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Biomedical subjects

M Beggs

Publications and source records attributed to M Beggs.

15 recordsLinked to original sources
PubMed1999

The High Throughput Screening Infrastructure: The Right Tools for the Task.

HTS is a key component of pharmaceutical lead identification process. Over recent years, the pharmaceutical industry has experienced significant increases in the throughput capabilities of its HTS functions. In those companies where HTS has been effectively deployed, it is now possible to screen the entire corporate compound collection against a pharmacological target within a timescale of several weeks to a few months. This capability has been realized, not as a result of the purchase of any one particular piece of hardware, but rather through the development of a truly effective HTS infrastructure that matches the needs of the parent organization. Central to this is the need to understand how to effectively combine the use of the different types of hardware available to the HTS specialist. The use of both modular workstations and single-arm robotic systems have underpinned most HTS groups operations. Recent advances in the field of multiple-arm robotic systems and dedicated automation systems offer even further potential for increasing productivity. This article describes our experience with the use of a dedicated automation system for HTS applications.

Journal Article↗
PubMed1999

Stacker Modules Used in a High-Capacity Robotics System for High Throughput Screening Compound Replication.

High throughput screening is now established as a key component of the pharmaceutical lead identification process in many pharmaceutical companies. Over recent years, thanks to advances in assay technology, process automation, and logistics control, the throughput capacity of HTS groups has increased significantly. It is now entirely possible to screen corporate compound collections against an individual pharmacological target within a timescale of several weeks. Despite these improvements, many HTS groups find that their capacity is limited by the rate at which they can provide test compounds in a "screen-ready" format. This limitation is usually imposed by the capacity and productivity of the single-armed robotic systems utilized. We have recently constructed a robotic system aimed at overcoming this particular problem. This system uses purpose-built microplate stacker units that provide high-capacity microplate storage and, importantly, provide an easy and fast interface between the robotic system and the human operators. This paper describes this automation project and the benefits that have resulted from its deployment.

Journal Article↗
PubMed1997

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110.

SETTING: Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES: To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN: The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS: The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION: In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Base Sequence↗
PubMed1995

Bioluminescence method to evaluate antimicrobial agents against Mycobacterium avium.

Plasmid pLUC10, carrying the firefly luciferase gene, was transformed by electroporation into Mycobacterium avium A5. Bioluminescence production by strain A5(pLUC10), as measured in a microdilution plate luminometer, was approximately 1 relative light unit per 2 x 10(6) viable bacilli, whereas it was 0.0005 relative light unit for an equal number of parental cells. The susceptibility of strain A5(pLUC10) to eight concentrations of each of eight antimicrobial agents was evaluated by the luciferase microplate assay in parallel with a conventional broth macrodilution method with antimicrobial agents. Decreases in bioluminescence to levels that were < or = 10% of those of drug-free controls were observed in microplate wells containing inhibitory concentrations of drugs in as few as 3 days. The close correlation of these inhibitory concentrations with the MICs determined by a conventional broth macrodilution method suggests that the luciferase microplate method may offer a convenient and reliable means of evaluating the in vitro activities of antimicrobial agents against the M. avium complex.

Anti-Bacterial Agents↗
PubMed1990

Interleukin-1 beta inhibits glucokinase activity in clonal HIT-T15 beta-cells.

Interleukin-1 beta (IL-1 beta) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. In the present study we have investigated the effects of IL-1 beta on glucose metabolism in clonal HIT-T15 beta cells. In the short-term (1 h), 25 U/ml IL-1 beta significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL-1 beta inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6-phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL-1 beta may be due to impaired glucokinase activity.

Adenine Nucleotides↗
PubMed1990

Insulin-secreting beta-cells possess specific receptors for interleukin-1 beta.

The effect of the cytokine interleukin-1 beta on the insulin secretory responsiveness of single beta-cells (HIT-T15) was investigated. In the short-term, IL-1 beta induced a dosage-dependent stimulation of insulin release. In contrast, in the long-term, IL-1 beta, inhibited both basal and secretagogue-stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL-1 beta on beta-cells. IL-1 beta, which has been implicated in the pathogenesis of insulin-dependent diabetes, may therefore mediate its opposing effects on beta-cells through a specific plasma membrane receptor.

Calcium↗
PubMed1989

Longer-term regulation of branched-chain-2-oxoacid dehydrogenase complex studied in rat hepatocytes in culture.

The effect of protein-free diet to decrease liver activity of branched-chain (-2-oxoacid) dehydrogenase (BCD) complex (active form) and increase BCD kinase activity was unaffected by preparation of hepatocytes, but partially reversed by 25 h of culture of hepatocytes in medium 199. Activation of BCD complex preceded loss of BCD kinase. The effect of culture on BCD complex was completely prevented by omission of branched-chain amino acids and partially prevented by 1 mM-alpha-cyano-4-hydroxycinnamate or 0.2 mM-pyruvate/2 mM-lactate. Protein-free diet decreased plasma branched-chain amino and oxo ('keto') acids and increased plasma pyruvate and lactate. It is concluded: (1) that branched-chain amino acids are involved directly in regulation of activities of BCD complex and BCD kinase; (2) that mitochondrial uptake of branched-chain oxo acids is necessary for regulation of BCD complex activity; and (3) that the stable increase in BCD kinase may function as a hysteresis mechanism.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1988

Activity of branched-chain 2-oxo acid dehydrogenase complex in rat liver mitochondria and in rat liver.

Four mitochondrial marker enzymes were used to show that: (1) high-protein (24%) diet increased the rat liver concentration and content of total branched-chain 2-oxo acid dehydrogenase complex (BCDC) by 31% by increasing mitochondrial specific activity of BCDC; (2) starvation increased the liver concentration of BCDC by 25% by decreasing liver weight; the liver content of mitochondria and the mitochondrial specific activity of BCDC were unchanged; (3) protein-free diet decreased rat liver BCDC concentration and content by 20%, by decreasing the liver concentration and content of mitochondria. Protein-free diet increased liver mitochondrial specific activities of L-glutamate, 2-oxoglutarate and NAD-isocitrate dehydrogenases. The validity of a mitochondrial method for the determination of the liver concentration of BCDC and the percentage in the active form in vivo is confirmed, and improvements are described. The experimental basis of criticisms of its use in this regard by Zhang, Paxton, Goodwin, Shimomura & Harris [(1987) Biochem. J. 246, 625-631] was not confirmed. The finding by Harris, Powell, Paxton, Gillim & Nagae [(1985) Arch. Biochem. Biophys. 243, 542-555], that starvation has no effect on the percentage of BCDC in the active form in rat liver, is confirmed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Temporal relationships in the effects of protein-free diet on the activities of rat liver branched-chain ketoacid dehydrogenase complex and kinase.

Feeding rats 0% casein diet decreased liver activities of branched-chain ketoacid dehydrogenase complex (active form) and of activator protein (complete within 4 days), and increased activity of branched-chain ketoacid dehydrogenase kinase (complete within 9-10 days). Refeeding normal diet to rats fed 0% casein diet for 10 days resulted in a rapid and partial (approx. 50%) reversal of the above effects within 24 h; complete reversal required 20-30 days of refeeding.

Animals↗
PubMed1986

Effects of low-protein diet and starvation on the activity of branched-chain 2-oxo acid dehydrogenase kinase in rat liver and heart.

The activity of branched-chain 2-oxo acid dehydrogenase kinase was 3-fold greater in extracts of heart mitochondria than in extracts of liver mitochondria from rats fed on normal diet. Feeding rats on a 0%-casein diet for 10 days increased the activity of branched-chain kinase 4-fold in extracts of liver mitochondria and in branched-chain dehydrogenase complex purified from such extracts; starvation (48 h) was without effect. In extract of heart mitochondria, kinase activity was increased 2-fold by feeding on 0%-casein diet and 1.5-fold by 48 h of starvation.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Effect of chlordane on influenza type A virus and herpes simplex type 1 virus replication in vitro.

The effect of chlordane on the susceptibility of Madin-Darby canine kidney cells and African Green monkey kidney cells to infection with influenza type A/PR/8/34 (HON1) virus and herpes simplex type 1 virus was determined. Exposure of both cell lines to various concentrations of chlordane for 24 h at 37 degrees C (acute exposure) effected a marked reduction in the efficiency of influenza type A virus infection, except at a dose of 0.025 ppm. Acute exposure of the monkey cells did not alter their susceptibility to herpes simplex virus infection. Viral adsorption studies at 4 and 37 degrees C revealed a marked reduction in the attachment of influenza type A virus to both cell lines following acute exposure to 10 ppm chlordane. Viral inactivation studies carried out at 4 and 37 degrees C failed to reveal differences in the level of influenza type A virus inactivation in the presence or absence of chlordane. Madin-Darby canine kidney cells exposed to 10 ppm chlordane for 60 d (chronic exposure) manifested a decrease in the efficiency of influenza type A virus infection, whereas cells chronically exposed to 0.025 ppm chlordane manifested an increase in the efficiency of influenza type A virus infection relative to mock-treated control cells. When chronically exposed cells were passaged six times in the absence of chlordane, these effects were reversed. Viral adsorption studies carried out at 4 and 37 degrees C on cells chronically exposed to 10 ppm chlordane revealed a decrease in the adsorption of influenza type A virus. Quantitation of the levels of cell-surface sialic acid, the essential terminal sugar on the receptor for influenza type A virus, indicated that the reduced adsorption of influenza type A virus to Madin-Darby canine kidney cells was not due to a loss of cell-surface sialic acid. Our findings indicate that chlordane alters the susceptibility of cells to infection with influenza type A virus but not to herpes simplex type 1 virus.

Adsorption↗