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Biomedical subjects

D B Olsen

Publications and source records attributed to D B Olsen.

At least 55 records · Page 3Linked to original sources
PubMed1994

Sensitivity of HIV-1 reverse transcriptase and its mutants to inhibition by azidothymidine triphosphate.

HIV-1 reverse transcriptase can catalyze the addition of either azidothymidine monophosphate (AZTMP) or thymidine monophosphate (dTMP) to a primer strand opposite template adenosine bases. The ratio of incorporation of AZTMP to dTMP as catalyzed by HIV-1 reverse transcriptase has been determined to be 0.4 using an RNA-DNA duplex substrate prepared from oligonucleotides with sequences taken from the HIV-1 genome sequence. Slight variations are found for the incorporation ratio of the two nucleotides on other substrates. Substrates containing more than one adenosine in the single-stranded part of the template allow for more chances to incorporate AZTMP and less full-length product. Variations in the intensity of bands on an autoradiograph of a DNA sequencing gel corresponding to different positions of incorporation of AZTMP suggest that not all template adenosine positions offer the same level of discrimination against incorporation of AZTMP. A reverse transcriptase containing a set of four mutations (D67N, K70R, T215Y, K219Q) known to cause resistance to AZT in cell culture assays has a ratio of incorporation that is 0.77 +/- 0.03 times the ratio for the wild-type reverse transcriptase opposite one specific template adenosine. In contrast, a hybrid mutant containing the same four mutations that cause resistance to AZT and an additional mutation, Y181C, which by itself causes resistance to the non-nucleoside inhibitor L-697,661 [Sardana et al. (1992), J. Biol. Chem. 267, 17526-17530], has a ratio of incorporation that is 1.34 +/- 0.01 times that of the wild-type, indicating that the hybrid mutant enzyme is more susceptible to inhibition by AZTTP than the wild-type reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗
PubMed1994

Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits.

Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.

Base Sequence↗
PubMed1993

Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives. Potency, binding characteristics, and effect of template sequence.

The inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by pyridinone compounds has been investigated using as templates synthetic RNA with sequences based on the HIV-1 genome sequence. In reactions catalyzed by the enzyme that incorporated more than one nucleotide per primer, inhibition by a representative pyridinone inhibitor, 3-[2-(1,3-benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2(1H)one (L-696,229), was noncompetitive against deoxynucleotide triphosphate. For reactions that incorporated one deoxynucleotide per primer, IC50 values ranged from 20 to 200 nM, depending on the position of incorporation of the incoming deoxynucleotide base on the template. Inhibition of synthesis on a set of four templates differing only at the template base complementary to the incoming nucleotide had similar IC50 values. These results demonstrate that inhibitory potency is dependent on the primary structure of the template and that inhibitory potency is largely independent of the identity of the incoming nucleotide base. The inhibition of HIV-1 reverse transcription by L-696,229 also displayed slow-binding characteristics. The slow-binding aspect was exploited to gauge the interaction between inhibitor and enzyme. By titrating the reduction in the extent of the burst of synthesis observed in a reaction incorporating dideoxythymidine monophosphate into poly(rA)-oligo(dT)18, the apparent equilibrium constant for dissociation of the reverse transcriptase-L-696,229 complex was estimated to be 400 nM.

Antiviral Agents↗
PubMed1993

Alprostadil: an effective antiplatelet agent for calves.

Aspirin and other nonsteroidal, antiinflammatory drugs inhibit human platelets, but their effect as an antiplatelet agent on bovine platelets is not certain. Since calves are used for cardiovascular implant research, need exists for an effective antiplatelet agent for this animal model. After screening a number of potential antiplatelet drugs for calves, Alprostadil (prostaglandin E1) appeared to be the most promising. Alprostadil was administered (intravenous continuous drip) to 8 calves. The concentration of the drug administered was gradually increased until 50% inhibition of platelet aggregation was obtained. Platelet function, blood pressure, body temperature, and hematologic parameters were closely monitored. For in vitro evaluation, Alprostadil was added to human or bovine blood, and the platelet aggregation and cyclic adenosine monophosphate levels were measured. Alprostadil inhibited both bovine and human platelets, although bovine platelets were relatively more responsive to this drug. At an infusion rate of approximately 0.20 micrograms/kg/min in vivo, Alprostadil showed 50% inhibition of platelet aggregation with slightly decreased blood pressure (7 +/- 6 mm Hg) but no adverse effects. Complete reversal of in vivo platelet inhibition was noted within 24 h after cessation of drug administration. This short half-life and the lack of significant adverse effects make Alprostadil an attractive antiplatelet agent for calves with cardiovascular implants.

Alprostadil↗
PubMed1993

In vitro hematological testing of rotary blood pumps: remarks on standardization and data interpretation.

Pump test procedures using blood will have to meet several standards not only to obtain reliable results in vitro but also to allow comparison of results of different investigators. This article reviews some of the issues that should be considered in pump testing, especially referring to the discussions held at the International Workshops on Rotary Blood Pumps in 1988 and 1991. The test loop itself should meet some requirements such as constant physiological temperature, standardized circulating volume, control of pressure and flow, and exact definition of the blood-contacting surface. Specifications have to be made concerning the test fluid blood, including sampling technique, anticoagulation, blood gases, pH, and glucose level. Only fresh blood should be used. Heparin is recommended for anticoagulation because it will be used also in vivo. Different procedures for cleaning and rinsing of plastic materials for reuse are mentioned. Bacterial overgrowth, which can lead to extreme oxygen consumption and acidosis, may be avoided through addition of antibiotics (e.g., gentamicin). To be able to compare data of the different working groups, a new modified index of hemolysis (MIH) has been defined.

Animals↗
PubMed1993

Measurement of oxygen consumption and arterial-venous oxygen saturation following total artificial heart implantation.

Current algorithms for control of the total artificial heart are directed at maintaining hemodynamic homeostasis. Future control systems will also need to modify cardiac output in response to metabolic needs. This study was undertaken to evaluate oxygen metabolism monitoring as an indicator of the adequacy of organ and tissue perfusion. Following recovery from implantation of the Utah-100 pneumatic total artificial hearts, five calves (85 to 95 kg) underwent placement of fiberoptic oximetry catheters to determine mixed venous and arterial oxygen saturations. By continuously measuring oxygen consumption with a gas analyzer, oxygen utilization and delivery were determined. In the awake calves, at-rest cardiac output was varied to produce hyperperfused and hypoperfused conditions while the adequacy of tissue perfusion was assessed with continuous mixed venous oximetry and confirmed with serum lactate (Lact) levels. Inadequate tissue perfusion (Lact > 1.0 mmol/L) was evidenced by a mixed venous oxygen saturation < 40%, oxygen delivery of < 200.0 milliliters/minute/m2), and oxygen delivery to utilization ratio of < 1.8 during the hypoperfusion conditions of the experiment. By accounting for oxygen consumption, the ratio of oxygen delivery to oxygen utilization was predictive of the adequacy of tissue perfusion. These results suggest that continuous oxygen metabolism monitoring may be useful as a physiologic control modifier to maintain total artificial heart output sufficient to meet physiologic needs, while avoiding hyperperfusion, unnecessary wear and deterioration of the implanted device due to excessive heart rates.

Animals↗
PubMed1992

Investigation of the substrate binding and catalytic groups of the P-C bond cleaving enzyme, phosphonoacetaldehyde hydrolase.

Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde (Ki = 1.6 mM), phosphonoacetate (Ki = 10 mM), phosphonoethanol (Ki = 10 mM), and fluorophosphate (Ki = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH4, acetonyl-phosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3-3H]acetonylphosphonate/NaBH4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the Vm/Km and Vm pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6-8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiol-sulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8.

Amino Acid Sequence↗
PubMed1992

Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus.

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

Amino Acid Sequence↗
PubMed1992

Potent agonists of growth hormone-releasing hormone. Part I.

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr1, or N-MeTyr1. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH2, [Dat1,Ala15]GH-RH(1-28)Agm (MZ-3-191) and [D-Ala2,Ala15]GH-RH(1-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met27. Their Nle27 analogs, [Dat1,Ala15,Nle27]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [D-Ala2,Ala15,Nle28]GH-RH(1-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met27 by Nle27 enhanced the GH-releasing activity of the analog when the molecule contained Dat1-Ala2 residues at the N-terminus, but peptides containing Tyr1-D-Ala2 in addition to Nle27 showed decreased potencies. Replacement of Ser28 with Asp in multi-substituted analogs of GH-RH(1-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser28-containing MZ-2-51, and [Dat1,Ala15,D-Lys21,Nle27]GH-RH(1-28)Agm, its Asp28 homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Agmatine↗
PubMed1992

Thrombus with infection in total artificial heart animals.

From January 1980 to December 1990, several types of total artificial hearts were implanted into 378 animals. In a retrospective study of these animals, 147 (39%) were found to have thrombus with infection (T&I). The criteria for diagnosis was thrombus formation in the artificial heart and a positive blood culture. The most common pathogen isolated from T&I animals was Pseudomonas species. Concurrent skin lesions and contamination from the pressure lines may be the primary sources of infection, but bacterial translocation from the intestine is another possible route. The main pathological findings at necropsies of artificial heart animals with T&I were associated with sepsis, congestive heart failure, infected thrombus, thromboembolism, and multiple organ infarctions. Most thrombi appeared to have originated from valve junctions and connectors. On the basis of these observations, a possible mechanism for pathogenesis of T&I has been proposed. The results suggest that design improvements and surface modifications to reduce thrombosis are important factors that should be carefully considered. Similarly, it is important to eliminate the route of entry of pathogenic microorganisms. These findings imply that bacterial interaction with thrombus, device related bacterial colonization, and host immunomodulation and gut barrier function following artificial heart implantation need further investigation.

Animals↗
PubMed1992

Wearable air supply for pneumatic artificial hearts and ventricular assist devices.

An experimental wearable air supply for pneumatic artificial hearts and ventricular assist devices has been built and tested. The unit eliminates the need for tethering to a large, stationery driver. The miniaturized air supply is designed for ambulatory patients with implanted pulsatile pneumatic total artificial hearts (TAH) or pneumatic left-ventricular assist devices (LVAD), to permit mobility in clinical and home settings. The device has major short-term utility as a supply for pneumatic TAH or VAD bridges in patients awaiting heart transplants. The system design for the wearable driver includes a novel, fast rotary compressor, driven by a brushless direct current (DC) motor to supply air to the ventricle through an electromagnetically actuated directional valve, all controlled by a microcomputer. Stroke volume from 0 to 200 cc; pulse rate from 60 to 160 bpm, and duty cycle from 33% to 50% are selected on a keyboard, and the selected or measured parameters can be shown on a liquid crystal display. For control of delivery from a single ventricular assist device, stroke volume is controlled by variation of compressor speed. In the wearable air supply for a TAH, a single compressor drives both ventricles alternately through a double-acting directional valve. Air volume delivered to the left ventricle is adjusted by variation of compressor speed, and air volume to the right ventricle by variation of ejection time. The effect on blood flow rate of the lower impedance to the right ventricle is compensated by provision of a two-stage compressor, in which a single stage drives the right ventricle, and both stages connected in parallel drive the left ventricle. The overall dimensions of the prototype air supply for driving either a TAH or LVAD are 4.5 by 7.8 by 4.5 inches, including an emergency battery with a duration of 15 to 30 min depending on load. The weight is presently 5.5 lb, but this will be reduced in a production design and for a dedicated LVAD air supply. The size and weight of a separately worn, rechargeable battery pack depend on the load and desired duration of operation.

Electric Power Supplies↗
PubMed1991

Study of a hammerhead ribozyme containing 2'-modified adenosine residues.

The improved synthesis of 2'-fluoro-2'-deoxyadenosine (2'-FA) starting from adenosine is described. This compound was converted to the phosphoramidite and incorporated into a hammerhead ribozyme RNA with the use of automated RNA synthesis techniques. Ribozymes containing 2'-deoxy-adenosine (2'-dA) were prepared in a similar manner. A kinetic rate comparison of the unmodified ribozyme with two ribozymes that had every adenosine replaced with 2'FA or 2'-dA revealed a large decrease in catalytic efficiency (kcat/Km) for the modified ribozymes resulting from a drop in kcat. The kinetic analysis of a number of partially substituted 2'-FA or 2'-dA containing hammerheads revealed that the decrease in activity was not associated with any particular residue but was the result of the accumulation of modified nucleosides within the structure.

Base Sequence↗
PubMed1991

Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes.

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.

Base Sequence↗
PubMed1991

The role of plasma proteins in formation of obstructive protamine complexes.

Formation of complexes between heparin and protamine (in saline), or heparin, plasma proteins, and protamine (in plasma) was assessed by measurements of light transmission through different test solutions. To examine the formation of these complexes, 125I-labeled protamine was used. Addition of 125I-protamine to plasma or blood resulted in the sedimentation of 125I-protamine in the form of insoluble complexes. This complex formation was not affected by the presence of heparin, suggesting that protamine-plasma protein interaction may be primarily responsible for precipitation of 125I-protamine. To assess the capability of these complexes to obstruct the pulmonary circulation, an in vitro experimental model was developed. Citrated serum, plasma, blood, or saline were allowed to flow through a glass bead column with the help of a peristaltic pump. A pressure transducer positioned before the column allowed pressure measurements at a constant flow rate during the experiment. Mixing of protamine with plasma or blood prior to their passage through the glass bead column resulted in a significant increase in pressure suggesting that the column was being clogged with insoluble complexes. The increase in pressure occurred both in the presence and absence of heparin in plasma or blood. Under identical experimental conditions, the increase in pressure was insignificant when protamine was added to saline or serum regardless of whether heparin was present or absent. This was further confirmed by the use of 125I-protamine. These observations suggest that protamine forms insoluble complexes with certain plasma proteins. Based on these observations, it is hypothesized that following intravenous administration, protamine immediately forms complexes in circulating blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗