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At least 973 records · Page 54Linked to original sources
PubMed2006

Synthesis and characterization of a novel paramagnetic macromolecular complex [Gd(TTDASQ-protamine)].

Adenocarcinomas in rats and humans frequently contain perivascular, degranulating mast cells that release heparin. Protamine is a low-molecular weight, cationic polypeptide that binds to heparin and neutralizes its anticoagulant properties. A novel magnetic resonance imaging (MRI) contrast agent containing protamine was synthesized. TTDASQ, the derivative of TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid), was also synthesized and the kinetic stability of [Gd(TTDASQ)]- chelate containing phosphate buffer and ZnCl2 to measure the relaxation rate (R1) at 20 MHz was studied by transmetallation with Zn(II). The water-exchange rate (k(ex)298) of [Gd(TTDASQ)]- is 6.4 x 10(6) s(-1) at 25.0 +/- 0.1 degrees C which was obtained from the reduced 17O relaxation rates (1/T(1r) and 1/T(2r)) and chemical shift (omega(r)) of H(2)17O, and it is compared with that previously reported for the other gadolinium(III) complex, [Gd(DO3ASQ)]. The binding affinity assay showed that the (TTDASQ)3-pro19 has higher activity toward heparin. On the other hand, the effect of heparin on the relaxivity of the [Gd(TTDASQ)3-pro19] conjugate shows the binding strength (K(A)) is 7669 dm3 mol(-1) at pH 7.4 and the relaxivity (r(b)1) of the [Gd(TTDASQ)3-pro19]-heparin adduct is 30.9 dm3 mmol(-1) s(-1).

Cyclobutanes↗
PubMed1967

Esters of serine and threonine in hydrolysates of histones and protamines, and attendant errors in amino acid analyses of proteins.

1. Partial acid hydrolysates of histones from various origins and of protamine were analysed by a two-dimensional ionophoretic procedure to reveal strongly acidic ninhydrin-positive components. 2. Histone fractions prepared by extraction with sulphuric acid gave rise to spots identified as serine O-sulphate and threonine O-sulphate. These two compounds, which were not found in hydrolysates of corresponding fractions prepared by extraction with hydrochloric acid, were artifacts. 3. Hydrolysis of proteins in the presence of traces of sulphate can lead to the formation of the O-sulphates of serine and threonine. This can cause errors, which may sometimes be serious, in amino acid analyses of proteins. 4. O-Phosphoserine was obtained in small amounts from some histone fractions and from protamine, but was undetectable in other histone fractions, notably those of lower lysine content.

Activation Analysis↗
PubMed1977

Mechanism of anticholinesterase activities of cardiotoxin, protamine and polylysine.

Cardiotoxin, protamine and polylysine are potent inhibitors of various cholinesterases. CaCl2 and MgCl2 overcome the inhibition. The order of addition of the inhibitor and the protecting agent (MgCl2) influences the final degree of the inhibition observed. These findings suggest that cardiotoxin, protamine and polylysine inhibit cholinesterases by the ionic binding of their basic groups with the anionic sites of cholinesterase molecules.

Animals↗
PubMed1988

Ca2+-independent binding of [3H]phorbol dibutyrate to protein kinase C is supported by protamine and other polycations.

The activity of the Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), can be modulated by diacylglycerols and phorbol esters. The association of these agents with PKC is, in turn, generally understood to be dependent on Ca2+ and phospholipids. Certain substrates, e.g. protamine sulphate, are known to undergo cofactor-independent phosphorylation by PKC. We report here that, in the presence of such substrates, PKC bound 1,2-dihexanoylglycerol and phorbol dibutyrate in a Ca2+-independent manner. Histone IIIs, which is phosphorylated by PKC only in the presence of Ca2+ and phospholipid, also supported Ca2+-independent binding of 1,2-dihexanoylglycerol and phorbol dibutyrate to PKC, but to a lesser extent than did protamine. Support for Ca2+-independent binding was also exhibited by non-peptide polycations (e.g. DEAE-cellulose DE52), indicating that recognition of the catalytic site is not a prerequisite for this effect. The natural polyamines spermine and putrescine did not have this property, however. The affinity of PKC for phorbol dibutyrate and 1,2-dihexanoylglycerol was found to be unchanged by the presence of substrates or DE52. It is proposed that, in the absence of Ca2+, certain polycations favour expression of the diacylglycerol/phorbol ester binding site by stabilizing the active conformation of PKC.

Animals↗
PubMed1991

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid.

Amino Acid Sequence↗
PubMed1991

[Delayed hypersensitivity to protamine and immediate hypersensitivity to insulin].

A 63-year-old female, with type II diabetes mellitus, diagnosed in 1967, was started on combination therapy with sulphonylureas and human depot insulin in May 1989, because of inadequate blood sugar control with sulphonylureas alone. Within 3 months she began to develop nodular skin reactions at the site of injection, 12-24 hours after insulin injections. Intradermal testing demonstrated delayed (Gell and Coombs type IV) hypersensitivity to protamine. No specific IgE or IgG antibodies were demonstrable. She was changed to protamine-free human delayed action insulin. After an initial reaction-free period, red urticarial lesions, attributable to immediate (Gell and Coombs type I) hypersensitivity to human insulin, appeared at the injection sites. There were no other complications with continued insulin therapy, and after about 6 weeks no further local reactions were detectable. When an allergic reaction to an insulin preparation is suspected, careful immunological investigation should be performed, to ensure adequate treatment without risk to the patient.

Delayed-Action Preparations↗
PubMed1969

Genetic control of immunological responsiveness in guinea pigs to 2,4-dinitrophenyl conjugates of poly-L-arginine, protamine, and poly-L-ornithine.

The ability of guinea pigs to respond immunologically to dinitrophenyl (DNP)-poly-L-arginine, DNP-protamine, DNP polyornithine, and to unsubstituted poly-L-arginine and protamine is linked to the presence of the poly-L-lysine gene. This gene has previously been demonstrated to control the immune response of guinea pigs to poly-L-lysine, to a copolymer of L-glutamic acid and L-lysine and to haptenic derivatives of these materials. Although the ability to respond to each of these highly charged compounds is linked to the presence of the poly-L-lysine gene, the cellular immune responses to these antigens is highly individually specific.

Animals↗
PubMed1975

Three-dimensional structure of herring sperm protamine Y-I with the aid of dark field electron microscopy.

High resolution electron micrographs of herring sperm protamine (clupeine) Y-I provide sufficient detail to constrain the folding of the known amino-acid sequence of the protein into a unique three-dimensional configuration. The structure consists of a loose helix of turns of various sizes held together at one edge, spread apart along the other. This form appears to represent the shape of several fish protamines.

Animals↗
PubMed1993

Analysis of the mouse protamine 1 promoter in transgenic mice.

Protamines are small arginine-rich proteins that package DNA in spermatozoa. The mouse protamine 1 (Prm-1) gene is transcribed exclusively in post-meiotic spermatids. To identify elements in the Prm-1 promoter required for spermatid-specific transcription, we generated transgenic mice by microinjection of transgenes containing Prm-1 5' flanking sequences with 5' truncations or internal deletions of conserved sequences linked to a marked Prm-1 gene. We also tested Prm-1 promoter regions with a heterologous human growth hormone reporter gene. We conclude that a 113-bp region can direct spermatid-specific transcription and we have defined sequences within this region that are essential for proper function. These results will facilitate the isolation and characterization of transcription factors essential for post-meiotic gene expression.

Animals↗
PubMed2004

Physicochemical characterization of protamine-phosphorothioate nanoparticles.

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90-200nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a > or = 1:2 ratio (w/w) of PTO and PFB. About 53-68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of -19 up to +32 mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20000 (PEG), which also led to an increase of stability in cell medium.

Chemical Phenomena↗
PubMed1981

Heparin assay and protamine titration.

By utilizing the fact that heparin and protamine sulfate directly neutralize each other, it is possible to quickly detect excess levels of protamine (anti-heparin activity) by back-titrating a plasma specimen with predetermined amounts of heparin. The approach suggested allows for the simultaneous definition of low levels of heparin, and incorporates an equally rapid and accurate measurement of high heparin concentrations. The methodology presented employs heparin assays using the "Protopath" technic. The concept appears to be applicable to other test systems currently designed to monitor heparin activity.

Blood Coagulation Tests↗
PubMed1994

Reversal of post-reperfusion coagulopathy by protamine sulphate in orthotopic liver transplantation.

We report details of two liver transplant procedures in which post-reperfusion coagulopathy was reversed by administration of protamine sulphate. Both procedures were uncomplicated until about 30 min after reperfusion of the graft, when the cut surfaces began to ooze blood. Failure of coagulation was confirmed by thrombelastography and in both cases routine coagulation tests revealed a prolonged kaolin cephalin clotting time (KCT). A heparin-like effect was suspected. Protamine sulphate 50 mg was given i.v., resulting in cessation of bleeding and normalization of the thrombelastogram and KCT.

Adult↗
PubMed2005

Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males.

BACKGROUND: A significant relationship exists between an abnormally high sperm protamine-1 (P1)/protamine-2 (P2) ratio and male infertility. In this study we investigate whether a decreased P1/P2 ratio is also linked to male infertility and we attempt to describe, at the protein expression level, the underlying cause of sperm P1/P2 deregulation. METHODS: P1 and P2 protein concentrations were quantified in sperm from 272 infertility patients and 87 fertile donors. P1/P2 ratios and protamine quantity were correlated with fertility status using semen analysis, sperm penetration capacity, and IVF data. RESULTS: We identified four distinct groups in the study: normal P1/P2 fertile donors, normal P1/P2 patients, low P1/P2 patients, and high P1/P2 patients. P1 and P2 were both under-expressed in patients with a normal P1/P2 ratio, but not in fertile donors. In patients with a low P1/P2 ratio, P1 was under-expressed while P2 was over-expressed; in patients with a high P1/P2 ratio, P1 was normally expressed and P2 was under-expressed. Patients with abnormal P1/P2 ratios displayed significantly reduced semen quality and sperm penetration ability. CONCLUSIONS: We have identified a novel population of infertile males with a reduced P1/P2 ratio. Aberrant P1/P2 ratios arise from an abnormal concentration of P1 and/or P2, either of which is associated with male infertility.

Female↗
PubMed2000

Expression of protamine-1 and -2 mRNA during human spermiogenesis.

During spermiogenesis, the histone-to-protamine replacement causes the compaction of the spermatid chromatin. The genes for protamines, PRM-1 and PRM-2, are transcribed in round and elongating spermatids. The transcripts are stored in a translationally-repressed state by the binding of protein repressors before being translated in elongating and elongated spermatids. RNA extracts from homogenized whole testis samples supply only average data, and cell-specific and stage-specific expression cannot be addressed. Therefore, we used UV-laser-assisted cell-picking (UV-LACP) to select spermatids of defined differentiation steps. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) with intron-spanning primer pairs allowed the detection of DNA-free and pseudogene-free PRM-1 and PRM-2 cDNA. Additional in-situ hybridization with digoxygenin-labelled cRNA probes exhibited PRM-1 and PRM-2 mRNA from step 1/2 spermatids to step 4 spermatids, but not in elongated spermatids. RT-PCR revealed amplicons for PRM-1 and PRM-2 in all spermatids except step 3 round spermatids. Applying proteinase K digestion, PRM-1 and PRM-2 transcripts were also detected in step 3 spermatids indicating that protein repressors may bind to both PRM-1 and PRM-2 mRNA in step 3 round spermatids. These data demonstrate that the combination of UV-LACP and non-radioactive in-situ hybridization appear to be a suitable approach for the study of cell-specific and stage-specific gene expression during spermiogenesis.

Adult↗
PubMed2003

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations.

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.

Amino Acid Sequence↗
PubMed2003

Decreased protamine-1 transcript levels in testes from infertile men.

Infertile men exhibit an aberrant protamine-1 (Prm1) to protamine-2 (Prm2) ratio at both the mRNA and protein level. We therefore investigated whether male infertility could be related to the amount of Prm1 and Prm2 mRNA by applying real time quantitative PCR following RNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies. Samples (n = 51) were normalized to the same amount and similar size of tissue sections. The threshold cycle (C(T)) representing a measure of the initial number of mRNA copies was significantly (P < 0.001) higher for Prm1, but not Prm2, and thus the amount of Prm1 mRNA was lower in men with at least qualitatively normal spermatogenesis (Prm1: 29.88 +/- 2.99; Prm2: 34.28 +/- 2.26) and impaired spermatogenesis (Prm1: 31.89 +/- 2.54; Prm2: 35.59 +/- 2.09) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (Prm1: 29.04 +/- 1.02; Prm2: 34.91 +/- 1.40). In addition, the Prm1 - Prm2 C(T) difference (deltaC(T)) was significantly (P < 0.001) decreased in these two groups. A negative correlation (r = -0.504; P < 0.001) was demonstrated between the score for efficiency of spermatogenesis and the C(T) for Prm1. These data suggest that the decreasing amount of Prm1 and, as a consequence, the aberrant Prm1:Prm2 mRNA ratio plays an important role for male infertility and may serve as a possible predictive factor for the outcome of ICSI.

Analysis of Variance↗
PubMed2005

Cellular expression of protamine 1 and 2 transcripts in testicular spermatids from azoospermic men submitted to TESE-ICSI.

Testicular sperm extraction (TESE) combined with ICSI is used to treat azoospermia. However, the factors that influence the outcome of ICSI in this situation are ill-defined. We sought to investigate the expression of protamine 1 (PRM1) and protamine 2 (PRM2) transcripts in testicular spermatids from obstructive and non-obstructive azoospermic men with impaired spermatogenesis. The relationship between PRM1 and PRM2 transcript levels and the TESE-ICSI outcome was evaluated. The cellular expression of PRM1 and PRM2 mRNAs in single testicular spermatids from 41 azoospermic patients (in whom testicular spermatozoa were subsequently recovered and submitted for TESE-ICSI) was determined by radioactive in situ hybridization. Group I contained seven men with congenital, obstructive azoospermia and whose testicular biopsies indicated quantitatively normal spermatogenesis. Group II consisted of 18 azoospermic men with moderately impaired spermatogenesis. Sixteen men with non-obstructive azoospermia and severely deranged spermatogenesis (i.e. mixed atrophy with small foci of spermatids and spermatozoa) constituted group III. The spermatids of men with severely deranged spermatogenesis exhibited significant lower PRM1 mRNA expression than in the other patient groups. There were no significant inter-group differences in PRM2 mRNA expression. Spermatid PRM1 expression was lower in non-pregnant couples than in pregnant couples. The low number of spermatids in cases of mixed atrophy with small spermatogenic foci is associated with significantly lower PRM1 expression and a lower pregnancy rate. These results emphasize the role of PRM1 as a potentially critical factor in post-ICSI embryonic development.

Adult↗
PubMed1982

The localisation of the 5'-termini of in vivo transcripts of a cloned rainbow trout protamine gene.

The mRNA start site of a cloned rainbow trout protamine gene (TPG-3) has been localised using S1-nuclease mapping and primer extension of in vivo synthesised trout testis poly A+-RNA. The presumptive cap site occurs within an AT-rich region, only 14 nucleotides from the start of the protein-coding sequence. Transcription of this protamine gene in vitro, using the Hela whole-cell extract system, generates products initiated at the same nucleotide as that used in vivo. In vitro transcription is abolished by deletion of sequences between -20 and -48, within which is a canonical TATA-box having an llbp homology with the strong chick conalbumin and Adenovirus-2 major late promoters (CTATAAAAGGG).

Amino Acid Sequence↗