared from recombinant plasmids containing segments of the guinea pig cytomegalovirus (GPCMV) genome were used to detect GPCMV nucleic acids in guinea pig cells by in situ hybridization. The time course of GPCMV infection was assessed in two cultured cell types, guinea pig embryo (GPE) cells and 104C1 cells, a transformed and cloned guinea pig cell line. Detection of GPCMV nucleic acids was accomplished in both cell types with individual GPCMV DNA fragments and with mixtures of GPCMV DNA fragments. When compared to other established methods of GPCMV detection, the method of in situ hybridization enabled the detection of a higher percentage of positive cells early during the course of the infection. In addition, differences in the replication cycle of GPCMV in the two cultured cell lines could be demonstrated. These findings will facilitate future studies of GPCMV tissue tropism in vivo.

Animals↗

PubMed1986

Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose.

We have developed a general procedure for the rapid and efficient enrichment of specific DNA, RNA or cDNA sequences. Biotinylated DNA or RNA is used as a hybridization probe in solution, avidin is then added to label both the probe and hybrid molecules, and the hybridization mixture chromatographed over cupric-iminodiacetic acid agarose beads. Avidin-probe and avidin-hybrid molecules are selectively retained on the column; non-hybridized sequences are contained in the flow-through fraction. Sequences retained on the column are recovered in high yield by the addition of ethylenediamine tetracetic acid in the buffer. The method can be used in both subtractive enrichment and positive selection protocols. Here we report its application to the isolation of Neisseria gonorrhoeae specific genomic DNA clones and the purification of a cDNA subpopulation representing mRNA sequences that are over-expressed in murine Friend cells after dimethylsulfoxide induction.

Avidin↗

PubMed1986

Effect of duodenal ulcer surgery and enterogastric reflux on Campylobacter pyloridis.

To assess the effect of duodenal ulcer surgery on Campylobacter pyloridis gastric biopsies were done and fasting bile acid concentrations in gastric aspirates were measured in 35 patients with active duodenal ulceration and 54 who had undergone surgery at some time. Biopsy specimens were assessed blind for the presence of C pyloridis and scored for severity of reflux gastritis by the use of a histological grading system. Among patients who had undergone highly selective vagotomy the proportion who were C pyloridis-positive was similar to that in the unoperated group, but among those who had undergone Billroth I partial gastrectomy, Billroth II partial gastrectomy, or truncal vagotomy and gastroenterostomy it was significantly lower (p less than 0.001). The absence of C pyloridis correlated strongly (p less than 0.001) with high reflux scores and increased bile acid concentrations in the stomach. Reflux scores and bile acid concentrations were significantly higher (p less than 0.01) after Billroth I and Billroth II partial gastrectomies and truncal vagotomy and gastroenterostomy than in the active duodenal ulcer or highly selective vagotomy groups. There was a highly significant correlation (p less than 0.001) between reflux scores and bile acid concentrations. These results suggest that reflux may disrupt mucus and thus cause the death of campylobacters that live beneath it. They also suggest that reflux may produce a reflux-specific gastritis. Highly selective vagotomy may protect against these changes in the gastric mucosa.

Adult↗

PubMed1986

Rapid plasmid library screening using RecA-coated biotinylated probes.

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

Adenosine Triphosphate↗

PubMed1986

Identification and characterization of a protein covalently bound to DNA of minute virus of mice.

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.

Animals↗

PubMed1986

Three splicing patterns are used to excise the small intron common to all minute virus of mice RNAs.

We identified three splicing patterns used to excise the small intron common to all three transcripts encoded by minute virus of mice. Sequence analysis of minute virus of mice-specific cDNAs indicated that two donor and two acceptor splice sites were used: in pattern 1, the most frequent, nucleotide 2280 was spliced to nucleotide 2377; in pattern 2, nucleotides 2317 and 2399 were joined. Oligonucleotide probes, each specific for one of the four possible splice junction sequences, were synthesized and hybridized to viral mRNAs immobilized on nitrocellulose filters. The probes specific for splice patterns 1 and 2 hybridized to all three viral mRNAs, as did a third oligomer specific for a splicing pattern in which nucleotides 2280 and 2399 were joined. The fourth potential splicing pattern, linking nucleotides 2317 and 2377, was not detected. The presence of three splicing patterns in the transcripts designated R2 and R3 would allow the translation of five distinct polypeptides from these two mRNAs.

Base Sequence↗

PubMed1986

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

Avidin↗

PubMed1986

Detection of bovine herpesvirus-specific nucleic acids by in situ hybridization with biotinylated DNA probes.

Biotin-labeled DNA probes for bovine herpesvirus type 1 (BHV-1) were used to detect viral nucleic acids in infected cell cultures and clinical specimens by in situ hybridization. Hybridization signal was detected 2 hours after inoculation in the cytoplasm of infected cells, presumably representing input virus. Hybridization was first detected in the nucleus at 4 hours after inoculation; by 10 hours after inoculation, hybridization signal was detected in both nucleus and cytoplasm of almost 50% of the cells. By 15 hours after inoculation, 95% of the cells were positive. Treatment of specimens with ribonuclease or deoxyribonuclease before hybridization allowed clear differentiation of virus-specified DNA and RNA within infected cells. The BHV-1 nucleic acid sequences were detected in nasal epithelial cells obtained from inoculated calves. Since in situ hybridization provides a rapid technique for the detection of BHV-1-specified nucleic acid sequences, it should facilitate studies on the replication, pathogenesis, and diagnosis of BHV-1 infections.

Animals↗

PubMed1985

Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication.

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogeneous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.

Animals↗

PubMed1985

Use of biotinylated probes for detecting sickle cell anemia.

Earlier, we reported that the 5' end of the normal beta-globin gene (beta) resides on a 1.14-kilobase DNA fragment, whereas the 5' end of the sickle cell gene (beta s) resides on a 1.34-kilobase fragment. In that blot hybridization analysis, we used genomic DNA digested with restricted endonuclease Mst II, and radioactive probes with short half-life. We demonstrate here that, if a biotinylated probe is used instead in a slightly modified procedure, sickle cell anemia can be quickly and directly detected if there is as much as 5 micrograms of total genomic DNA in the sample. This procedure obviates the special precautions necessary when radioactive materials are used.

Anemia, Sickle Cell↗