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PubMed1990

Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system.

Transcriptionally active nuclear extracts were prepared from mouse testes to study the transcription of the testis-specific mouse protamine 2 (Prm-2) gene in vitro. The testicular system is unique among mammalian in vitro transcription systems in regard to its temperature optimum. In extracts made from prepuberal testes, the temperature optimum for in vitro transcription of Prm-2 is 30 degrees C, similar to somatic in vitro systems. However, in adult testis extracts, the optimum temperature for Prm-2 transcription is 20 degrees C. The different temperature optima seen in vitro for prepuberal and adult testes extracts parallels in vivo physiological temperature sensitivities of the differentiating male germ cells. The testis system also differs from other in vitro transcription systems in its divalent metal cation and ionic strength requirements for optimal transcription. The mouse Prm-2 gene is maximally transcribed at a MgCl2 concentration of 3-5 mM and over a KCl concentration range of 40-100 mM. By using the testis in vitro transcription system to study the Prm-2 gene by deletion analysis, we have determined that positive promotion for the gene lies within the region -170 to -82 from the start of transcription. This region contains a putative Sp-1 binding site. Additional upstream sequences appear to repress Prm-2 transcription in a heterologous transcription system.

Animals↗

PubMed1991

Protamine transcript sharing among postmeiotic spermatids.

Sharing of cytoplasmic constituents through intercellular bridges connecting postmeiotic spermatids can allow for functional equivalence of genetically nonequivalent spermatids. The technique of in situ hybridization was used to study postmeiotic distribution of transcripts from the mouse protamine 1 (Prm-1) gene among spermatids of mice with chromosomally unbalanced gametes. The Prm-1 gene is located on chromosome 16 and is expressed exclusively in haploid spermatids. Mice doubly heterozygous for two Robertsonian translocations involving chromosome 16 were used for the study of postmeiotic accumulation of transcripts of the Prm-1 gene in spermatogenic cells. The meiotic segregation pattern of chromosomal homologues in these mice produces some spermatids that are chromosomally unbalanced; some spermatids lack chromosome 16 while others have two. In situ hybridization with a cDNA probe for the Prm-1 gene transcript performed on both whole testis sections and spermatogenic cell suspensions showed that there was no statistical difference in distribution of grains over step-5 to step-10 spermatids from Robertsonian-translocation heterozygous mice and from control mice of normal karyotype. These results are consistent with sharing of transcripts of the Prm-1 gene among spermatids within a syncytium.

Animals↗

PubMed1995

Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice.

Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines.

Animals↗

PubMed1997

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells.

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.

Alleles↗

PubMed1995

Phosphorylation of eukaryotic protein synthesis initiation factor 4E by insulin-stimulated protamine kinase.

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.

Animals↗

PubMed1997

The histamine releasers crotamine, protamine and compound 48/80 activate specific proteases and phospholipases A2.

Crotamine, a basic, myonecrotic, histamine-releasing neurotoxin, was isolated from Crotalus durissus terrificus venom. Carboxypeptidase A was shown to be activated by crotamine when acting upon N-carbobenzoxyglycil-L-phenylalanine. However the activity of carboxypeptidase B upon the substrate hippuryl-L-arginine was not enhanced by this toxin. Teh basic histamine releasers protamine and compound 48/80 also activated carboxypeptidase A. These three agents activated both alpha-chymotrypsin when acting upon acetyl-L-tyrosine ethyl ester and also five snake venom phospholipase-like myotoxins acting upon egg yolk phosphatidylcholine. These findings suggest that the action of these agents during histamine release may involve the participation of specific intermediary hydrolases which, upon activation, would enhance their cytolytic effects on the sequence of events which lead to granule extrusion and histamine release from mast cells.

Carboxypeptidases↗

PubMed2003

Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs.

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.

Binding, Competitive↗

PubMed1989

Complex dielectric constant of arginine-DNA and protamine-DNA aqueous systems at 10 GHz.

The complex dielectric constant of arginine and protamine from herring sperm (clupeine) and their complexes with herring sperm DNA was measured at 10 GHz in the temperature range -20 to +45 degrees C by a microwave cavity perturbation method. The experimental results were analysed in terms of a three-component equation (solute molecules, interfacial water and bulk water) to calculate the fractional volume of modified water and hence the specific hydration of the samples. A fourfold reduction of the specific hydration is observed for the clupeine molecule as compared to the free monomers. This is consistent with a folded conformation of the protein in solution. The specific hydration of the complex between clupeine and DNA is reduced by 50% with respect to the weighted average for the uncomplexed components. This result indicates an intimate contact between clupeine and DNA with exclusion of water molecules and is consistent with the highly condensed form of nucleoprotamines which is known in vivo.

Animals↗

PubMed1997

Evaluation of a new protamine titration method to assay heparin in whole blood and plasma.

When unfractionated heparin is used for therapeutic anticoagulation, the heparin effect must be monitored to avoid thrombotic or hemorrhagic complications. The ability of a factor Xa inhibition (XaI) assay was compared with that of a low-level heparin protamine titration (LLHPT) assay to measure the concentration of heparin after heparin was added in vitro to specimens of plasma and whole blood. Heparin effect on the activated partial thromboplastin time also was assessed in the same specimens. The XaI and LLHPT assays had comparable precision and provided linear results over a wide range of heparin concentrations. Both assays slightly underestimated the total amount of heparin added to the specimens. The most rapid test was the whole blood LLHPT assay; this test therefore may be useful for bedside monitoring of heparin. A significant disadvantage of the LLHPT assay was the large sample size required to perform it. These results provide in vitro evidence that the XaI and LLHPT assays can provide equally precise monitoring of heparin concentration.

Anticoagulants↗

PubMed1976

Semiquantitative determination of soluble fibrin monomer complexes by chromatography and serial-dilution protamine sulfate test.

Plasmas were prepared from blood obtained from rabbits and from healthy individuals and patients with thrombotic disorders. Gel filtration (Sepharose-4B) chromatography and the serial-dilution protamine sulfate (SDPS) test were performed on each sample. For healthy individuals and rabbits, a narrow fibrinogen peak was obtained on chromatogram and was associted with a negative SDPS test. After administration of thrombin or endotoxin to rabbits and in plasmas from patients with intravascular coagulation, major shifts to earlier effluent volumes occurred. These higher-molecular-weight derivatives corresponded to soluble fibrin monomer complexes (FM). Positive SDPS tests were found in these plasmas. There was a highly significant correlation between percentage area occupied by FM on chromatogram and SDPS test titer. It is concluded that the SDPS test is a reliable semiquantitative measure of FM that can be used clinically as well as in experimental animal models.

Animals↗

PubMed1976

Laboratory suggestion. Interpretation of plasma protamine paracoagulation test.

The reliability of the plasma protamine paracoagulation test has been questioned because of the controversy surrounding its interpretation and because of false positives reported with high frequency in some series. To minimize the frequency of false positives, it is recommended that when the test is done at room temperature, clot or fibrin strand formation be considered a positive result and precipitates be considered negative. Use of the wire hook to distinguish fibrin strands from precipitates is suggested. Nascent clotting and blood drawn from indwelling catheters should be avoided.

Blood Coagulation Tests↗

PubMed1983

Classical pathway activation during an adverse response to protamine sulphate.

A severe anaphylactoid reaction was observed following the i.v. administration of protamine sulphate in a 66-year-old male undergoing coronary artery catheterization. Serial measurements of complement factors (C3 and C4) and C3 split products (C3c and C3d) were performed by conventional electro-immunoassays and by double zone rocket immunoelectrophoresis. A parallel decrease in circulating C3 and C4 concentrations was seen for 1 h, reaching a nadir at 1.3 h. Markedly increased concentrations of C3c and C3d were observed in the first sample examined after exposure to the drug. C3c and C3d concentrations gradually declined to the normal range within 5.9 h and 21.8 h, respectively. These observations are discussed in relation to the mechanism of this adverse reaction.

Aged↗

PubMed1980

The release of high mobility group protein H6 and protamine gene sequences upon selective DNase I degradation of trout testis chromatin.

Limited digestion of trout testis nuclei with DNase I selectively degrades the protamine genes. Concomitant with the degradation of transcribed DNA sequences a series of chromosomal proteins are released; among these, the major species corresponds to the high mobility group protein H6. The amounts of H6 released from chromatin by limited DNase I action and that in the residual nuclear pellet have been determined. A very high proportion of H6 is associated with DNase I sensitive chromatin regions.

Animals↗

PubMed1992

Primary structures of sardaines Z1 and Z2, protamines isolated from striped bonito (Sarda orientalis).

Striped bonito protamine, sardaine, was isolated from the sperm of striped bonito (Sarda orientalis) by extraction with sulfuric acid followed by ion-exchange chromatography. The preparation gave a single band upon polyacrylamide gel electrophoresis. Sardaine consists of 34 amino acid residues, and its sequence is: Pro-Arg-Arg-Arg-Arg-Arg-Ser(Ala)-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg-Tyr -Arg- Arg-Ser-Thr-Ala-Ala-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. At position 7, serine (sardaine Z1) is partially replaced by alanine (sardaine Z2). The ion spray mass spectrum shows that sardaines Z1 and Z2 have molecular masses of 4,612.49 and 4,596.09 Da, respectively. The sequence of sardaine Z1 is 100% identical with that of thynnine Z2 from tuna fish (both fish belong to Scombridae, Perciformes).

Amino Acid Sequence↗

PubMed1993

Primary structure of scombrine gamma, protamine isolated from spotted mackerel (Scomber australasicus).

Spotted mackerel protamine, scombrine, was isolated from the sperm of a spotted mackerel (Scomber australasicus) by extraction with sulfuric acid and fractionated into one major (scombrine II) and one minor (scombrine I) components by chromatography on CM-Sephadex C-25. Scombrine II gave a single band, whereas scombrine I gave three bands upon PAGE. Scombrine II (scombrine gamma) consists of 34 amino acid residues, and its sequence is: Pro-Arg-Arg-Arg-Arg-Arg-Ala-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg- Ala-Arg-Arg-Ser-Thr-Ala-Val-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. The ion spray mass spectrum shows that scombrine gamma has a molecular mass of 4,532.13 Da. The other minor component (scombrine I) is considered to be a mixture of degradation products of scombrine gamma based on the results of PAGE and ion spray mass spectrometry. Scombrine gamma has a similar sequence to sardaine Z2 from striped bonito except for two positions.

Amino Acid Sequence↗

PubMed1975

Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands.

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.

Amino Acids↗

PubMed2003

Concerted changes in the YB2/RYB-a protein and protamine 2 messenger RNA in the mouse testis under heat stress.

Translation of a number of mRNAs is under strict regulation via RNA-binding proteins in the spermatogenic cells of testes. A family of Y-box binding proteins represents promising candidates for these presently uncharacterized RNA-binding proteins. The effects of heat stress on the expression of a Y-box binding protein, YB2/RYB-a, and mouse protamine 2 (mP2) were investigated in cultured spermatogenic cells and mouse testes by immunoblot and Northern blot analyses. Localization and alterations in the expression of the YB2/RYB-a protein and the mP2 mRNA in heat-stressed testes were examined by immunohistochemistry and in situ hybridization, respectively. Levels of the YB2/RYB-a protein in spermatogenic cells decreased rapidly as the result of exposure to higher temperature, 37 degrees C or 43 degrees C, compared with the scrotal temperature, 32.5 degrees C, under the culture conditions used. In experimental cryptorchidism, levels of the YB2/RYB-a protein were decreased after Day 10, while the mRNA levels were affected only slightly. The levels of the mP2 mRNA were also decreased and about comparable with those of the YB2/RYB-a protein. Exposure of the lower abdomen to a high temperature, 43 degrees C for 15 min, also damaged the testis and led to a decrease in YB2/RYB-a protein and the mP2 mRNA levels in a coordinated manner. Because YB2/RYB-a is proposed to function as a stabilizer of mP2 mRNA, the perturbation of YB2/RYB-a by heat stress could account for the decline of the mP2 mRNA in elongated spermatids.

Animals↗

PubMed1994

Testis-specific and ubiquitous proteins bind to functionally important regions of the mouse protamine-1 promoter.

To understand the spermatid-specific regulation of the protamine-1 (Prm-1) gene, we examined the nuclear proteins that bind to regions of the Prm-1 promoter known to regulate transcription. We focused on the Prm-1 promoter region between bp -224 and -37 because this region directs spermatid-specific expression of a heterologous reporter gene in transgenic mice and because regulatory function has been demonstrated for several subregions of this fragment. Testis nuclear proteins that bind to this promoter region were identified by means of gel mobility shift assays, and the tissue distribution of these proteins was examined through nuclear extracts derived from several mouse tissues. Nuclear extracts prepared from prepubertal and mutant mouse testes were used to demonstrate the developmental appearance of these DNA-binding proteins during spermatogenesis. These studies indicate that a testis-specific protein that appears after Day 12 interacts with a sequence (between bp -37 and -77) shown to be essential for Prm-1 transcription in vivo. In addition, a number of proteins that are not restricted to the testis interact with other functionally important regions of the Prm-1 promoter.

Animals↗