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SV-IV (seminal vesicle protein no. 4) is a potent immunomodulatory and anti-inflammatory secretory protein (Mr 9758) produced in large amounts by the rat seminal vesicle epithelium. Here we show that this protein possesses the ability to upregulate in J774 macrophages the expression of the gene coding for the inducible nitric oxide synthase (iNOS). The increase in NO production consequent on the marked enhancement of iNOS activity was not associated with apoptotic damage of the SV-IV-treated cells. In the same experimental model, however, LPS induced upregulation of iNOS coupled with an increase in NO production and marked apoptotic death. Differences in the ability of SV-IV and LPS to control the life/death signal balance in target cells via trans-membrane activation of apoptotic (mediated by TNF-alpha and NO/iNOS system) and anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways are suggested to be the basis of the apoptotic fate of the experimentally treated cells. In addition, considering the important role played by NO in the process of mammalian reproduction, SV-IV may be involved in the fine tuning of NO concentration in the female genital tract mucosa via an SV-IV-mediated control of iNOS gene expression in local macrophages.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln(16) which was identified as the amino acceptor substrate. Different gamma(glutamyl(16))amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry, When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys(21), was demonstrated to be involved in both inter- and intramolecular cross-link formation.

The pretreatment of epididymal spermatozoa with SV-IV, one of the major secretory protein produced by the epithelium of adult rat seminal vesicles, was found to markedly decrease their ability to induce in vivo peritoneal macrophage activation, measured as class II major histocompatibility complex surface antigen expression, superoxide anion production, phagocytic activity, and antigen presentation. In addition, the treatment of spermatozoa with SV-IV produced a significant decrease of their immunogenicity evaluated in vitro by [H-3]thymidine incorporation in splenocyte/spermatozoon co-culture. The concurrent presence of SV-IV and transglutaminase, an enzyme secreted in large amounts from the rat anterior prostate, amplified these phenomena. The suppression of the epididymal sperm immunogenicity is suggested to be of crucial importance for the prevention of the immune response to the sperm introduced in the immunocompetent female genital tract during coitus.

The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium , on human blood coagulation was investigated. It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro. Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin. A possible binding of porins with thrombin has been suggested to be the basis of this effect. The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, antiinflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure- function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, hall of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.

Retinoic acid (RA) and sodium butyrate (NaB) are regulators of cell growth and differentiation. We studied their effect on normal (SVC1) or v-Ki-ras-transformed (Ki-SVC1) rat seminal vesicle (SV) epithelial cell lines. The treatment of these cells with 10(-((7( M RA did not produce significant changes in the morphological and biochemical parameters analyzed. When RA was used in combination with 2 mM NaB, the treatment induced substantial morphological changes, apoptosis-independent growth arrest, up-regulation of tissue transglutaminase (tTGase), and down-regulation of beta and gamma RA receptor (RAR) mRNA expression. The same cells did not express RAR alpha either before or after NaB/RA treatment. A similar treatment did not change the amount of mRNA coding for the protein SV-IV (a typical differentiation marker of the SV epithelium) in normal or ras-transformed cells nor the level of v-Ki-ras mRNA in Ki-SVC1 cells. These findings suggest that a defective RA/RARs signaling pathway is probably the biochemical condition that underlies the unresponsiveness to RA of our in vitro culture system, and indirectly points to the possibility that the NaB/RA-induced effects were brought about by a cooperation at the transcription level between the histone deacetylase inhibitory activity of NaB and the ability of RA/RAR to modulate the expression of various genes involved in the control of cell growth and differentiation.

The protein SV-IV, a major protein secreted from the rat seminal vesicle epithelium, is a basic protein with immunomodulatory, anti-inflammatory, and procoagulant activity. Predictions suggested that this protein is very flexible, with its three tyrosyl residues presumably located in water-exposed segments of the primary structure. The solution behaviour of the protein was investigated by two types of spectroscopic techniques. Modifications of the spectral characteristics of tyrosyl residues induced by changes of protein concentration were demonstrated by absorption and fluorescence experiments. In addition, secondary structure rearrangements associated with a possible self-association equilibrium were highlighted by far-UV CD spectra. The equilibrium, confirmed by chromatographic techniques, appears to control some biological properties of the protein.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.

Two transglutaminase-mediated modifications of the rat epididymal spermatozoon surface were demonstrated in vitro. Transglutaminase was effective in promoting the binding of spermidine to the sperm. Moreover, the enzyme, by reacting with one of the major proteins secreted by the rat seminal vesicle epithelium, produced a modified form of the protein with a higher molecular weight and the capability of binding to the sperm cells. A specific physiological role for the enzyme, bringing about modifications of the rat sperm surface in the seminal fluid environment, is suggested.

In the seminal plasma of normal men a protein, immunologically related to a major protein (RSV-IV) secreted from the rat seminal vesicle epithelium, was detected by competition with RSV-IV in a specific radioimmunoassay using a rabbit anti RSV-IV antiserum. The protein was partly (80%) purified by column chromatography; characterization by gel electrophoresis indicated that the protein is slightly basic and has a molecular weight of 140 000. The protein was present on the surface of human spermatozoa as well as in the ejaculates of azoospermic men and it is believed to be a sperm-binding protein.

A comparison of both amino acid composition and sequence of the rabbit uteroglobin (UG) subunit and the rat seminal vesicle sperm-binding protein (rSBP) by computer analysis indicates homology between the two polypeptide chains. These findings are supported by immunological studies showing the occurrence of similar antigenic determinants. In addition, our data indicate the glutamine-9 of the rat seminal vesicle sperm-binding protein and glutamine-40 of UG as the possible glutamine residues involved when the proteins act as transglutaminase (TGase) substrates. The latter results represent an interesting approach to determining the general structural features of the acyl donor site in the TGase-catalyzed reaction.

The synthesis of a thyroglobulin precursor directed by endogenous messenger RNA has been achieved in a heterologous cell-free system containing porcine thyroid membrane-bound ribosomes and rat brain cortex cell sap. The main features of the system, such as ion and nucleotide requirements, temperature, dependence, inhibitors sensitivity, and ability to reinitiate, were studied. The nature of the product has been determined by specific immunoprecipitation and its size has been estimated by sucrose density gradient and sodium dodecyl sulfate acrylamide gel electrophoresis. Under our conditions, the thyroglobulin precursor was mainly synthesized by membrane-bound ribosomes.

The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.

In the pre-vitellogenic oocytes of the hibernating lizard Lacerta sicula, a fraction of the ribosomal population crystallizes in the form of ribosomal bodies (RB). In these oocytes the rate of protein synthesis is 35–75 % lower than in the oocytes of the summer animals in which the RB have dissolved. The isolated RB are able to respond to Poly(U) and are also active in the endogenous protein synthesis in a cell-free system. This contrasts with the in vivo situation, where they appear to be completely inactive in protein synthesis. The possibility that RB represent a regulatory mechanism of protein synthesis is discussed.

The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.

In human seminal plasma a family of proteins that is immunologically related to the RSV-IV protein secreted under androgen control from the epithelium of the rat seminal vesicles was detected by a radioimmunoassay. Evidence for the origin of these antigens from human seminal vesicle is presented. Quantitative measurements of this family of proteins were performed in men with low levels of serum testosterone (idiopathic hypogonadotropic hypogonadism) and in individuals having serum testosterone in the normal range of values but carrying sex chromosome aberrations (Klinefelter's syndrome). In the first case we have found a marked decrease in the total amount of the RSV-IV-related proteins. An increase of about 40% in the total amount of these antigens was obtained in these subjects by gonadotropin treatment. A decreased amount of these proteins was also detected in the subjects affected by Klinefelter's syndrome. The possibility that some factor(s) under genetic control is involved, in addition to testosterone, in the regulation of this family of proteins is discussed.

The proteins secreted by the rat seminal vesicle can be separated into five denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and RSV-V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant of RSV-IV that can be expressed only in rats homozygous for a given allele at the svp-2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology of RSV-IV and RSV-V and of their corresponding gene.

Poly/A)+mRNA, prepared from rat seminal vesicles (RSV), was translated in a rabbit reticulocyte cell-free system. Among the translation products, three proteins (A, B and C), immunologically related to a major RSV secretory protein (RSV-IV), were detected. Recombinenta plasmids, harbouring specific cDNA sequences for RSV-IV, were used to positively select the mRNAs for antigens A and B. Phosphorylation sites were mainly detectable in the antigen B.

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical co¬relates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 x 10-7 M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

Avian myeloblastosis virus reverse transcriptase has been shown to use human globin mRNA as an in vitro template.

Protein synthesis in reuroblastoma cells has been studied in a cell-free system. The activity of lysates from cells grown insuspension and monolayer has been compared. A higher level of activity has been found in monolayer cells. The activity of some components of the lysate that are involved in protein synthesis has been analyzed.The data suggest that the controlling step of protein synthesis in this system might be in the initiation process. The correlation between activation of protein synthesis and neurite outgrowth in monolayer cultures is discussed.

Primary cell cultures were established from explants of rat seminal vesicle. The establishment of primary cell cultures required, among other factors, the presence of testosterone. Two cell populations were detected in such primary cultures: fibroblast-like cells and epithelial-like cells; the latter encompassed a subtype of small cells and a subtype of large squamous cells (most likely the result of a degenerative process acting upon the former). Histochemical, as well as electron-microscopical observations, indicated the presence of a persistent secretory activity in the small epithelial cells; fibroblast and large squamous epithelial cells were inactive in this respect. Staining of the cells with a peroxidase-conjugated antibody and analysis of the proteins produced in the presence of labelled methionine, showed that one of the major rat seminal vesicle secretory proteins, namely RSV-IV, was also produced. Conditions which favoured the growth of epithelial cells, rather than of fibroblasts, were determined. The use of nearly homogeneous cell populations and the use of collagen-coated Petri dishes, allowed the cloning of two independently obtained permanent cell lines, namely SVC-1 and SVC-2. The in vitro growth rate of both cell lines was modulated by the amount of testosterone in the medium. Both cell lines were able to synthesize a significant amount of RSV-IV protein under testosterone control.

The presence in the human seminal plasma (h-SP) of a protein immunologically related to the RSV-IV, protein secreted by the rat seminal vesicles (RSV), is shown. We also report that this protein (hereafter termed the h-SCA protein) binds the human sperm cell surface. This protein has been partially characterized by chromatography and gel electrophoresis, and could represent a good marker to study the human function of seminal vesicles and their androgen regulation.

The synthesis of a thyroglobulin precursor directed by endogenous messenger RNA has been achieved in a heterologous cell-free system containing porcine thyroid membrane-bound ribosomes and rat brain cortex cell sap. The main features of the system, such as ion and nucleotide requirements, temperature, dependence, inhibitors sensitivity, and ability to reinitiate, were studied. The nature of the product has been determined by specific immunoprecipitation and its size has been estimated by sucrose density gradient and sodium dodecyl sulfate acrylamide gel electrophoresis. Under our conditions, the thyroglobulin precursor was mainly synthesized by membrane-bound ribosomes.

Following NaF treatment of rabbit reticulocytes, polyribosomes dissociate to monoribosomes with concomitant cessation of protein synthesis. mRNA was isolated from different fractions of the cell before and after NaF treatment and quantitated by assay for globin synthesis in a cell-free system prepared from Krebs ascites tumor cells. mRNA activity present in polyribosomes of control cells was recovered quantitatively from monoribosomes following NaF treatment. On removal of NaF, polyribosomes reform with mRNA activity similar to that of control cells. During polyribosome dissociation, no significant amount of mRNA is recovered from ribosomal subunits, the supernatant fraction or is lost by degradation.

Upon fertilization of Paracentrotus lividus eggs, the ability of their ribosomes to form a ternary complex with poly(U) and phenylalanyl-tRNA is enhanced. In addition, it is possible to extract from ribosomes of the unfertilized egg a protein factor that inhibits polyphenylalanine synthesis by decreasing the binding of mRNA and aminoacyl-tRNA to the ribosomes. The observed increase in the rate of protein synthesis that occurs at fertilization could be accounted for by the removal of this inhibitory protein from the ribosomes.

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human α and β chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qβ, and viral RNA-directed protein synthesis.

Platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a mediator of inflam¬mation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) generated from 1-O-alkyl-2-acyl-¬sn-glycero-3-phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid-¬induced anti-(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen-induced anti-inflammatory protein, SV-IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV-IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl-CoA: lyso-PAF acetyltransfe

Increase of VPAC receptor s binding to the (16)gamma-glutamyl diaminopropane vasoactive intestinal peptide (VIP-DAP) agonist, a vasoactive intestinal polypeptide (VIP) structural analogue containing a positive charge at position 16, has confirmed the importance of a positive charge at this site. By investigating the effect of distance from the peptide backbone Calpha of a positive charge in position 16, data are reported here concerning: (i) a novel chemical method used for the synthesis of a new family of (16)gamma-glutamyl diamine VIP derivatives differing among them for single carbon atoms and including diaminoethane (VIP-DAE2), diaminopropane (VIP-DAP3), diaminobutane (VIP-DAB4), diaminopentane (VIP-DAP5), and diaminohexane (VIP-DAH6); (ii) functional characterization of these compounds on human VPAC1 and VPAC2 receptors. In more detail, the EC50 and IC50 values, when measured as a function of the alkylic chain length, show in more detail, that the use of VIP-DAB4 derivative changes the IC50 but not the EC50, thus indicating on hVPAC2 receptor an unexpected relationship between binding and activity that differs from that obtained on hVPAC1.

We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti-inflammatory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self-association process induces conformational change mainly in the 1-70 region, which appears to be without secondary structure in the monomer but contains alpha-helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the monomeric and trimeric forms represent a crucial aspect of activity modulation.