Identification of galectins as novel regulators of platelet signaling and function

Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis. However, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Platelets can be activated by soluble molecules including thrombin, TXA2 , adenosine diphosphate (ADP), and serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor and collagen. In this article, we review recent advances on the role of galectins in platelet physiology. By acting in either soluble or immobilized form, these glycan-binding proteins trigger platelet activation through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.     

Creatine kinase and ATPase in human seminal fluid and prostatic fluid

A creatine kinase assay based on estimation of creatine liberated from creatine phosphate was accurate and reproducible for use with seminal or prostatic fluid, after allowance was made for acid phosphatase interference. Comparison of this method with one which relies on enzymic coupling of ATP formation to NADP+ oxidation shows that the latter under-estimates creatine kinase activity by a factor of about 3. This discrepancy could be due to the high ATPase activity found in prostatic and seminal fluid. Uncritical use of the NADP+ assay might account for different seminal creatine kinase values reported in the literature. Interrelationships between ATPase, creatine kinase and zinc suggest that seminal ATPase is a prostatic secretory product while creatine kinase may be multiglandular in origin.     

Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2

Presence or absence of three distinct bovine seminal heparin-binding proteins (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility-associated antigenin bovine seminal fluid as a unique DNase I-like protein. We now report purification and identification of a 24 kDa seminal heparin-binding protein (HBP-24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identity to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding proteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm membranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin-binding protein increased under nonreducing SDS-PAGE to approximately 21 kDa, characteristic of the reported molecular mass of TIMP-2. To our knowledge, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate previous reports regarding the site of production of heparin-binding proteins that are related to bull fertility, and suggest that TIMP-2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition.     

Immunoreactive vasopressin in bull seminal fluid

Immunoreactive arginine vasopressin (irAVP) was measured in seminal fluid with and without extraction using a specific radioimmunoassay (RIA). A large fraction of irAVP was removed after extraction on octadecasilylsilica cartridges. The measured amount of irAVP corresponded to the levels found in blood plasma. Dilutions of seminal plasma extracts were parallel with the RIA standard curve. On reversed phase HPLC the extracted material coeluted with synthetic AVP. These findings suggest an identity of this immunoreactive material with intact AVP. During incubations of synthetic AVP and its analogue 8-D-arginine vasopressing (8-DAVP) in seminal plasma, immunoreactivity decreased considerably with the former peptide, while the concentration of 8-DAVP was not significantly altered.     

Differential proteolysis of sigma regulators controls cell-surface signalling in Pseudomonas aeruginosa

Cell-surface signalling systems are widespread in Gram-negative bacteria. In these systems gene expression occurs following binding of a ligand, commonly a siderophore, to a receptor protein in the outer membrane. The receptor interacts with a sigma regulator protein that extends from the periplasm into the cytoplasm to control the activity of a cognate sigma factor. The mechanisms of signal transduction in cell-surface signalling systems have not been determined. Here we investigate signal transduction in the pyoverdine, ferrichrome and desferrioxamine siderophore systems of Pseudomonas aeruginosa. When pyoverdine is present the sigma regulator FpvR undergoes complete proteolysis resulting in activation of two sigma factors PvdS and FpvI and expression of genes for pyoverdine synthesis and uptake. When pyoverdine is absent subfragments of FpvR inhibit PvdS and FpvI. Similarly, subfragments of the sigma regulators FoxR and FiuR are formed in the absence of desferrioxamine and ferrichrome. These are much less abundant when the siderophores are present and downstream gene expression takes place. In all three systems RseP (MucP/YaeL) is required for complete proteolysis of the sigma regulator and sigma factor activity. These findings indicate that regulated proteolysis is a general mechanism for signal transduction in cell-surface signalling.     

Identification in human seminal fluid of an inhibin-like factor which selectively regulates FSH secretion.

Human seminal plasma obtained by centrifugation of human semen contains a factor capable of selectively inhibiting the secretion of FSH both in vivo (reduction of the levels of FSH in rats 24 h after castration) and in vitro (reduction of the FSH released by LH-RH in rat pituitary cell culture). This effect is not due to testosterone, oestradiol-17 beta or progesterone present in the active fractions. The factor has the characteristics of a protein in that its biological activity is destroyed by heat and trypsin digestion. It does not resemble androgen-binding protein. The biological action is not completely specific for FSH as inhibition of LH can be seen with doses usually higher than those which produce inhibition of FSH alone. There is no effect on TSH or prolactin levels in vitro. The factor clearly acts on the release and synthesis of gonadotrophins by gonadotrophs but an effect on the hypothalamus is not excluded. This factor fits the definition of inhibin.     

Identification of the acid proteinase in human seminal fluid as a gastricsin originating in the prostate

Summary On the basis of a) kinetic data obtained with a synthetic substrate and two peptide inhibitors and b) immunological cross-reactivity, it is shown that the aspartic proteinase of human seminal fluid is a gastricsin. The source of the precursor (progastricsin) in the male genital tract is identified to be the prostate.     

Quantification of prostaglandins in human seminal fluid

A method is described for measurement of the prostaglandins present in human seminal plasma. The PGEs and the 19-hydroxy-PGEs are converted to the respective PGB-compounds. They are determined with UV-absorbance after purification with two chromatography steps. The PGFs and the 19-hydroxy-PGFs are determines by gas chromatography, which also allows measurement of the 8β-isomers. Recovery of added PG was in the average 99.2% (range 89.9–107.7). Mean variation between duplicate analyses was 5.6% (range 0.9–9.1). The method has been simplified to allow at least limited clinical use.     

Antifertility factors of mammalian seminal fluid

The fertilizing ability of spermatozoa is inhibited by certain substances present in the seminal fluid. Most of these antifertility factors are proteinaceous in nature and differ in their physical characteristics. They inhibit fertilization by inhibiting either motility, capacitation, acrosome reaction or penetration of the ovum investments by the spermatozoa. This review describes and discusses the properties of these factors and their possible role, individually and collectively, in the regulation of fertility.     

Haemolytic factor in bovine seminal vesicle fluid

The haemolytic factor of bovine seminal fluid has a protein nature; it is present in the precipitate obtained by treating the fluid with alcohol-ether, ammonium sulphate and rivanol; it occurs in three out of five protein fractions obtained by chromatographic separation on Sephadex G 100.
of Enzymatic treatment of seminal vesicle fluid by pronase caused total inactivation of the haemolytic factor. Chymotrypsin caused considerable damage, while a number other enzymes did not affect the activity of the haemolytic factor.