Biomedical subjects

M Gorecki

Publications and source records attributed to M Gorecki.

At least 19 recordsLinked to original sources

PubMed2006

Influence of rapeseed phospholipids on ibuprofen dissolution from solid dispersions.

The dissolution profiles of ibuprofen (IB) from solid dispersions prepared by the solvent evaporation method, containing the rapeseed lecithin ethanol soluble fraction (LESF) or rapeseed phosphatidylcholine (RPC) have been determined. The effect of incorporation of PEG 4,000 or PEG 8,000 in the solid dispersions on the controlled-release of IB was also investigated. Dissolution studies conducted in double-distilled water using the paddle dissolution apparatus showed that the initial dissolution rate (IDR) within the first 5 min and the maximum percent of dissolved IB of IB/LESF were double of those of IB/RPC (both in ratio 4:1 w/w). The low amounts of LESF markedly increased dissolution of IB. Increasing of LESF concentration from 0 to 10 and 20% in solid dispersions produced 60 and 100% improvement of IB maximum dissolution level respectively, to compare with that of IB alone. PEG 4,000 caused the slightly decreasing in IB dissolution rate, while PEG 8,000 markedly improved the dissolution of IB in examined conditions.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles.

Recombinant hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells were stored at various conditions for 12-18 months in their naked form or adsorbed to alum (HBV vaccine). The physical, chemical and immunological parameters during storage at -20 degrees C, 4 degrees C, room temperature and 37 degrees C were investigated. HBsAg particles fully retained the original peptide composition when stored for 6 months, as a dispersion, at -20 degrees C and 4 degrees C; and as lyophilized powder, at all four temperatures. Ten percent sucrose preserved the size, shape and protein content of the naked particles stored at 4 degrees C and -20 degrees C for 18 months as a dispersion or lyophilized. Lyophilization in the presence of glucose, sucrose and trehalose, but not mannitol, further improved the 4 degrees C stability of size, shape and the protein content during 2 years of storage. Stability of the particle's lipid components was inferior to that of the protein components. Dispersions of naked HBsAg and of particles lyophilized in the presence of sucrose and stored at -20 degrees C were the only forms in which the lipid content and composition, including the lipid polyunsaturated acyl chains, were preserved for at least 18 months storage. The level of phospholipid and free cholesterol were most stable in the HBV vaccine which was stored at 4 degrees C; they did not change after 1 year of storage. Preservation of immunogenicity was evaluated according to dose-dependent changes in S-specific antibody titers in sera obtained from immunized BALB/c mice. The ED50 for achieving seroconversion was 0.07 microg/ml/mouse, indicating that the vaccine is very immunogenic. Freezing or freeze-drying of the HBV vaccine results in the total loss of vaccine immunogenicity (in spite of the good chemical stability), while full immunological potency was retained for at least 2.5 years at 4 degrees C. Storing formulated vaccine at 25 and 37 degrees C for 4 and 2 weeks, respectively, did not alter the vaccine potency. This study suggests that the vaccine's physical, chemical and immunological characteristics are sufficiently stable at high temperatures to reduce the need for 'cold chain' transportation.

Animals↗

PubMed1997

Heparan sulfate proteoglycan-mediated uptake of apolipoprotein E-triglyceride-rich lipoprotein particles: a major pathway at physiological particle concentrations.

We explored potential mechanisms of non-low-density lipoprotein (LDL) receptor-mediated uptake of triglyceride-rich particles (TGRP) in the presence of apolipoprotein E (apo E). Human fibroblasts were incubated with model intermediate-density lipoprotein- (IDL-) sized TGRP (10-1000 microg of neutral lipid/mL) containing apo E. The extent of receptor-mediated uptake of TGRP was assessed with (a) an anti-apo E monoclonal antibody, which blocks receptor interaction; (b) incubation with heparin; (c) normal vs LDL receptor-negative fibroblasts; and (d) receptor-associated protein (RAP) to determine the potential contribution of LDL receptor-related protein (LRP). Cell surface heparan sulfate proteoglycan- (HSPG-) mediated uptake was examined with or without the addition of heparinase and heparitinase to cell incubation mixtures. At low particle concentrations (</=100 microg of neutral lipid/mL), almost all apo E-TGRP uptake was via the LDL receptor. At higher particle concentrations, within the physiologic range (>250 microg of neutral lipid/mL), most (>/=60%) particle uptake and internalization was via HSPG-mediated pathways. This HSPG pathway did not involve classical lipoprotein receptors, such as LRP or the LDL receptor. These data suggest that in peripheral tissues, such as the arterial wall, apo E may act in TGRP as a ligand for uptake not only via the LDL receptor and LRP pathways but also via HSPG pathways that are receptor-independent. Thus, at physiologic particle concentrations apo E-TGRP can be bound and internalized in certain cells by relatively low affinity but high capacity HSPG-mediated pathways.

Antibodies, Monoclonal↗

PubMed1997

Recombinant polypeptides derived from the fibrin binding domain of fibronectin are potential agents for the imaging of blood clots.

Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa ("2 fingers"), 18.5 kDa ("3 fingers") and 31 kDa FBD ("5 fingers"), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C-putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111In]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [111In]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Animals↗

PubMed1996

Molecular analysis of the second template switch during reverse transcription of the HIV RNA template.

The molecular events leading to the second template switch during reverse transcription of the HIV genome were studied in a defined in-vitro system. In order to investigate displacement of the tRNA(lys) primer from the primer binding site (PBS) of the viral genomic RNA, following DNA synthesis, we produced an HIV RNA/DNA substrate that resembles the intermediate reverse transcription complex formed prior to the second template switch. Partial tRNA(lys) primer displacement was observed during plus (+) strand DNA synthesis and during minus (-) strand DNA elongation. We found two determinants that may serve as a stop signal for (+) DNA strong stop synthesis, the A(m) at position 19 of the natural tRNA(lys) and the secondary structure at the PBS sequence. The later signal appears to constitute a stronger terminator in-vitro. The 3' end of the nascent (-) DNA strand prior to the second template switch was also determined. It was mapped to the U5-PBS junction at the site for the first endonucleolytic cut introduced by the RNase H activity of the HIV reverse transcriptase (RT). Thus, different signals dictate the arrest of (-) and (+) nascent DNA synthesis. These stop signals appear to be required for the subsequent second template switch. However, an excess of (-) DNA "acceptor" molecules, having a 18-base sequence complementary to the (+) DNA "donor" template, was required to demonstrate the actual template switch in the in-vitro system. Taken together these results indicate that the reverse transcriptase can catalyze all the steps leading to the second template switch and auxiliary viral proteins may act to enhance the efficiency of this step during the reverse transcription process.

Base Sequence↗

PubMed1996

Ribonuclease H activity during initiation of reverse transcription using tRNA(lys)/RNA primer/template of human immunodeficiency virus.

The specificity of the initial cleavage by the RNaseH activity of HIV-1 reverse transcriptase (RT) during minus strong-stop DNA synthesis was studied using the authentic primer/template tRNA(lys)/HIV RNA. We observed that concomitant with the initiation of DNA synthesis, RNaseH activity of HIV RT introduced the first endonucleolytic cuts within the U5 region of the HIV RNA template, mainly 1 and 3 bases away from the primer binding site. To analyze whether the cleavage sites were determined by sequence specificity, the authentic U5 region at one of the cleavage sites was mutated. The change of sequence did not alter the initial cleavage pattern of RNaseH. In order to determine the size of the RNA/DNA hybrid that is required for RNaseH activation during reverse transcription initiation, DNA synthesis was limited by dideoxynucleotides. DNA extension of the tRNA(lys) primer by 17 deoxyribonucleotides but not by 6 deoxyribonucleotides was sufficient to activate the RNaseH site of HIV RT. Taken together, our results indicate that during initiation of minus strongstop DNA synthesis by HIV RT, the first RNaseH-mediated endonucleolytic cut of the genomic RNA is dictated mainly by the length of the nascent DNA and not by sequence preference.

Base Sequence↗

PubMed1996

Adhesion of blood platelets is inhibited by VCL, a recombinant fragment (leucine504 to lysine728) of von Willebrand factor.

VCL, fragment Leu504 to Lys728 of von Willebrand factor (vWF) expressed in Escherichia coli, contains the glycoprotein (GP) Ib-binding domain of vWF. This fragment inhibited ristocetin-induced platelet aggregation with an IC50 of 0.2 mumol/L and botrocetin-induced platelet aggregation with an IC50 of 0.08 mumol/L. We studied the antiadhesive profile of VCL by adding it to blood that was circulated over various adhesive surfaces. VCL inhibited adhesion to endothelial cell matrix, which served as a model of the vessel wall. Maximal inhibition at a high shear rate of 1600 s-1 was stronger (60%) than at a low shear rate of 300 s-1 (40%). Half maximal inhibition was found to be 1.5 mumol/L at both shear rates. The role of various adhesive molecules was investigated in more detail by coating glass coverslips with collagen type I, laminin, fibronectin, or vWF. Fibrinogen was studied as well. Platelet adhesion to laminin and vWF was not inhibited by VCL. Adhesion to collagen, fibronectin, and fibrinogen was particularly inhibited at a high shear rate. VCL coated to a coverslip caused a concentration-dependent adhesion that was blocked by antibodies against GPIb, which block interaction with vWF. Binding studies showed a nonsaturable ristocetin binding of VCL to platelets that was blocked by vWF or inhibitory antibodies against GPIb. Binding to collagen was weak, and VCL did not inhibit binding of vWF at a 5000-fold excess. From these data, we conclude that VCL inhibits adhesion in all cases in which adhesion is vWF dependent by competing for vWF binding to activated GPIb. The lack of inhibition of adhesion to vWF as a single molecule may be explained by assuming that this adhesion is determined by interaction of nonactivated GPIb with vWF that has been changed in conformation by adsorption. Studies investigating thrombus formation on the connective tissue of an atherosclerotic plaque in a human coronary artery showed that VCL was able to partially prevent this thrombus formation. VCL may be of value in preventing adhesion and thrombus formation under conditions in which these processes are dependent on vWF.

Animals↗

PubMed1995

Effects of apoprotein E on intracellular metabolism of model triglyceride-rich particles are distinct from effects on cell particle uptake.

Apoprotein E (apoE) enhances uptake of triglyceride-rich lipoprotein particles (TGRP). We questioned whether apoE would also modulate intracellular metabolism of TGRP in addition to its effects on particle uptake. We prepared model TGRP with triolein and cholesteryl oleate (1:1, w/w) as the core lipids, emulsified by egg yolk phosphatidylcholine, and containing a non-degradable marker, [3H]cholesteryl hexadecyl ether. Particles were intermediate density lipoprotein-sized as determined by core lipid/phospholipid ratios (2.0-3.0/1) and gel filtration chromatography on Sepharose CL-2B. Emulsions were incubated with J774 macrophages for 5 min to 6 h at core lipid concentrations of 300-1200 micrograms/ml and 0-0.2 microgram recombinant apoE/mg core lipid. Particle uptake was determined by [3H]cholesteryl ether uptake and fluorescence microscopy in the absence and presence of apoE. Similar uptake of particles with and without apoE was achieved by utilizing a 4 times higher particle concentration in the absence of apoE. At equivalent levels of uptake, particles with apoE lead to one-half of the triglyceride mass accumulation and twice the triglyceride utilization as compared to particles without apoE. Further, apoE doubles cell cholesteryl ester hydrolysis and to a lesser extent (approximately 30%) increases cholesteryl ester resynthesis by acyl-CoA cholesterol acyltransferase. Particles, both with and without apoE, reach the lysosomal compartment as determined by colocalization with fluorescein-labeled alpha 2-macroglobulin. These results suggest that, in addition to its role in enhancing TGRP uptake, apoE has additional effects on modulating the cellular metabolism of both triglyceride and cholesteryl ester, after particle internalization.

Animals↗

PubMed1995

Recombinant human acetylcholinesterase expressed in Escherichia coli: refolding, purification and characterization.

A large-scale preparation of a recombinant human acetylcholinesterase (rhAChE) mutant harbouring a CyS580-->Ser substitution, expressed in Escherichia coli, was refolded following solubilization of the inclusion bodies. Refolded active rhAChE was purified by DEAE-Sepharose and affinity chromatography to apparent homogeneity with a specific activity (4572 units/mg) similar to that of erythrocyte AChE. The stability of the purified enzyme at 22-37 degrees C was dependent on the presence of 0.5 mg/ml BSA, and the optimum pH for stability was 9.0. rhAChE has a UV-absorbance spectrum typical of a tryptophan-rich protein, with a distinct shoulder at 290 nm and a high absorption coefficient at 280 nm (epsilon 1% = 23.1). The tryptophan residues in active rhAChE are located in an apolar environment, characteristic of a globular molecule. The difference in amino acid composition between red-blood-cell-derived and recombinant hAChE is probably reflected in their different pI values, namely 5.5-5.8 and 4.6-5.2 respectively. The CD spectrum of rhAChE is typical for an alpha/beta protein, indicating 39% alpha-helix and 22% beta-sheet. This secondary structure is similar to that determined for the Torpedo (electric fish) AChE, by both CD and X-ray crystallography. On the other hand, a purified misfolded and inactive molecule displays a decrease in alpha-helical content to 24%, accompanied by an increase in beta-sheet up to 42%, indicative of extensive changes in the conformation of the protein. On the whole, the recombinant enzyme has been refolded into a native-like conformation possessing full activity, and is thus similar to the naturally occurring red-blood-cell-derived hAChE.

Acetylcholinesterase↗

PubMed1994

Effects of particle size on cell uptake of model triglyceride-rich particles with and without apoprotein E.

The effect of apoprotein E on cellular uptake of "VLDL-size" and "IDL-size" triacylglycerol-phospholipid emulsion particles was studied in J-774 macrophages and fibroblasts. In the absence of apoprotein E (apo E), uptake of the smaller IDL-size particles was up to 2-fold higher by mass and 100-fold higher as calculated by particle number. Apo E enhanced the uptake of both VLDL-size and IDL-size emulsion particles, but the effect was greater on the uptake of larger particles (4-5-fold) as compared to up to a 2-fold increase in the uptake of IDL-size particles. In fibroblasts, particle uptake was less than in macrophages (30-50%), but preferential uptake of smaller particles was similarly observed. Particle internalization was demonstrated by 125I-apo E degradation and resistance to particle release by heparin-suramin. In the absence of apo E, cholesteryl ester of emulsion particles (prepared with trace amounts of [3H]cholesteryl ester) was hydrolyzed to free cholesterol, proving internalization and intracellular metabolism. Double-label experiments using DiI-labeled emulsion particles, in the absence and presence of apo E, showed that emulsion particles are rapidly targeted to perinuclear lysosomes. Thus, at physiological concentrations of triglyceride-rich particles, non-receptor-mediated uptake is a mechanism for the uptake of VLDL-size and IDL-size particles into cells.

Apolipoproteins E↗

PubMed1994

Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-S1 and pre-S2 antigens as compared with conventional yeast-derived vaccines.

The widely used hepatitis B virus (HBV) vaccines consist of the small hepatitis B surface (SHBs) protein produced in transfected yeast cells. The frequency of non-responders, especially among immunocompromised patients, has increased the demand for a more immunogenic vaccine. We evaluated the immunogenicity of recombinant HBs 20 nm particles secreted by transfected Chinese hamster ovary (CHO) cells, Bio-Hep-B (BioTechnology General Ltd, Israel), and compared it with yeast-derived vaccines. The CHO-derived vaccine contains the small hepatitis B surface antigen (SHBs protein) as the major component, as well as the middle HBs (MHBs, pre-S2) and the large HBs (LHBs, pre-S1) antigens. Nine groups of ten female Balb/c mice, 4-6 weeks old, were injected once intraperitoneally (i.p.) with 0.09, 0.27 or 0.81 micrograms of each of three vaccines: Bio-Hep-B or two conventional yeast-derived recombinant vaccines, Engerix-B (SmithKline Beecham, Belgium) and H-B-Vax II (Merck, Sharp & Dohme, USA) containing only non-glycosylated SHBs antigen. After 30 days, 40% of the mice injected with 0.09 microgram Bio-Hep-B had seroconverted, but none of the mice receiving the same dose of the other vaccines. The immunogenic dose in 50% of the mice at day 14 after injection was 0.13 microgram for Bio-Hep-B, but over 0.81 microgram for the other two vaccines. Mice of the strain B10/M (which are unresponsive to SHBs and MHBs antigens at the T-cell level) developed 100-fold higher anti-HBs titres after immunization with 1 microgram of Bio-Hep-B i.p., as compared with mice receiving the same amount of yeast-derived HBsAg vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Blockade of platelet membrane glycoprotein Ib receptors delays intracoronary thrombogenesis, enhances thrombolysis, and delays coronary artery reocclusion in dogs.

Von Willebrand factor and platelet membrane glycoprotein Ib receptors interact to mediate platelet adhesion and thrombogenesis in stenosed and endothelium-injured arteries. We wished to determine whether blocking glycoprotein Ib receptors with a recombinant von Willibrand factor binding domain (VCL) increases the time required for thrombus formation after injury to the coronary arteries. We also wished to determine whether, after thrombolysis with tissue plasminogen activator (TPA), VCL delays or protects against coronary artery reocclusion. Twenty-seven dogs were treated with either saline, VCL, or aspirin before thrombosis was induced in their coronary arteries by electrical injury. The time from injury to the formation of occlusive thrombi was significantly greater with VCL (70 +/- 10 minutes) and aspirin (69 +/- 20 minutes) than with saline (18 +/- 3 minutes, P < .001 and P < .05). Thrombosis was induced in 30 other dogs that then received thrombolytic treatment in four groups. Our major finding was that coronary artery reocclusion occurred in 72 +/- 11 minutes after treatment with TPA (80 micrograms/kg + 8 micrograms.kg-1.min-1) and heparin (200 U/kg) (n = 7); in 142 +/- 24 minutes after TPA, heparin, and VCL (4 mg/kg + 2 mg.kg-1.h-1) (n = 7) (compared with TPA and heparin, P < .05); in 74 +/- 13 minutes after TPA, heparin, and aspirin (5 mg/kg) (n = 8); and in 173 +/- 8 minutes after TPA, heparin, VCL, and aspirin (n = 8) (compared with TPA and heparin, P < .001). Thus, VCL increases the length of time required for thrombus formation in coronary arteries, and, when given with TPA and heparin, delays coronary artery reocclusion more effectively than aspirin.

Animals↗

PubMed1994

Abolition of cyclic flow variations in stenosed, endothelium-injured coronary arteries in nonhuman primates with a peptide fragment (VCL) derived from human plasma von Willebrand factor-glycoprotein Ib binding domain.

BACKGROUND: Platelets play an important role in the pathophysiology of acute coronary syndromes. The interaction between the platelet glycoprotein Ib receptor and von Willebrand factor is a critical event allowing platelet adhesion and aggregation and subsequent thrombus formation in vessels with high shear rates and damaged endothelium. Therefore, we tested the hypotheses that VCL, an antagonist of von Willebrand-glycoprotein Ib binding domain, (1) attenuates/abolishes cyclic flow variations in stenosed, endothelium-injured coronary arteries in nonhuman primates and (2) reduces botrocetin-induced platelet aggregation in vitro after intravenous in vivo administration. METHODS AND RESULTS: Cyclic flow variations were established in anesthetized, open-chest baboons (n = 18). The baboons were divided into three groups. One group (n = 8) received a bolus of VCL (4 mg/kg IV) followed by an infusion (6 mg.kg-1.h-1) for 90 minutes (schedule A). Another group (n = 6) received a 2-mg/kg bolus followed by an infusion of 3 mg.kg-1.h-1 for 90 minutes (schedule B). The third group received a placebo infusion of normal saline. Under dosing schedule A, cyclic flow variations were abolished in 7 of 8 baboons after 33 +/- 18 minutes and markedly attenuated in 1. The frequency of cyclic flow variations fell from 18 +/- 9.4 per hour during the control period to 1 +/- 2.5 per hour after VCL infusion, P < .002. After cessation of infusion, cyclic flow variations remained abolished in 5 of 7 animals for > 3 hours and returned in 2 of 7 after 2 to 2.5 hours. Under schedule B, cyclic flow variations were abolished in 3 of 6 baboons and markedly reduced in the remainder. The number of cyclic flow variations fell from 17 +/- 4.8 per hour during the control period to 5 +/- 4.9 per hour after the VCL infusion, P < .001. The cyclic flow variations returned spontaneously at 38 +/- 40 minutes under this dosing schedule. Placebo infusion of saline had no effect on cyclic flow frequency or severity. VCL administration was associated with slight prolongation in bleeding time and a reduction in botrocetin-induced platelet aggregation. The bleeding time increased from a control time of 88 +/- 32 to 276 +/- 204 seconds, P < .03, and from 142 +/- 28 to 176 +/- 36 seconds, P = .056, for schedules A and B, respectively. VCL decreased platelet aggregation in response to botrocetin (20 micrograms/mL), from a control value of 66 +/- 30.3 to 33 +/- 31.3 omega, P < .05, and from 64 +/- 23.5 to 46 +/- 15.8 omega, P = .006, for dosing schedules A and B, respectively. CONCLUSIONS: Therefore, administration of a peptide fragment corresponding to von Willebrand-glycoprotein Ib binding domain (1) is effective in abolishing cyclic flow variations in stenosed, endothelium-injured coronary arteries and (2) reduces platelet aggregation in vivo in response to botrocetin in nonhuman primates.

Animals↗

PubMed1993

RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome.

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.

Avian Myeloblastosis Virus↗

PubMed1993

Studies on fibronectin and its domains. I. Novel recombinant cell-binding domain of fibronectin--a modulator of human platelet functions.

The DNA sequences encoding for two proteins of the cell-binding domain (CBD) of human fibronectin (FN), namely a 33-kDa protein (aa 1329-1722) and a 40-kDa (aa 1380-1851) protein, were cloned and expressed in Escherichia coli. The interactions of the resulting rCBD proteins, refolded and purified to homogeneity, with human platelets were studied in comparison with those of the pentapeptide GRGDS. The binding of both the 33-kDa and the 40-kDa proteins to washed platelets appeared to be dependent upon platelet activation. In the case of the 33-kDa protein, binding to stimulated platelets was shown to be saturable, with Kd = 2 microM (thrombin as agonist). Moreover, both the 33-kDa and the 40-kDa proteins inhibited fibrinogen binding (at 0.1 microM) to ADP- or thrombin-stimulated platelets with IC50 values in the same concentration range. Binding seemed therefore to occur mainly at the GPIIb/IIIa receptor, and accordingly monoclonal antibodies against this receptor prevented up to 85% of the binding of the 33-kDa protein to platelets. With most stimuli the 33-kDa and the 40-kDa proteins inhibited platelet aggregation at concentrations 15- to 25-fold lower than those required by GRGDS and, in the case of the 33-kDa protein, this was shown to occur in either platelet-rich plasma, washed platelets, or whole blood. The 33-kDa protein also inhibited platelet aggregation and thromboxane A2 (TXA2) generation on the subendothelial extracellular matrix, whereas the GRGDS peptide inhibited only matrix-induced platelet aggregation, but not TXA2 formation. Furthermore, the 33-kDa protein, which is derived from the human FN CBD, seemed to be highly selective, since it inhibited the aggregation of platelets from primates only, and not from other animals tested. Finally, the 33-kDa protein did not promote fibroblast cell attachment, as was observed for both whole FN and the 40-kDa protein, thus displaying a selectivity toward platelets. In conclusion, the unique properties of the 33-kDa protein, and, in particular, its special affinity directed only toward activated primate platelets, seem to hold a promising potential for the further development of an antithrombotic agent.

3T3 Cells↗

PubMed1993

Expression and reconstitution of biologically active human acetylcholinesterase from Escherichia coli.

1. Authentic human acetylcholinesterase (AChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermo-inducible lambda PL promoter. 2. To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein. 3. rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins. A partially purified inactive recombinant protein was recovered from inclusion bodies. 4. Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine. 5. The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity.

Acetylcholinesterase↗

PubMed1993

Active oxygen species play a role in mediating platelet aggregation and cyclic flow variations in severely stenosed and endothelium-injured coronary arteries.

A canine model with cyclic flow variations (CFVs) in stenosed and endothelium-injured coronary arteries was used to examine the role of active oxygen species in platelet aggregation in vivo. We studied 90 anesthetized dogs in which the pericardial cavity was opened and the heart was exposed. The velocity of blood flow in the left anterior descending coronary artery (LAD) was monitored by a pulsed Doppler flow probe. In 67 dogs, the LADs were stenosed by applying external constrictors at the site where the endothelium was mechanically injured. CFVs developed in all 67 dogs. Treatment with the antioxidants recombinant human copper-zinc superoxide dismutase (r-h-CuZnSOD), recombinant human manganese superoxide dismutase (r-h-MnSOD), and catalase eliminated platelet aggregation-associated coronary CFVs in 63%, 62%, and 64% of animals, respectively. Intravenous infusion of epinephrine restored CFVs in most dogs. Ketanserin, a serotonin (5-hydroxytryptamine2) receptor antagonist, abolished epinephrine-restored CFVs and eliminated CFVs in dogs in which CFVs had not been eliminated by free radical scavengers. In an additional 23 dogs, the LADs were stenosed but not mechanically injured. For control studies, saline was infused into the LADs of 5 dogs. Xanthine/xanthine oxidase was infused into the LADs of 8 dogs and induced CFVs in 4. Hydrogen peroxide was infused into the other 10 dogs and induced CFVs in 9. Histological analysis of the coronary artery revealed that the intima was significantly injured by the infusion. In ex vivo platelet aggregation studies, the in vivo treatment with r-h-CuZnSOD, r-h-MnSOD, and catalase significantly inhibited platelet aggregation induced by platelet-activating factor. Thus, active oxygen species are involved in mediating platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries in vivo.

Animals↗