Biomedical subjects

D Dennis

Publications and source records attributed to D Dennis.

At least 37 records · Page 2Linked to original sources

PubMed1997

AIDS and the new medical gaze: bio-politics, AIDS, and homosexuality.

The essay argues that the contemporary resurgence of homophobia and the remedicalization of homosexuals in the wake of AIDS is, in part, an unintended but predictable effect of a quarter century of fractious identity-politics. Prominent gay and lesbian political strategies of the 1970s and 1980s borrowed heavily from increasingly discredited, if once politically correct, discourses that valorized individuals on the basis of membership in governmentally constructed bio-bureaucratic categories. Drawing on the work of prominent gay intellectuals, such as Foucault, Watney, and Richard Rodriguez, and locating their insights within the context of contemporary cultural and political conflicts, the essay argues that gay advocates who essentialize homosexual identity, however benignly, unwittingly participate in constituting the ground for an emergence of a neoeugenic movement at millennium's end. The essay concludes with the observation that escaping the conceptual prison of bio-bureaucratic categories is not a uniquely gay or lesbian task. It is a human task.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene.

The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.

Bacterial Proteins↗

PubMed1996

Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene in Nocardia corallina.

To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 x 10(6) - 3 x 10(6) transformants/microgram plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.

Acyltransferases↗

PubMed1996

"In vivo" pose estimation of artificial knee implants using computer vision.

This paper describes an algorithm to estimate the position and orientation (pose) of artificial knee implants from fluoroscopy images using computer vision. The resulting information is used to determine contact position from " in vivo" bone motion in implanted knees. This determination can be used to support the development of improved prosthetic knee implants. Current generation implants have a limited life span due to premature wear of the polyethylene material at the joint surface. To get "in vivo" motion, fluoroscopy videos were taken of implant patients performing deep knee bends. Our algorithm determines the full 6 degree of freedom translation and rotation of knee components. This is necessary for artificial knees which have shown significant rotation out of the sagittal plane, in particular internal/external rotations. By creating a library of images at known orientation and performing a matching technique, the 3-D pose of the femoral and tibial components are determined. By transforming the coordinate systems into one common system contact positions can be determined. The entire process, when used at certain knee angles, will give a representation of the positions in contact during normal knee motion.

Algorithms↗

PubMed1995

Regulated expression of the Alcaligenes eutrophus pha biosynthesis genes in Escherichia coli.

A novel poly-beta-hydroxybutyrate (PHB) production system in which the expression and gene dosage of the Alcaligenes eutrophus pha biosynthetic operon were effectively regulated by cultivation temperature was constructed in Escherichia coli. The pha operon was fused to the negatively regulated tac promoter and cloned into a vector in which the copy number is temperature dependent. A two-phase process was employed to produce PHB during fed-batch growth. In the growth phase, the culture was maintained at a low temperature. Under this condition, the plasmid copy number was depressed and the number of LacI proteins was sufficient to repress tacupha transcription. The production phase was initiated by temperature upshift. At the elevated temperature, the number of plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tacupha transcription, synthesis of poly-beta-hydroxyalkanoate-specific proteins, and polymer synthesis. During the production phase, the PHB production rate was 1.07 g of PHB liter-1 h-1 under optimized conditions. This rate is comparable to that of bacteria which naturally produce this polymer.

Alcaligenes↗

PubMed1995

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.

Acetyl Coenzyme A↗

PubMed1995

The function of ackA and pta genes is necessary for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha+ E. coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA+ plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+ E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.

Acetate Kinase↗

PubMed1995

1,3-Dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine, a potent, specific and selective A1 adenosine receptor antagonist in the guinea pig heart and brain and in DDT1MF-2 cells.

The objective of this study was to characterize the adenosine receptor (AdoR) antagonistic properties of a newly synthesized alkylxanthine, 1,3-dipropyl-8[2-(5,6-epoxy)norbornyl]xanthine (ENX), and compare them to those of 1,3-dipropyl-8-(cyclo-pentyl)xanthine (CPX), 1,3-dipropyl-8-(3-noradamantyl)xanthine (NAX) and (+/-)-N6-endo-norbornan-2-yl-9-methyladenine (N-0861). The potencies and selectivities of ENX, CPX, NAX and N-0861 were determined by functional studies of guinea pig isolated perfused hearts, and by radioligand binding assays for A1 and A2a AdoRs in the guinea pig forebrain and striatum. ENX competitively antagonized A1 AdoR-mediated prolongations of atrioventricular nodal conduction time caused by Ado or by 2-chloro-N6-cyclopentyladenosine, but not those caused by carbachol (0.14 microM) or MgCl2 (3 mM). Schild analysis of 2-chloro-N6-cyclopentyladenosine-antagonist competition curves yielded pA2 values for ENX, CPX and NAX of 8.45 +/- 0.19, 8.55 +/- 0.28 and 8.79 +/- 0.15, respectively. ENX (30 microM) and N-0861 (30 microM) did not attenuate the A2 AdoR-mediated increase in coronary conductance caused by adenosine. CPX and NAX attenuated the coronary vasodilation caused by adenosine with IC50 values of 1.5 and 7.1 microM, respectively. Radioligand binding assays revealed that ENX, CPX and NAX and N-0861 had a 400-, 209-, 110- and 10-fold greater affinity, respectively, for A1 than for A2a AdoRs of guinea pig brain membranes. Thus, ENX was equipotent with CPX and NAX and more potent than N-0861 (pA2 = 6.2) as an antagonist at A1 AdoRs, but had lower affinity for A2 AdoRs in guinea pig hearts and brain striatum than did either CPX or NAX. In DDT1 MF-2 cells, all three alkylxanthines had similar affinities for A1 AdoRs, whereas the affinity of N-0861 for A1 AdoRs was significantly lower. ENX appears to be the most A1 AdoR subtype-selective of the alkylxanthine class of AdoR antagonists reported to date.

Adenosine↗

PubMed1994

Production of polyhydroxyalkanoates in sucrose-utilizing recombinant Escherichia coli and Klebsiella strains.

The cloned poly-3-hydroxybutyrate (PHB) synthesis pathway from Alcaligenes eutrophus has been introduced into sucrose-utilizing strains of Escherichia coli, Klebsiella aerogenes, and Klebsiella oxytoca. The plasmid-borne genes were well expressed in these environments and were able to mediate the production of significant amounts of PHB when the bacteria were grown with sucrose as the sole carbon source. The molecular weight of the PHB polymer made in K. aerogenes and E. coli was approximately 1 x 10(6) to 2 x 10(6). Sucrose uptake in K. aerogenes was measured and found to be similar to that found for other Klebsiella strains, but sucrose uptake in the E. coli strain was not detectable. K. aerogenes is able to utilize sugarcane molasses as the sole carbon source to accumulate PHB at the rate of approximately 1 g of PHB per liter of culture fluid per h. A K. oxytoca fadR strain was able to incorporate 3-hydroxyvalerate into a poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-V) polymer to levels as high as 56 mol% when grown in a medium containing propionate. Total PHB-co-V levels could be enhanced by adding propionate at the beginning of stationary phase rather than at the time of inoculation.

Acyltransferases↗

PubMed1994

Allosteric enhancer PD 81,723 acts by novel mechanism to potentiate cardiac actions of adenosine.

The 2-amino-3-benzoylthiophene derivative PD 81,723 is an allosteric enhancer of agonist binding to brain A1 adenosine receptors. One aim of this study was to characterize and contrast the effects of PD 81,723 on the A1 receptor-mediated negative dromotropic and A2a receptor-mediated vasodilatory actions of adenosine and of a nonmetabolizable and unselective N6-(3-pentyl)adenosine derivative. A second aim was to determine the mechanism of action of PD 81,723. In guinea pig isolated hearts, PD 81,723 potentiated the adenosine and the N6-(3-pentyl)adenosine derivative-induced prolongations of the stimulus-to-His bundle (S-H) interval in a concentration-dependent manner. PD 81,723 (30 mumol/L) decreased the EC50 value for adenosine to prolong the S-H interval by ninefold from 7.4 +/- 1.2 to 0.8 +/- 0.1 mumol/L but did not increase the content of adenosine in cardiac effluent. PD 81,723 (30 mumol/L) increased the specific binding of the A1 agonist [3H]cyclohexyladenosine ([3H]CHA) to human atrial and guinea pig atrial and brain membranes by 38%, 78%, and 300%, respectively. PD 81,723 also increased the fraction of A1 receptors in the high-affinity binding state by an average of 56 +/- 13%. The dissociation rate of [3H]CHA from guinea pig brain membranes was decreased in the presence of PD 81,723 (10 mumol/L) from 0.55 +/- 0.01/min to 0.35 +/- 0.01/min. PD 81,723 did not alter the binding of the A1 antagonist [3H]cyclopentyldipropylxanthine to guinea pig brain membranes. The IC50 values for 5'-guanylylimidodiphosphate to reduce specific binding of [3H]CHA to guinea pig cardiac and brain membranes were increased from 1.5 +/- 0.2 and 2.0 +/- 0.2 mumol/L in the absence of PD 81,723 to 10 +/- 3.3 and 18 +/- 0.5 mumol/L, respectively, in the presence of PD 81,723 (30 mumol/L). PD 81,723 did not potentiate the coronary vasodilatory actions of the N6-(3-pentyl)adenosine derivative. Specific binding of the A2a agonist [3H]CGS 21680 to brain membranes and the nucleoside transporter ligand [3H]nitrobenzylthioinosine to cardiac membranes was unchanged in the presence of PD 81,723. The results suggest that PD 81,723 specifically potentiates the action of adenosine on A1 receptors by stabilizing receptor-G protein interactions in the presence of agonists.

Adenosine↗

PubMed1994

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias.

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Animals↗

PubMed1994

The cardiac effects of a novel A1-adenosine receptor agonist in guinea pig isolated heart.

Adenosine increases atrioventricular (AV) nodal conduction time and is used for termination of AV nodal re-entrant tachycardias, but it is rapidly metabolized. The purposes of the present study were to characterize the cardiac actions and effects of an orally active and stable adenosine analog, N6-cyclohexyl-2-O-methyladenosine (SDZ WAG-994) and to evaluate its potential as an antiarrhythmic agent. Guinea pig hearts were isolated and perfused with oxygenated Krebs-Henseleit solution. SDZ WAG-994 slowed the atrial rate and prolonged the AV nodal conduction time of spontaneously beating hearts in a concentration-dependent manner. The EC50 values for the negative chronotropic and dromotropic effects of SDZ WAG-994 were 0.69 +/- 0.04 and 1.49 +/- 0.54 microM, respectively. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.2 microM) significantly antagonized SDZ WAG-994-induced stimulus-to-His bundle (S-H) interval prolongation. The negative dromotropic effect of SDZ WAG-994 showed very strong frequency dependence. In hearts paced at an atrial cycle length of 300 msec (200 beats/min), the EC50 value of SDZ WAG-994 to prolong the S-H interval was 3.7-fold lower (0.40 +/- 0.02 microM) than in unpaced hearts, and at atrial pacing cycle lengths of 500 and 250 msec, 0.3 microM SDZ WAG-994 prolonged the S-H interval by 8 and 26 msec, respectively. SDZ WAG-994 also decreased coronary perfusion pressure (EC50 = 1.50 +/- 0.80 microM); this effect of SDZ WAG-994 was attenuated by adenosine deaminase and by 8-cyclopentyltheophylline (2 microM). Radioligand binding assays revealed that SDZ WAG-994 had a 280-fold greater affinity for A1- than for A2a receptors of the guinea pig brain. The marked frequency dependence of the negative dromotropic effect of SDZ WAG-994 suggests that this A1 agonist may be highly effective in the termination of AV nodal re-entrant tachycardias.

Adenosine↗

PubMed1993

Active site labeling of HIV-1 reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Wheat germ and yeast RNA polymerase II: photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound [32P]-containing transcript.

A stable ternary transcription complex was formed with either wheat germ or yeast RNA polymerase II using a ribotrinucleotide primer (GpCpG) to initiate transcription on a short synthetic single-strand DNA template. The template was designed to limit the incorporation of a photoprobe S4-UMP (4-thio-UMP) to a unique position at the 3' terminus of the transcript. The resulting stable ternary transcription complex was photolyzed to cross-link the bound transcript ([32P]-labeled by the incorporation of [alpha-32P]CMP) with the protein domain at or near the active site. Separation of the protein components by electrophoresis in polyacrylamide gel containing SDS and analysis by autoradiography and silver staining revealed that for either enzyme only the largest subunit was [32P] labeled.

Affinity Labels↗

PubMed1992

Polyhydroxyalkanoate production in recombinant Escherichia coli.

The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).

Escherichia coli↗

PubMed1992

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.

Alcaligenes↗

PubMed1992

Evidence of spare A1-adenosine receptors in guinea pig atrioventricular node.

In normoxic, isolated perfused guinea pig hearts instrumented for measurement of atrioventricular nodal conduction time (AVCT), an analysis utilizing the irreversible A1-adenosine (Ado) antagonist, meta-1,3-phenylene diisothiocyanate xanthine amine cogener (m-DITC-XAC), a novel isothiocyanate derivative of 1,3-dialkylxanthine, was used to investigate whether spare A1-Ado receptors exist in the guinea pig atrioventricular (AV) node and the degree of amplification (reserve) between A1-Ado receptor occupancy and dromotropic response (e.g., AVCT slowing). The potency, dose dependency, and kinetic profile (time dependence of washout and washin) of m-DITC-XAC was determined and compared with those of known competitive (reversible) A1-Ado receptor antagonists. In the presence of m-DITC-XAC, Ado and N6-cyclopentyladenosine (CPA) produced submaximal dromotropic responses. In a series of 19 hearts, m-DITC-XAC caused 100% apparent antagonism of the effect of Ado on AVCT even after 60 min of washout. In contrast, greater than 90% of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and XAC-induced antagonism of the effect of Ado on AVCT dissipated within 35 min. Unlike XAC, which caused maximal attenuation of Ado's AVCT effect within 5 min and remained constant thereafter, m-DITC-XAC showed marked time- and concentration-dependent behavior. It was found that 5 min of 0.5 microM m-DITC-XAC pretreatment irreversibly inactivated 72% of the A1-Ado receptors mediating the dromotropic effect, and the estimated agonist equilibrium dissociation constant for CPA was 84 +/- 4 nM. The percent of spare A1-Ado receptors at the EC50 and extrapolated maximal S-H interval prolongation levels was 20 and 54%, respectively, and the reserve (coupling amplification) varied from 1 to 2.3 within the 0-50% maximal response range. In summary, m-DITC-XAC appears to specifically and irreversibly antagonize the negative dromotropic effect of Ado and CPA, and guinea pig AV nodal tissue possesses spare A1-Ado receptors.

Adenosine↗

PubMed1991

Open versus laparoscopic cholecystectomy: an initial analysis.

Laparoscopic cholecystectomy is a new procedure in the armamentarium of the general surgeon. Its utility was investigated by comparison to open cholecystectomy in terms of procedure time, complications, hospital stay, and total hospital cost. Procedure time was approximately 200% longer with a higher incidence of intraoperative stone and bile spillage (17%) in the laparoscopic group. Hospital stay was reduced by 60% using the laparoscopic technique. No difference in total hospital cost existed between the two groups. The learning curve had an affect on hospital costs, which will decrease as more experience is gained with this procedure. Although laparoscopic cholecystectomy, at least initially, has no cost advantage over open cholecystectomy, laparoscopic cholecystectomy may be preferred by patients seeking shorter hospital stays and presumably shorter total recovery time.

Adult↗