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A new procedure for estrogen receptor assay in breast cancer tissue is discussed. According to the method cytosol receptor level is interpreted as the estradiol binding capacity of protein which is precipitated by protamine sulfate from the cytosol fraction of tumor.
The synthesis of peptides containing blocks of arginyl residues is proposed through amidination of the corresponding ornithyl analogs. In order to test this strategy the ornithyl analog of the C-terminal sequence 52--65 of galline was synthesized by the conventional method. The amidination reaction, performed on fragments of different length and ornithyl-residue content, quantitatively converts ornithines into arginines. The strategy proposed may represent a powerful tool for the synthesis of protamines and other basic proteins.
A double blind study testing the effect of long-acting zinc-protamine-glucagon (7.5 mg every 12 hours for 4--5 days) was carried out in acute pancreatitis. There were 32 patients in ZP-glucagon- and 39 patients in the placebo group. The results show that glucagon had a slightly favourable effect on the general clinical course of the disease but they do not give enough evidence for routine use of glucagon in pancreatitis.
The interaction of basic oligopeptides--F2aI histone, iridine and salmine protamines synthetic fragments with DNA has been studied by the methods of thermal denaturation, circular dichroism and equilibrium dialysis. All the peptides investigated are albe to stabilize the DNA helix and change its conformation. This ability grows with the increase of a number of basic residues in the peptide molecule and decreases when there is a serin residue on the end. The binding of the peptide with DNA obtains a cooperative character if 4--6 arginine clusters are present in the molecule. The comparison of the binding coefficient with the thermal denaturation data allowed to propose an important role of nonelectrostatic forces on the binding of the peptides to DNA.
The Mg2+-dependent factor capable of introducing the superhelical turns into circular closed DNA in the presence of core histones H2a, H2b, H3, H4 or protamine in solution at physiological conditions has been isolated from Drosophila melanogaster embryos. The factor does not work with histone H1, cytochrome c, poly-L-lysine or tetralysine. It is assumed that the factor is responsible for folding of core histones into nucleosomes.
The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques. The dynamic behaviour, examined through the evaluation of the spin lattice relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone alpha-carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 +/- 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side-chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase. Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.
When mouse mammary carcinoma (FM3A) cells were treated with egg agglutinin of Rana catesbiana for 15 min at 25 degrees C, percent total particle number of both cell aggregates and single cells was in direct proportion to the cell electrophoretic mobility. Phytohemagglutinin P mediated agglutination proceeded with biphasic kinetics: the higher the concentration of phytohemagglutinin P the shorter was the lag period between the first and second stages of agglutination. In protamine-mediated agglutination, the percent total particle number was reduced at low concentrations, while the electrophoretic mobility reduced only at high concentrations. Agglutinating and cytotoxic activities of these three reagents were in an intimate relation: the higher the agglutinating activity, the greater was their cytotoxic activity.
The role of heparin structure in neutralization by the neutralizing substances (NS) platelet factor 4 (PF4) and protamine sulfate (PS) was investigated using a thrombin clotting assay and a series of more homogeneous heparin fractions varying systematically in charge density (Z). For a given heparin, plotting inverse clotting times measured without NS, and in the presence of PF4 or PS, vs heparin concentration yielded approximately parallel straight lines displaced horizontally according to the amount of NS. Potencies of heparin fractions in the absence of NS, or in the presence of PF4 of PS, depended almost identically upon Z2. Small but significant quantitative differences in potency among equivalent fractions from different heparins showed both PF4 and PS had a slight preference for the least active subfraction of decolorized heparins, but for the most active subfraction of undecolorized heparins. Neutralization of heparin by PF4 and PS probably proceeds by similar mechanisms, but the details of structure outside the antithrombin III-binding oligosaccharide of heparin may enter in differently.
The circular dichroism (CD) spectra of DNA complexes with stellins A and B, protamines from Acipenser stellatus, in 2,5 x 10(-4) M EDTA were studied. The CD spectrum of the stellin B--DNA complex practically coincides with that of original DNA. In the CD spectrum of the stellin A--DNA complex the positive band amplitude increases at 275 nm, while the negative band amplitude decreases at 245 nm at increasing of protein/DNA ratio. The resulting A-like CD spectrum is apparently coupled with the changes in the parameters of DNA double helix. The changes in the CD spectra in the stellin A--DNA complex are correlated with a higher capacity of stellin A for helix formation in ethanol. The data obtained suggest that individual components of stellin play a specific role in the structural organization of nucleoprotamine.
A single diluted protamine sulphate test has been employed by the Authors in several respiratory diseases in order to define its diagnostic usefulness. Such a test, easily performed in all clinical chemistry laboratories and very cheap, has resulted to be positive mostly in the pleuropulmonary pathology following microembolic spread to the lung (pulmonary infarction), with a higher significance the earlier the test has been made. The test has appeared to be also able to differentiate infarctual active pleurites (positive) from tubercular eudates and transudates (negative).
Results are presented of individualized anticoagulatory management during open heart surgery in two groups of 20 patients comparing colloid versus non-colloid priming of the extracorporeal circuit. Advantages of individualized dosages of heparin according to a constructed dose response curve and controlled by measuring Activated Coagulation Times (ACT) were safer adaptation to individual variability in heparin sensitivity, reduction of incremental heparin doses during cardiopulmonary by-pass and nearly 50% reduction of protamine dosage as compared to conventional heparinprotamin protocols. No significant influence of the type of priming used in the extracorporeal circuit on coagulatory behaviour could be detected.
The authors report the new protocol the apply for prevention of central venous catheter-related infections in haemodialysis. The used preparation is a combination of protamine sulphate and rifampicin and gives better results that povidone iodine in the same conditions.
Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodinated and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivatized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.
The histone H3 (H3), metallothionein-B (MT-B), protamine (PT) promoters of sockeye salmon were cloned by PCR using primers based on the DNA sequences of the rainbow trout promoters. Comparison of the DNA sequences of the sockeye salmon and the rainbow trout histone H3, MT-B, and PT promoters revealed that their DNA sequences and putative transcriptional cis-elements are remarkably conserved. Promoter activity of the sockeye salmon H3, MT-B, and PT promoters were examined by transfection studies using cell lines from fish and human. The H3 and MT-B promoters were shown to be active in all fish cell lines but were only weakly active in HeLa and GM637 cells. The MT-B promoter was also inducible by zinc and cadmium in RTH-149 cells. In contrast, the PT promoter was inactive in all fish cell lines. Transfection experiments also established that the HCMV IE promoter has very strong activity in the various fish cell lines. Our results suggest that the HCMV IE promoter and the sockeye salmon H3 and MT-B promoters are useful for the construction of expression vectors for transgenic and gene transfer studies in fishes.
Using fluoresceine isothiocyanate labeled protamine sulfate it is possible to stain rat mast cell fractions of isolated periotoneal cells in a selective way. The cells are not degranulated, though they have lost their histamine completely. The method shows the dissociation of the mediator release and the extrusion of the granules.
Growth factors, such as bFGF, have been shown to exert autocrine and paracrine effects on the growth of Kaposi's Sarcoma (KS)-derived cells, which suggested that the inhibitors of angiogenesis may be promising for KS treatment. However, KS lesions have been found to continue to enlarge after patients had been treated with FGF binding antagonists such as suramin. We investigated the effect of protamine and suramine on the growth of KS derived cells in vitro. Although both of these agents which are FGF binding antagonists were found to inhibit the incorporation of 3H thymidine in KS-derived cultured cells, increased expression of bFGF, FGF5 and the FGF receptor was observed after the KS cells were exposed to these substances. These results might explain the clinical observation that FGF binding antagonists such as suramin caused an apparent stimulation of KS tumor growth when administered systemically to patients with AIDS-related KS.
PURPOSE: A bladder injury model was developed using protamine sulfate (PS) and endotoxin lipopolysaccharide (LPS) administered intravesically to female Sprague-Dawley rats. MATERIALS AND METHODS: Experimental and control animals were catheterized and intravesically exposed to PS-LPS, PS, LPS, or phosphate buffered saline. After 4, 24 or 72 hours, rats were sacrificed. Urines and bladder tissues were then obtained. Bladder mucosal permeability was evaluated by measuring 14C-urea uptake 24 hours after injury. Repeated instillations of PS/LPS were also made in another group of rats over a period of 5 weeks to attempt to establish a more serious mucosal injury, possibly reflected by altered staining of the collagen IV component of the urinary basement membrane (UBM). RESULTS: Histological examination of the tissues indicated a maximal inflammatory response in the mucosa 4 hours after instillation of PS/LPS. Neutrophils and macrophages in close proximity to the UBM and intraepithelially could be demonstrated. Bladder permeability was significantly altered (26.9% 14C-urea uptake) in rats assayed 24 hours after the PS/LPS treatment, but not after exposure to PS or LPS alone (11.9 and 17.5%, respectively). Protease activity detected in urines from experimental, but not control, animals coincided with the appearance of inflammatory cells in the lamina propria. Inflammatory injury did not appear to alter the collagen IV staining of the UBM. CONCLUSIONS: This rat bladder injury model is useful for examining controversial issues regarding bladder wall structure-function alterations induced by inflammation and possibly important in the pathobiological mechanisms involved in some patients with interstitial cystitis.
The protamine sulphate test (PST) was studied as a screening test for intravascular coagulation in experimental Babesia bovis infections. The test became positive in 39 of 42 splenectomised calves and two of six intact cattle tested during acute infections. Haematological studies during the course of infection in two splenectomised and six intact cattle indicated that a positive PST was reliable evidence for intravascular coagulation. Of four splenectomised and one intact cattle that were positive to the test and were subsequently autopsied, all had histopathological changes indicative of disseminated intravascular coagulation (DIC). It was concluded that the PST could be used as a screening test for DIC in acute bovine babesiosis.