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At least 937 records · Page 52Linked to original sources
PubMed1983

In vivo complement activation by polyanion--polycation complexes: evidence that C5a is generated intravascularly during heparin--protamine interaction.

In vitro studies have established the complement-activating potency of polyanion-polycation interaction. By transferring the heparin-protamine model to an animal (rabbit) system that picks up ongoing intravascular complement activation by documenting acute granulocytopenia due to margination, evidence that intravascular polyanion-polycation complexing leads to instantaneous activation of the C system in vivo that goes beyond C1, C4, and C2, and results in generation of biologically highly reactive products, such as C5a, is provided. Dependence of the phenomenon on an activable complement system is documented by its complete abolishment when animals were complement depleted by cobra venom factor administration. Therefore, activation of the complement system may play a causative role for untoward effects following heparin neutralization by protamine under clinical conditions. Furthermore, these studies suggest an in vivo role of nonimmune generation of inflammatory mediators by interaction of naturally occurring polyions.

Agranulocytosis↗
PubMed1981

An E. coli gene coding for a protamine-like protein.

Several open reading frames exist in the region of the tRNATyr/1 gene of the E. coli genome. One such sequence encodes a polypeptide 33 amino acids in length that, on the basis of size and amino acid sequence, bears a striking resemblance to the protamines found in trout sperm. DNA from the transducing phage phi 80 tRNATyr/1 su3+ directs in vitro synthesis of two small basic proteins that are not made when homologous DNA containing a deletion that overlaps both the su3+ gene and the gene for the putative protamine-like protein is used. DNA from phage that have regained the parental su3+ phenotype again direct in vitro synthesis of the basic proteins. Synthesis of basic protein is inhibited by the presence of ppGpp, as would be expected if the mRNA is part of a large RNA transcript that starts at the promoter for the tRNATyr/1 gene.

Escherichia coli↗
PubMed1990

Prevention of calcification of glutaraldehyde pretreated bovine pericardium through controlled release polymeric implants: studies of Fe3+, Al3+, protamine sulphate and levamisole.

Calcification is the principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. The present study investigated controlled-release implants for prevention of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model. Either Al3+ and Fe3+ (inhibitors of the growth and dissolution rate of hydroxyapatite crystals), levamisole (alkaline phosphatase inhibitor) or protamine sulphate (charge modifier) were individually incorporated into various polymeric carriers (either silicone rubber, polyurethane or silicone rubber-polyurethane copolymer). Polymeric implants were evaluated for in vitro release kinetics, which revealed that sustained drug release was obtained from 21 d to more than 90 d from various drug matrices. In vivo efficacy was studied by co-implanting the polymeric delivery systems with glutaraldehyde pretreated bovine pericardium for 21 d using a subdermal rat model; glutaraldehyde pretreated bovine pericardium calcium levels were quantitated by atomic absorption spectroscopy in the explanted tissues. Fe3+ and Al3+ polymeric implants were the most effective for inhibiting deposition of calcium mineral. Al3+ demonstrated 82% inhibition of calcification compared to controls and Fe3+ resulted in 80% inhibition of calcification. Specific histologic staining methods showed that Fe3+ and Al3+ were localized within the devitalized cells of the explanted glutaraldehyde pretreated bovine pericardium. No adverse effects on somatic growth or recipient bone morphology were noted following controlled-release drug administration. Controlled release of protamine sulphate or levamisole did not significantly inhibit glutaraldehyde pretreated bovine pericardium calcification. It is concluded that regional controlled release of Fe3+ or Al3+ inhibits glutaraldehyde pretreated bovine pericardium calcification in the rat subdermal model without adverse effects.

Aluminum↗
PubMed1993

Haploid specific activations of protamine 1 and hsc70t genes in mouse spermatogenesis.

Protamine 1 and heat shock cognate 70 kDa protein (hsc70t) are known to be synthesized in haploid cells during spermatogenesis, and the mRNAs of these proteins have also been shown to accumulate in the haploid cells. However, it is unknown at which stage of spermatogenesis the genes for these proteins are actually activated. To examine this problem, we fractionated mouse adult testes cells at four different developmental stages, extracted their nuclei and carried out run-off assays with hsc70t and protamine 1 DNA probes. Results showed that both genes are mainly activated at the round spermatid stage. As the protein products of these genes accumulate at the later stage, it is interesting that these genes are regulated at the transcriptional and translational levels during spermatogenesis.

Animals↗
PubMed1993

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Amino Acid Sequence↗
PubMed1983

Structural heterogeneity of reconstituted complexes of DNA with typical and intermediate protamines.

The binding of the intermediate proteins phi 1 and phi 3 from the mussel Mytilus edulis to DNA was studied in comparison with the typical protamine from the squid Loligo vulgaris using precipitation curves, thermal denaturation and X-ray diffraction techniques. The properties of protein phi 1 appear to be very close to those of typical protamines while the properties of protein phi 3 are notably different. The method of reconstitution influences the structural properties of the complexes. This effect is most pronounced in the case of protein phi 3. The structural heterogeneity of the protein component in the complexes is discussed in the light of these observations.

Animals↗
PubMed1988

Heparin-, dextran sulfate- and protamine-induced release of extracellular-superoxide dismutase to plasma in pigs.

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomena in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. When given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.

Animals↗
PubMed1995

Amino acid sequences of P1 protamines and the phylogeny of eutherian mammals: a cladistic study.

Amino acid and cDNA sequences of eutherian P1 protamines, known from publications of other authors, were compared by a cladistic method. Fish, toad and bird protamines were used for the pertinent "outgroup comparisons", i.e. they provided relevant data for the comparative alignment of the sequences and for the recognition of evolutionary trends. In the sequence positions compared, each amino acid was individually assigned as a plesiomorphic or apomorphic character state (qualitative treatment). The resulting phylogenetic tree (Fig. 2) is only partially in accordance with common ideas on eutherian phylogeny. Disagreements refer to the branching points of Perissodactyla, Lagomorpha and Rodentia.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Stp-1 gene coding for rat spermatid nuclear transition protein 1 (TP1): homology with protamine P1 and assignment of the mouse Stp-1 gene to chromosome 1.

Spermatid transition protein 1 (TP1) is a 54 amino acid (aa), highly basic chromosomal protein found in mammals during the brief period when histones are being replaced by protamines in the haploid phase of spermatogenesis. Using a cDNA clone as probe, we have isolated the gene (Stp-1) coding for rat TP1 from a population of recombinant bacteriophage lambda. The nucleotide (nt) sequence was established from a point 126 nt upstream from the mRNA cap site to a point about 30 nt downstream from the predicted site of polyadenylation. The gene contains a single intron separating the codon for aa 45 of the mature protein. Comparison of the nucleotide sequences for Stp-1 and the mouse gene coding for protamine P1 suggests a possible evolutionary relationship. Southern blot hybridization to genomic DNA isolated from a panel of mouse-hamster somatic cell hybrids unambiguously mapped Stp-1 to mouse chromosome 1.

Amino Acid Sequence↗
PubMed1993

Identification of conserved potential regulatory sequences of the protamine-encoding P1 genes from ten different mammals.

In order to detect regulatory conserved DNA elements within the protamine 1-encoding gene (P1) promoter, we have sequenced this region from the rat, guinea pig, gorilla, orangutan, anubis baboon and red monkey P1 genes and compared it to the homologous human, bull, boar and mouse nucleotide (nt) sequences. We demonstrate the presence of a consensus sequence, HSMCYTCAYAAT (Prot1C: protamine 1 consensus), from nt position -64 to position -53 in all P1 genes whose promoter sequences are now known. We also show that sequences similar to Prot1C are found in the promoter region of other testis-specific genes, such as the transition protein 1-encoding gene promoter which is thought to have derived from the P1 genes. The relevance of this conserved element in the expression of P1 genes is strongly supported by the recent demonstration of a mouse testis trans-acting factor [Tet-1; Tamura et al., J. Biol. Chem. 267 (1992) 4327-4332] which binds and matches in the mouse the first 11 bp of the corresponding consensus Prot1C sequence reported here. Another highly conserved element (TGTGAGG) has been identified 20 +/- 3 nt upstream from Prot1C. This sequence forms a perfect palindrome with the central 7 nt of Prot1C and is absent in the homologous region of other genes. Further upstream, at positions -113 to -132, a third highly conserved region is present (MATGCCCATATWTGGRCAYG) which is similar to the c-fos SRE (serum-response element) and contains the central core common to all SREs. This element has not been found in the homologous region of other sperm-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Chromosomal localization and structure of the human P1 protamine gene.

The human P1 protamine gene and mRNA were amplified with the use of the polymerase chain reaction and cloned into PTZ19R. The sequences were determined which revealed the presence of an intron. Southern and Northern hybridization analyses showed that the gene was single copy and that the mRNA was approximately 450 bases long. The gene was mapped to chromosome 16 with the use of a somatic cell hybrid panel and localized to the 21 region of the q arm by in situ hybridization of the human P1 protamine probe to human metaphase chromosomes.

Amino Acid Sequence↗
PubMed1994

Determination of immunoglobulin M concentration by piezoelectric crystal immunobiosensor coated with protamine.

In the present study, the specific binding between protamine and immunoglobulin M (IgM) has been exploited to construct a piezoelectric crystal based immunobiosensor for the determination of concentration of IgM. The system consisted of highly stable IC based oscillator, 8-digit frequency counter and modified piezoelectric crystal device. The crystal surface was physically modified and chemically treated (refluxed) with strong acid to produce stable hydroxylic groups of silicon oxide. This modified surface reacted strongly with coupling reagents for binding of protein molecules. The protamine was immobilized by using either gamma-aminopropyltriethoxy silane (gamma-APTES) or 2.2.2-trifluoroethanesulfonyl chloride (tresyl chloride). Scanning electron microscope images of piezo crystal revealed that tresyl activated surface presented more surface area for binding than gamma-APTES modified surface and showed better sensitivity. This immobilization technique also improved the reproducibility and long term stability of the detection system. Using the system described, the IgM concentration up to the level of 10 ng/ml could be detected without interference of IgG.

Biosensing Techniques↗
PubMed1995

The ISFET based heparin sensor with a monolayer of protamine as affinity ligand.

The ion-step measuring method was used to determine absolute heparin concentrations in PBS and blood plasma with a Ta2O5 ISFET on to which a monolayer of protamine had been immobilized. Heparin is a highly negatively charged polysaccharide, which is used clinically to delay the clotting of blood. Protamine acts as an affinity ligand for heparin. The response of the ISFET system on a step-wise increase in the electrolyte concentration (a so-called ion-step) is a transient change of the output voltage, which is related to the surface charge density of the ISFET gate oxide. After 2 mins of incubation in a plasma sample containing heparin, the amplitude of the transient ISFET response to an ion-step showed a linear relation to the heparin concentration. In blood plasma, heparin concentrations between 0.3 and 2.0 Units/ml could be determined with an accuracy of +/- 0.08 Units/ml. Heparin concentrations in different plasma samples of heparinized patients were determined and compared with the APTT. No direct relation was found between the APTT and the heparin concentration, but this result was not surprising.

Biosensing Techniques↗
PubMed1990

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography.

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.

Amino Acid Sequence↗
PubMed2004

Bivalirudin versus heparin and protamine in off-pump coronary artery bypass surgery.

BACKGROUND: Bivalirudin is a short-acting direct thrombin inhibitor, with advantages over unfractionated heparin for anticoagulation in cardiac surgery. We hypothesized that bivalirudin is not associated with a clinically important increase in blood loss compared with heparin with protamine reversal in patients undergoing off pump coronary artery bypass (OPCAB) surgery. We also assessed flow with angiography at 3 months using a modified Thombolysis in Myocardial Infarction (TIMI) grade in the grafted coronary arteries. METHODS: One hundred patients were randomly assigned to receive bivalirudin (0.75 mg/kg bolus, 1.75 mg/kg/h infusion) or heparin (150 to 300 U/kg bolus) with protamine reversal. RESULTS: A median of 3 (range, 1 to 5) grafts were inserted per patient. Blood loss for the 12 hours after study drug initiation in the bivalirudin group (median, 793 mL; interquartile range, 532 to 1,214 mL; range, 320 to 4,909 mL; n = 50) was not significantly greater than in the heparin group (median, 805 mL; interquartile range, 517 to 1,117 mL; range, 201 to 2,567 mL; n = 50; p = 0.165). Median graft flow was 3.0 in the bivalirudin group (n = 40) and 2.67 in the heparin group (n = 39; p = 0.047). The bivalirudin group had more patients with grade 3 (ie, full) flow in at least 1 graft (100% versus 90%; p = 0.04), a trend toward more patients with grade 3 flow in all grafts (60% versus 38%; p = 0.06), and more grafts with grade 3 flow (82% versus 67%; p = 0.03). CONCLUSIONS: Anticoagulation for OPCAB surgery with bivalirudin was feasible without a clinically important increase in perioperative blood loss. Graft flow was better in the bivalirudin patients; the impact of this on clinical outcomes requires a larger study.

Aged↗
PubMed2005

Albumin-protamine-oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect.

Antisense oligonucleotides have been used as a specific tool to inhibit the expression of disease associated genes for many years. Unfortunately, oligonucleotides are polyanionic macromolecules which have a weak permeability through biological membranes and are rapidly degraded by nucleases. The purpose of this work is to characterise a new drug delivery system developed by [V. Vogel, D.Lochmann, J. Weyermann, G. Mayer, C. Tziatios, J.A. van der Brock, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which allows an increased cellular uptake and an intracellular dissociation of the oligonucleotides. The new system based on nanoparticles (NPs) consists of human serum albumin, protamine sulphate and antisense-oligonucleotides (AlPrO). We tested these new nanoparticles on mouse fibroblasts which were stably transfected with a N-methyl-D-aspartate (NMDA) receptor (NR). This cell line enabled us to perform in vitro studies of cellular uptake, intracellular dissociation and effect of the antisense-oligonucleotide in a simple excitotoxicity model. We compared our findings with free oligonucleotides and a commercial available liposomal preparation (DOTAP). We found a 12-fold increased cellular uptake of oligonucleotides in comparison to free oligonucleotides while 100% of the cells were transfected. The AlPrO-NPs showed very low cytotoxic side effects during a 24 h application. We saw an antisense effect of about 35% in a functional assay as well as on the protein level (western blot). The results of the cell penetration and the antisense assay demonstrated that AlPrO nanoparticles are promising carriers for oligonucleotide administration.

Animals↗
PubMed2005

Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular distribution.

Oligodesoxynucleotides (ODNs) or the corresponding phosphorothioates (PTOs) spontaneously form spherical nanoparticles ("proticles") with protamine in aqueous solutions. The proticles can cross cellular membranes and release the ODNs within the cells. Thus, they represent a potential drug delivery system. The major disadvantages of this system are a lack of stability in salt solutions and its inability to also release PTOs. The present study shows, using PTOs and protamine free base, that these shortcomings can be eliminated by the addition of human serum albumin (HSA) as a third component to the starting mixture. The "ternary" proticles thus obtained contain maximally a few percent of the HSA that was originally present. Nevertheless, they differ from the previously studied "binary" proticles: (1) They are stable in salt solutions for at least several hours. (2) They show a high cellular uptake into murine fibroblasts, and they readily release the PTOs after uptake. The ternary proticles therefore represent a considerable improvement over binary proticles for use as drug delivery systems.

Albumins↗