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Biomedical subjects

D R Smith

Publications and source records attributed to D R Smith.

At least 217 records · Page 12Linked to original sources
PubMed1990

Amplification of large artificial chromosomes.

Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome. We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes. The vector includes a conditional centromere that can be turned on or off by changing the carbon source. Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene. When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold. The selective conditions did not induce obvious rearrangements in any of the clones tested. Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations. Applications of copy number control to various aspects of artificial chromosome analysis are addressed.

Base Sequence↗
PubMed1990

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.

Animals↗
PubMed1990

Evaluation of antiproliferative agents using a cell-culture model.

Chinese hamster ovarian (CHO) cells in culture were used to evaluate the relative antiproliferative potential of drugs. These agents have been used to improve the clinical response after glaucoma filtering surgery. The following drugs were evaluated: 5-fluorouracil (5-FU) as the benchmark, 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUDR), 5'-deoxy-5-fluorouridine (DFUR), bleomycin, and cytarabine (ARA-C). In addition to cell counting, a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to follow growth response. The MTT assay was found to be extremely convenient and an indirect measure of cell activity, offering an alternate or addition to a measure of cell number. All agents tested were shown to inhibit cellular proliferation. Dose-response curves for each agent indicate the following absolute potency: FUDR greater than FUR greater than ARA-C greater than 5-FU = bleomycin greater than DFUR. Besides absolute potency, an evaluation of the effects of equivalent inhibitory concentrations of each drug on growth rate was assessed. Several agents affected the proliferation rate patterns differently. Based on these studies, it is suggested that the in vitro model can identify potential agents through an assessment of their overall activity profile in CHO cells, which includes not only their potency based on dose response, but their onset of activity, duration of effect, and potential for toxicity.

Animals↗
PubMed1990

Bacterial endocarditis of the tricuspid valve after insertion of a central venous catheter.

Central venous cannulation is a commonly used technique in the management of seriously ill patients. In experienced hands, the complication rate is low and primarily includes sepsis, thrombosis and local trauma. For chronic indwelling central venous catheters, thrombus formation within the right atrium has been described, particularly in premature infants, and a recent case has been reported in an adult. Valvular damage in the right heart is recognised and more often diagnosed at autopsy. This report describes the presence of tricuspid vegetations in a diabetic woman, related to the insertion of a central venous cannula.

Catheterization, Central Venous↗
PubMed1990

Fatal right ventricular thrombus secondary to Hickman catheterisation.

Complications arising from central venous cannulation include sepsis, thrombosis and trauma. Total parenteral nutrition via a Hickman catheter is an accepted therapeutic technique. Cases of catheter-related thrombus within the right atrium have been described, primarily in premature infants, although a recent case has been reported in an adult. We describe the presence of a fatal right ventricular thrombus in a female with an infected Hickman catheter.

Adolescent↗
PubMed1990

Paragordius varius.

We describe a 30-year-old man who presented with a tangled knot of worms that he "passed per rectum." The worms were identified as Paragordius varius. This is an uncommon pseudoparasite that is ingested from contaminated food or water. Identifying characteristics and the life cycle of Gordian worms are briefly discussed. Identification of this worm is important to distinguish it from true parasitic worms.

Adult↗
PubMed1989

Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences.

The Columnea latent viroid (CLV) occurs latently in certain Columnea erythrophae plants grown commercially. In potato and tomato, CLV causes potato spindle tuber viroid (PSTV)-like symptoms. Its nucleotide sequence and proposed secondary structure reveal that CLV consists of a single-stranded circular RNA of 370 nucleotides which can assume a rod-like structure with extensive base-pairing characteristic of all known viroids. The electrophoretic mobility of circular CLV under nondenaturing conditions suggests a potential tertiary structure. CLV contains extensive sequence homologies to the PSTV group of viroids but contains a central conserved region identical to that of hop stunt viroid (HSV). CLV also shares some biological properties with each of the two types of viroids. Most probably, CLV is the result of intracellular RNA recombination between an HSV-type and one or more PSTV-type viroids replicating in the same plant.

Base Composition↗
PubMed1989

Neotropical Africanized honey bees have African mitochondrial DNA.

Non-indigenous African honey bees have invaded most of South and Central America in just over 30 years. The genetic composition of this population and the means by which it rapidly colonizes new territory remain controversial. In particular, it has been unclear whether this 'Africanized' population has resulted from interbreeding between African and domestic European bees, or is an essentially pure African population. Also, it has not been known whether this population expanded primarily by female or by male migration. Restriction site mapping of 62 mitochondrial DNAs of African bees from Brazil, Venezuela and Mexico reveals that 97% were of African (Apis mellifera scutellata) type. Although neotropical European apiary populations are rapidly Africanized by mating with neotropical African males, there is little reciprocal gene flow to the neotropical African population through European females. These are the first genetic data to indicate that the neotropical African population could be expanding its range by female migration.

Animals↗
PubMed1989

A 12 megabase restriction map at the cystic fibrosis locus.

We have constructed a physical map of the chromosomal region containing the cystic fibrosis locus using seven DNA markers and pulsed-field gel electrophoresis methods. The map includes cleavage sites for 8 rare-cutting restriction enzymes and spans over 12 megabases (Mb) of DNA, with one unlinked probe covering an additional 5 Mb. To our knowledge, this is the largest segment of human DNA which has been restriction-mapped to date. We can identify thirteen putative HTF islands spaced at intervals of 0.3-3.2 Mb. The region between loci D7S8 and MET, where the CF gene lies, includes 1.4-1.9 Mb of DNA.

Blotting, Southern↗
PubMed1989

Hypoxia-reoxygenation induced increase in cellular Ca2+ in myocytes and perfused hearts: the role of mitochondria.

Reoxygenation of isolated rat cardiac myocytes following a period of hypoxia and substrate deprivation resulted in a 1.5-2-fold increase in the total Ca2+ content which could be inhibited by 1 microM antimycin A or ruthenium red (50% inhibition at 2.5 microM). This increase in Ca2+ content was not accompanied by any release of creatine kinase into the medium. Treatment of reoxygenated cells with digitonin also resulted in an antimycin A-sensitive increase in Ca2+ but this was inhibited by a lower concentration of ruthenium red (50% inhibition at 0.25 microM) and was associated with a substantial release of creatine kinase from the cells. It is concluded that the reoxygenation-stimulated increase in Ca2+ is dependent on functioning mitochondria and does not occur as a result of physical damage to the sarcolemma. In a parallel series of experiments, the effects of antimycin A and ruthenium red on the reoxygenation-induced increase in Ca2+ and release of cytosolic contents in the perfused heart (the oxygen paradox) were also investigated. As was observed with the isolated myocytes, each of the compounds significantly reduced the magnitude of the Ca2+ increase that occurred on reoxygenation: the compounds also reduced the extent of release of cell contents in the perfused heart. The implications of these results for the series of events occurring on reoxygenation of the hypoxic myocardium are discussed.

Animals↗
PubMed1989

The v-sea oncogene of avian erythroblastosis retrovirus S13: another member of the protein-tyrosine kinase gene family.

The cloning and sequencing of the oncogene of the avian erythroblastosis virus S13 is described. The oncogene, termed v-sea, was found to be another member of the protein-tyrosine kinase gene family. The oncogene was fused in frame with the retrovirus S13 envelope gene, thus generating a fusion protein with a structure resembling that of a growth factor receptor. Sequence comparisons revealed that the v-sea gene was most closely related to the insulin receptor family of protein-tyrosine kinases, the greatest similarity being with the human MET oncogene.

Alpharetrovirus↗
PubMed1989

The Xenopus laevis ribosomal gene terminator contains sequences that both enhance and repress ribosomal transcription.

A DNA segment approximately 200 base pairs upstream of the Xenopus laevis ribosomal promoter acts both as an upstream promoter element that augments transcription and as a transcription terminator. It is, however, unclear to what extent these two activities are related. A segment of the X. laevis ribosomal DNA, containing the terminator and the upstream promoter element, was subjected to point mutation, and the effects of the resulting mutations were investigated by oocyte microinjection. Analysis of 26 point mutants revealed not only sequences that augment 40S transcription but also those that repress it. The sequences that augmented transcription lay within the T3 homology box and also near the site of 3'-end formation. These sequences also played a role in termination. The sequences that repressed transcription lay within the G+C-rich DNA flanking the T3 box. It can be concluded that termination is probably essential but may not be sufficient for the activity of the upstream promoter element.

Animals↗
PubMed1989

Oxygen and reperfusion damage: an overview.

The ischaemic myocardium shows a number of distinct oxygen dependent responses to reperfusion. In the case of myocardial stunning and reperfusion arrhythmias there appears to be a beneficial effect of scavenging radicals in the extracellular space. This result is supported by the finding that free radicals can be detected extracellularly after reperfusion. The source of these oxidants and site of action is as yet unclear. In contrast hypoxic myocytes shown an oxygen dependent Ca2+ uptake on reoxygenation which is not affected by externally applied antioxidants. This Ca2+ uptake may in turn lead to the cell lysis characteristic of the oxygen paradox in the perfused heart. As yet there is no compelling evidence to suggest that this aspect of reperfusion damage is due to oxidative stress. It appears more likely that mitochondrial electron transport and ion movement play a central role. In the open chested dog model of ischaemia reperfusion, in which the volume of infarcted tissue is measured, considerable evidence suggests that oxidants derived from activated neutrophils contribute to cell death. This is not however the sole mechanism for cell damage in this model since an inhibitor of mitochondrial Ca2+ uptake, ruthenium red, can improve recovery after reperfusion. We conclude that a number of mechanisms may contribute to the observed oxygen dependent dysfunctions which occur on reperfusion of ischaemic tissue.

Animals↗
PubMed1989

Cystic fibrosis: diagnostic testing and the search for the gene.

Cystic fibrosis (CF) is a lethal genetic disorder inherited as an autosomal recessive at a frequency of about 1/2000 in Caucasian populations. A DNA marker genetically linked to CF was identified through a collaborative effort by random screening with a collection of RFLP markers on a set of CF families. The marker (CRI-L917) was mapped to chromosome 7. Construction of a genetic linkage map spanning the entire chromosome has led to the identification of a subset of 11 markers close to and flanking the CF locus. Using techniques of pulsed-field gel electrophoresis, which allow very large DNA fragments to be separated, we used seven probes to generate a long-range restriction map covering 12 million base pairs surrounding the CF locus. Information from the map is being used to isolate new probes closer to the CF gene. Methods being developed will allow candidate genes to be tested for their ability to correct defects in ion transport in cultured CF cells.

Chromosome Mapping↗
PubMed1989

Digital signal processing of ultrasonic signals for blood flow measurement.

This paper describes the application of advanced digital signal processing techniques in a noninvasive ultrasonic Doppler flowmeter used to measure extracorporeal blood flow during open heart surgery. The use of ultrasound to determine blood flow rates started in the 1950's with much of this work focused on measurement of blood flow in a patient by a variety of means, both invasive and noninvasive. Although the use of ultrasonics to measure blood flow is not in itself a new concept, the application of advanced digital signal processing techniques in the system being described has resulted in a unique product for accurate and reliable blood flow measurements. The flowmeter system is intended for use with a centrifugal blood pump and will measure blood flow in the flexible tubing used during surgery to an accuracy of better than +/- 10%. This paper describes the development and implementation of the digital flowmeter and its application to flow measurement.

Blood Flow Velocity↗
PubMed1989

Oral complications associated with neonatal oral tracheal intubation: a critical review.

This paper summarizes and evaluates the oral complications associated with orotracheal intubation in neonates. The palatal defect resulting from orotracheal intubation is best described as palatal grooving, rather than clefting since no oral nasal communication has been demonstrated. Palatal grooving may be caused by the inhibition of the molding tongue forces on the lateral palatine shelves. The incidence of palatal grooving increases with duration of intubation and reportedly resolves following extubation. However, posterior cross-bites, high palatal vaults, and poor speech intelligibility have been reported in children who previously have been intubated. Impingement of an orotracheal tube on the alveolus rather than on the palate may cause alveolar grooving which can cause dilaceration of primary teeth. Bilateral linear enamel hypoplasia in premature neonates is caused by an interruption in amelogenesis from intrauterine disturbances. However, gross unilateral incisal enamel hypoplasia in children who have been intubated is probably due to traumatic intubation. Avoiding excessive pressure on the maxillary alveolus during intubation is suggested. An appliance is available which secures oral tubes and protects the palate and alveolus.

Alveolar Process↗