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PubMed1997

Tissue-specific protein-DNA interactions of the mouse protamine 2 gene promoter.

During spermiogenesis, the haploid phase of spermatogenesis, the genome is packaged into a highly compacted form and this process requires replacement of histones by protamines. In the mouse, protamines are encoded by two genes, which are transcriptionally regulated in testis. To understand the regulation of transcription of the mouse protamine 2 (mP2) gene, the tissue-distribution of sequence-specific interactions between nuclear proteins and promoter DNA sequences have been analyzed. Protein binding to the promoter region from -370 to +65 was studied using DNase I footprinting and gel shift assays. Five protein binding sites were identified, which are recognized by nuclear proteins from either testis or liver. Site 1 from -64 to -48, contains part of a cAMP responsive element (CRE), which in testis is recognized by CREM tau, an activator of post-meiotic transcription. Testicular protein(s) also binds to three other promoter domains: site 2, -87 to -67, a region containing a CAAT box, and sites 4 and 5, -239 to -210 and -328 to -311, sequences with similarity to consensus steroid hormone responsive elements (HRE). In contrast, interactions between the mP2 promoter and nuclear factors from liver, a tissue in which the mP2 gene is not transcribed, are observed at sites 1, 2, and 4, as well as at an additional region at site 3, -202 to -175. Because occupancy at site 3 appears to correlate with inactivation of the gene in non-testicular tissues, whereas testicular protein binding at site 5 appears to be associated with active transcription, we conclude that the mP2 promoter displays intricate tissue-specific patterns of protein/DNA interactions at key regulatory elements.

Animals↗
PubMed1998

Safety issues in heparin and protamine administration for extracorporeal circulation.

This article reviews past approaches to heparin and protamine dosing and summarizes current practice. The author elucidates his experience with the Celite activated coagulation time (ACT), with attention to his adoption of a value of 400 seconds for this time; the adoption of an ACT of 480 seconds by Bull et al (J Thorac Cardiovasc Surg 69:674-684, 1975) and Young et al (Ann Thorac Surg 26:231-240, 1978); the proposed use of heparin response curves by Bull et al; the author's experience with a unitized dosing system to individualize dosing of heparin; and the use for this purpose by Despotis et al (J Thorac Cardiovasc Surg 110:46-54, 1995) of a system based on protamine titration. In more than 270 adult cardiac surgery patients, the unitized dosing system identified patients with high sensitivity or resistance to heparin and facilitated exact individualized doses to be given to produce the desired effect. Thus, less heparin was used in short bypass runs. Patients received less protamine than they would have with any other formula, and there was less blood loss and fewer transfusions required. Currently, no claims for efficacy or safety can be made for maintaining heparin concentrations greater than 3 U/mL. Pending further clarification, heparin dosage cannot be safely reduced when using heparin-bonded circuits. Aprotinin is not a procoagulant during cardiopulmonary bypass. Emerging studies suggest that graft patency is not affected by aprotinin use. The Celite ACT should not be used to monitor heparin effect and safety when using aprotinin; the kaolin ACT should be used instead.

Adult↗
PubMed1976

Effect of sex hormones on protamine inactivated and resistant postheparin plasma lipases.

Effects of estrogens and the synthetic anabolic-androgenic steroid, Oxandrolone, on extrahepatic (protamine inactivated) and hepatic (protamine resistant) lipases in postheparin plasma, were assessed in 15 subjects with familial hypertriglyceridemia. In six women receiving conjugated equine estrogen (1.25 mg/day), mean (+/- SE) postheparin protemine inactivated triglyceride lipase (PI-TGL) was depressed to 0.23 +/- 0.10 mumol FFA/ml/hr, and protamine resistant lipase was depressed to 5.3 +/- 0.5 mumol FFA/ml/hr. In the 2-wk period after estrogens were discontinued, PI-TGL remained depressed, 0.54 +/- 0.24, while PR-TGL increased to 7.3 +/- 0.88, p=less than 0.05. Mean triglycerides fell insignificantly from 628 +/- 136 to 447 +/- 44 mg/100 ml when estrogens were discontinued. There was no significant correlation between changes in PR-TGL and triglycerides when estrogens were stopped. In four women with familial hypertriglyceridemia, Oxandrolone significantly increased PR-TGL in two, increased PI-TGL in three, and reduced triglycerides in two. In five men with familial hypertriglyceridemia, Oxandrolone reduced triglycerides in four, increased PR-TGL in four, but had no effect on PI-TGL. For the nine hypertriglyceridemic subjects increments in PR-TGL failed to correlate significantly with decrements in triglyceride, (r=0.309, p is greater than 0.1). Selective alteration of PR-TGL and PI-TGL by estrogens and Oxandrolone may provide an approach to better understanding of the interaction of lipases and triglycerides in familial and acquired hypertriglyceridemia.

Adult↗
PubMed1987

A comparison of the pharmacokinetics of human protamine sodium insulin with human isophane insulin following subcutaneous injection in normal subjects.

The pharmacokinetics of human protamine sodium insulin 0.6 mg% were compared with those of human isophane zinc insulin using the glucose clamp technique. The insulin preparations were administered subcutaneously at a dose of 0.5 U/kg in 7 normal subjects; a placebo injection served as control. Plasma insulin and C-peptide concentrations were then measured over 24 hours. After correction of the results to allow for endogenous insulin secretion the two insulin preparations gave similar values throughout the study except that protamine sodium insulin achieved an earlier peak in plasma insulin concentration (90 minutes) compared to isophane (120 minutes). When measured by an index of the dextrose infusion rate required to maintain euglycaemia the two insulin preparations gave similar values for the first 16 hours, maximum values being obtained during the fourth hour after administration. Using this index isophane zinc insulin gave higher values during the 17th, 19th and 21st hours following administration suggesting it may have a slightly more prolonged action than protamine sodium insulin.

Adult↗
PubMed1979

[Haemodynamic effects of depot zinc protamine glucagon in heart failure (author's transl)].

In contrast to intravenously-administered crystallene glucagon, which acts for 20 minutes only, the depot form, zinc protamine glucagon, shows a prolonged haemodynamic action. Fourteen patients with pre-existing heart failure received a single dose of 20 mg Zn protamine glucagon intramuscularly. The stroke volume and cardiac output were increased, whereas the mean and end-diastolic pulmonary pressure were decreased, indicating a positive inotropic action of the administered drug. Heart rate and mean arterial pressure remained almost unchanged. The haemodynamic changes started 60 minutes after intramuscular administration of the drug, reached a maximum effect at 3 hours and started to decrease after the fourth hour. Zn protamine glucagon can, therefore, be considered a beneficial drug in the treatment of digitalis-resistant heart failure on the basis of its long duration of action and easy route of administration.

Aged↗
PubMed1984

Purification and some properties of protamine kinase from rabbit brain.

A protein kinase of high specificity towards protamine and ATP was isolated from rabbit brain and purified about 200-fold. The enzyme does not catalyse phosphorylation of acidic proteins, e.g. casein and phosvitin, nor is it susceptible to cyclic AMP or protein inhibitors of the cAMP-dependent kinase. The enzyme shows the highest activity in the presence of 10 mM-Mg2+ and 2 mM-dithiothreitol at pH 8. The only phosphorylated amino acid found in protamine phosphorylated by the enzyme was phosphoserine.

Adenosine Triphosphate↗
PubMed1988

Influence of blood flow and the effect of protamine on the thromboresistant properties of a covalently bonded heparin surface.

Polyethylene tubings, 2-mm inner diameter and the length of 1 m, untreated or furnished with a covalently bonded heparin surface layer, were inserted as arteriovenous shunts bilaterally in dogs. By compressing the middle part, the initial blood flow was adjusted to 10 or 40 mL/min. The thrombogenicity of the tubings was assessed by the patency of the shunts and by assaying the generation of fibrinopeptide A (FPA) in arterial blood and in blood after its passage through the shunts. In untreated shunts clotting rapidly occurred preceded by high FPA generation in blood passing through the shunts. The blood flow in heparinized shunts remained unchanged throughout the test period. At the low flow rate a certain degree of FPA generation in the shunts occurred. At the high flow rate no changes in FPA levels occurred. The function of the heparin surface is thus flow rate dependent. Systematic heparinization and subsequent neutralization with protamine or administration of protamine alone did not interfere with the function of the heparin surface.

Animals↗
PubMed1991

Polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells.

We investigated the effects of the basic peptides polyarginine, protamine, and polylysine on dispersed bovine parathyroid cells. All three peptides produced a dose-dependent inhibition of dopamine-stimulated cAMP accumulation, with half-maximal inhibition at 4 x 10(-8), 1.5 x 10(-7), 3 x 10(-7), and 2 x 10(-6) M, respectively, for polyarginine, protamine, and two preparations of polylysine of molecular weights 10,200 and 3800. The inhibition of cAMP accumulation was reversible and was blocked by preincubating the cells overnight with 0.5 micrograms/ml of pertussis toxin. The same peptides also inhibited PTH release at similar concentrations, markedly stimulated the accumulation of inositol phosphates at two- to threefold higher concentrations, and produced transient increases in the cytosolic Ca2+ concentration (Cai) in fura-2-loaded parathyroid cells. The polylysine-evoked spike in Cai persisted despite the removal of extracellular Ca2+, indicating that it arose from intracellular Ca2+ stores. Exposure of the cells to elevated extracellular magnesium (Mg2+) concentrations elicited a similar spike in Cai but blocked the Cai transient in response to subsequent addition of polylysine, or vice versa. Thus, Mg2+ and polylysine mobilize Ca2+ from the same intracellular store(s). These results indicate that highly basic peptides closely mimic the effects of polyvalent cations on parathyroid function, suggesting that both agents may regulate parathyroid function via similar biochemical pathways.

Animals↗
PubMed1990

Protamine-histone replacement and DNA replication in the male mouse pronucleus.

The protamine to histone replacement in fertilized mouse eggs was studied by using antibodies to these proteins. Its course was followed with respect to DNA replication by autoradiography of 3H-thymidine-labeled fertilized eggs. It was found that protamines were replaced by histones before the onset of DNA replication.

Animals↗
PubMed1991

Analysis of protamines isolated from alcohol preserved epididymides.

Protamines 1 and 2 have been isolated from the sperm of frozen and isopropanol preserved Syrian hamster (Mesocricetus auratus) epididymides and analyzed by gel electrophoresis, high-performance liquid chromatography (HPLC), and amino acid analysis and sequencing. The results show that alcohol preservation does not alter the primary structure of the two sperm nuclear proteins and that the preservation of mammalian reproductive organs in alcohol is a viable alternative to freezing tissues collected in the field. Sperm were isolated from tissues fixed in isopropanol for as long as 7 months without detectable adverse effects on either the isolation of sperm or the primary structure of the protamines.

1-Propanol↗
PubMed2005

Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells.

Two FDA-approved agents, ferumoxides (Feridex), a suspension of superparamagnetic iron oxide (SPIO) nanoparticles and protamine sulfate, a drug used to reverse heparin anticoagulation, can be complexed and used to label cells magnetically ex vivo. Labeling stem cells with ferumoxides-protamine sulfate (FePro) complexes allows for non-invasive monitoring by MRI. However, in order for stem cell trials or therapies to be effective, this labeling technique must not inhibit the ability of cells to differentiate. In this study, we examined the effect of FePro labeling on stem cell differentiation. Viability, phenotypic expression and differential capacity of FePro labeled CD34 + hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) were compared with unlabeled control cells. Colony-forming unit (CFU) assays showed that the capacity to differentiate was equivalent for labeled and unlabeled HSC. Furthermore, labeling did not alter expression of surface phenotypic markers (CD34, CD31, CXCR4, CD20, CD3 and CD14) on HSC, as measured by flow cytometry. SDF-1-induced HSC migration and HSC differentiation to dendritic cells were also unaffected by FePro labeling. Both FePro-labeled and unlabeled MSC were cultured in chondrogenesis-inducing conditions. Alcian blue staining for proteoglycans revealed similar chondrogenic differentiation for both FePro-labeled and unlabeled cells. Furthermore, collagen X proteins, indicators of cartilage formation, were detected at similar levels in both labeled and unlabeled cell pellets. Prussian blue staining confirmed that cells in labeled pellets contained iron oxide, whereas cells in unlabeled pellets did not. It is concluded that FePro labeling does not alter the function or differentiation capacity of HSC and MSC. These data increase confidence that MRI studies of FePro-labeled HSC or MSC will provide an accurate representation of in vivo trafficking of unlabeled cells.

Biomarkers↗
PubMed1995

Demonstration of trans-acting factors binding to the promoter region of the testis-specific rat protamine P1 gene.

Potential regulatory DNA elements within the rat protamine P1 promoter region have been identified using gel retardation assays and DNase I footprinting analysis with rat nuclear extracts obtained from different tissues. Distinctive gel shift bands are generated by rat nuclear extracts from mature testis, kidney, brain, spleen and liver, which bind to the cis-acting SRE (Serum response element) and Prot1C (Protamine 1 Consensus) oligonucleotide sequences [Queralt R. and Oliva R. Gene. 133 (1993) 197-204]. In vitro DNase I footprinting analysis demonstrates protection of the SRE region at positions -124 to -115. In addition, we have detected protection in two new regions adjacent to the SRE that we called SAP (SRE Adjoining Protection; nt -153 to -141) and SEP (SRE Extended Protection; nt-114 to -100), respectively. These sequences, SAP and SEP, show no apparent consensus homology with cis-acting elements of other known transcription factors. Two additional DNase I protected regions have also been found at positions +27 to +46 and +61 to +88, the first of which contains the sequence +42 TCGNNNNNGCCAA +30 recognized by nuclear factor I (NFI).

Animals↗
PubMed1993

Chromatin fibers with different protamine and histone compositions.

Previous studies have established that chromatin fibers which contain histones have variable diameters in different biological materials. In this paper we show that chromatin fibers are also present in spermatozoa and spermatids of bivalve molluscs in which some or all histones have been replaced by protamines. In all cases thick chromatin fibers are formed, with a diameter in the range 25-50 nm, depending on the species. We conclude that the formation of chromatin fibers is determined by the partial neutralization of the DNA charges with any histone or protamine, rather than due to a precise association of nucleosomes. This conclusion is in agreement with current theoretical work which shows that the diameter of complexes of DNA with counterions is determined by an overall balance of the interaction forces present.

Animals↗
PubMed1994

Rat spermatid basic nuclear protein TP3 is the precursor of protamine 2.

A second protamine, P2, synthesized as a precursor in late spermatids, is present in the sperm of some mammals such as the mouse. On the other hand, the sperm of other mammals such as the rat do not contain the mature P2, although they have the gene for the protein. Here we report that the P2 precursor is synthesized in late spermatids of the rat and is identical to the protein named TP3. TP3 and four other proteins present in the sonication-resistant spermatids of rat (steps 13-19) immunoreact with an antibody to human P2. By microsequencing the amino terminal ends of the immunoreacting bands, we show that TP3 is the primary translational product of the rat P2 gene and that at least two of the other bands are cleavage products of the precursor molecule. The cleavages occur between residues 10 and 11 and between 19 and 20, as is the case in the mouse. Basic nuclear protein extract of rat epididymal sperm also contains a protein that weakly immunoreacts with the antibody. This protein migrates along with the fastest immunoreacting band from late spermatids. By quantitating the amount of the precursor molecules, we conclude that appreciable levels of P2 precursors are present in rat late spermatids (about 9% of total protamines), but the processing appears to be impaired and most of the protein is lost from the chromatin by the spermatozoa stage.

Amino Acid Sequence↗
PubMed1995

Regulation of neuronal nitric oxide synthase by histone, protamine, and myelin basic protein.

We examined the effects of endogenous basic proteins rich in the amino acid L-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.

Amino Acid Oxidoreductases↗
PubMed1981

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

Animals↗
PubMed1983

[Individual heparin and protamine dosage in heart surgery].

Several methods of heparin administration before and heparin neutralization after cardiopulmonary bypass (CPB) have been proposed, based on individual dose-response curves of the activated clotting time (ACT). We investigated the use of a regression line, established from simultaneous double determinations of the ACT before and after serial doses of heparin (240 and 360 IU/kg), calculated by computer to determine the doses of heparin and protamine needed. Administration of protamine doses calculated from ACT measurements at termination of CPB in 31 patients resulted in ACT measurements which were not significantly different from pre-heparin levels, however. ACT values after 360 IU/kg heparin varied widely. Among 351 patients studied 62.9% had ACT values greater than 450 s, 27,9% had ACT values between 450 and 350 s, and 9.1% had ACT values less than 350 s. Our studies produced an interesting finding. Mean ACT values of women after both doses of heparin were significantly smaller (p less than 0.0000) (359.7 +/- 6 s, n = 144 and 452.4 +/- 9 s, n = 160) than those of men (393.8 +/- 3.7 s, n = 501 and 501.0 +/- 5.1 s, n = 549).

Cardiopulmonary Bypass↗
PubMed1989

Histamine 2 receptor blocker in the treatment of protamine related anaphylactoid reactions: two case reports.

Two case reports are described of acute anaphylactoid reactions following the administration of protamine to reverse the anticoagulation effect of heparin in patients undergoing coronary artery bypass graft surgery. The administration of cimetidine seemed to reverse the anaphylactoid reaction after conventional treatment with epinephrine, H1 receptor blocker, and steroids had failed. We recommend that H2 receptor blockade be included with other drugs in the treatment of anaphylactoid reactions following protamine, and possibly after anaphylactoid reactions associated with other substances.

Adult↗