Antiproteinase Activity Research Articles

In vivo molecular target assessment of matrix metalloproteinase inhibition.

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.

Inhibition of Angiogenesis in Vivo by Plasminogen Activator Inhibitor-1

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.

Complexing of basic pancreatic proteinase inhibitor with soybean phospholipid multilamellar vesicles.

The formation of complexes of basic pancreatic proteinase inhibitor (BPTI) with multilamellar vesicles (MLV) from six preparations of soybean phospholipids of various composition was studied. When BPTI, a non-membrane protein, interacts with MLV, the vesicles aggregate, forming a precipitate of protein-lipid complexes. The BPTI content in the protein-lipid complexes increases with decreasing pH of the medium and on addition of negatively charged components into the lipid mixture. The protein-induced aggregation of the phospholipid vesicles is suggested to be mainly determined by electrostatic forces. The antiproteinase activity of BPTI in the complexes was rather low but increased up to 70% of the initial activity on addition of an ionic detergent (sodium deoxycholate).

Airflow Limitation in Japanese Smokers: Significance of Serum Neutrophil Elastase/α1-Proteinase Inhibitor Ratio and FEV1 (%pred) Adjusted by Pack-Years

Background: In spite of the known role of cigarette smoking in the development of airflow limitation (AL), fewer than 20% of smokers actually develop chronic obstructive pulmonary disease (COPD). Objectives: We examined how smoking histories and indices in blood are related to the degree of AL in asymptomatic smokers in order to determine whether they can predict the development of AL. Methods: Spirometry and peripheral blood tests were examined in 433 Japanese asymptomatic current smokers at the initial examination. Forced expiratory volume in 1 s (FEV₁) was measured periodically for 2 or more years (2–13 years) in 66 of the subjects. Results: AL defined as an FEV₁/vital capacity of less than 0.7, was found in 11.3% (49 of 433) of the smokers. Pack-years of smoking, serum amounts of α₁-proteinase inhibitor, and serum procollagen III peptide activities were correlated with the degree of AL. Fifteen percent (10 of 66) of subjects underwent rapid declines in FEV₁ that were found to be related not with smoking amounts or initial FEV₁, but with low FEV₁ (%pred) adjusted by pack-years and an elevated serum neutrophil elastase/α₁-proteinase inhibitor ratio. These results suggest that smokers with a low FEV₁ out of proportion to pack-years are susceptible smokers at a high risk of developing COPD, and further, that increased proteinase burden relative to antiproteinase activity may contribute to the development of COPD. Conclusions: We conclude that the serum neutrophil elastase/α₁-proteinase inhibitor ratio and FEV₁ (%pred) adjusted by pack-years can be reliable predictors of the development of COPD.

Characterization of rainbow trout milt collected with a catheter: semen parameters and cryopreservation success

Collection of fish milt by stripping risks the danger of milt contamination by urine. This may seriously influence milt characteristics and quality, including usefulness for cryopreservation. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objectives of this study were to provide basic characteristics of milt collected with a catheter, to test the usefulness of this milt for cryopreservation, and to correlate characteristics of fresh and cryopreserved semen with sperm fertility rates. Milt from 25 rainbow trout Oncorhynchus mykiss (Walbaum) males were used. All samples were cryopreserved using the pellet method within 1 h of collection, using 0.6 m sucrose and 10% dimethyl sulphoxide (DMSO) as an extender. Catheterization resulted in semen of very good motility (> 90% motile spermatozoa) and high fertilization rates after cryopreservation (mean fertilization rate 81.8 ± 13.3% of control, at a sperm/egg ratio of 2.4 ± 0.3 × 106). Osmolality of seminal plasma and concentrations of sodium, potassium and magnesium ions had low variability, which suggests that they are important for creating a stable environment for sperm storage in the sperm duct. Higher variability of certain seminal plasma characteristics, such as protein concentration and antiproteinase activity, suggests that these characteristics are related to individual semen features of particular males. A strong correlation of seminal plasma zinc concentration with protein concentration may reflect an importance of zinc in semen biology. Cryopreservation caused a significant release of protein and acid phosphatase from spermatozoa. Our results did not reveal any single characteristic of semen collected by catheter that could be used as a powerful predictor of cryopreservation success, presumably because all samples were of high quality.

Inflammation-induced systemic proteolysis of inter-α-inhibitor in plasma from patients with sepsis

Inter-α-inhibitor (IαI) is a human plasma serine proteinase inhibitor. It contains one light peptide chain called bikunin that exerts antiproteinase activity and other anti-inflammatory functions. Bikunin is covalently linked to two heavy chains that, after tissular diffusion, stabilize the extracellular matrix. Owing to its negative acute-phase reactant character and its susceptibility to proteolysis, IαI has been implicated in the pathophysiology of sepsis. Moreover, IαI has been shown to exert a protective effect on a pig model of endotoxic shock. Twenty patients admitted to the intensive care unit (ICU) for a septic syndrome were included in the present study. IαI and antithrombin III (ATIII) levels were measured on admission. Sequential measurements of IαI could be done in 4 patients. We demonstrate that IαI levels are significantly decreased in plasma samples collected on admission from patients with sepsis (59 ± 32 mg/L vs 241 ± 70 mg/L; P <.0001). This decrease was greater in severe sepsis and septic shock than in sepsis. Death was not predictable from initial IαI levels. In 2 patients with a favorable course, IαI values regularly increased during the ICU stay. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot analysis and microsequencing, we characterized IαI-related components in plasma from several patients; they obviously arise from IαI through proteolytic cleavage. Thus, systemic proteolysis and decreased biosynthesis both contribute to the fall in the plasma level of IαI. Because IαI is very sensitive to proteolysis by polymorphonuclear granulocytes (PMNs) that are stimulated during sepsis, we suggest that IαI plasma level would be a useful marker for neutrophil proteinase activity. ATIII, as well as IαI, is considered a negative acute phase protein. Because in vitro ATIII is less susceptible than IαI to proteolysis by PMNs and because their relative levels weakly correlated, we suggest that an unspecific systemic proteolysis is not significantly involved in the ATIII deficiency occurring in sepsis. (J Lab Clin Med 2000;135:188-98)

Genetic selection of poliovirus 2Apro-binding peptides.

The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2Apro). Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library. In addition, five clones encoding short peptides that interact with poliovirus 2Apro were also identified. The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue). This sequence is invariably located just at the carboxy terminus of each peptide. This approach raises the possibility of designing substrate analogue inhibitors of 2Apro. Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2Apro in vitro. A more general method for identifying peptides with antiproteinase activity is discussed.

The trypsin-inhibitory efficiency of human alpha 2-macroglobulin in the presence of alpha 1-proteinase inhibitor: evidence for the formation of an alpha 2-macroglobulin--alpha 1-proteinase inhibitor complex.

The inhibition of bovine pancreatic trypsin was studied at pH 7, 25 degrees C, using mixtures of purified human alpha 2-macroglobulin (alpha 2M) and alpha 1-proteinase inhibitor (alpha 1 PI). The partitioning of the enzyme between the two inhibitors was determined by comparing control esterase activity, assayed with N-benzoyl-L-arginine ethyl ester as substrate, with that remaining after incubation with inhibitory mixtures. (At [I]0 > [E]0, remaining esteratic activity reflects the concentration of alpha 2M-associated enzyme (alpha 2M-E*) and the concentration of alpha 1PI-associated, inactive enzyme (alpha 1PI-E*) is given by the difference, [E]0-[alpha 2M-E*].) The pattern of product distribution was found to be incompatible with an inhibitory model involving parallel, second-order reactions of E with alpha 2M and alpha 1PI. The data pointed to complex formation between the two inhibitors, limiting the level of alpha 2M readily available for reaction with E. Analysis based on the binding equilibrium, alpha 2M (dimeric unit) + alpha 1PI reversible alpha 2M-alpha 1PI, yielded Kd = 2.1 +/- 0.3 microM. Complex formation between alpha 2M and alpha 1PI was verified by gel permeation experiments. alpha 2M was found to restrict the volume of distribution of alpha 1PI in Sephadex G200 beds. Kd, deduced from gel permeation behaviour, was 0.8 +/- 0.32 microM. Preliminary kinetic experiments with dialyzed plasma suggested that the alpha 2M-alpha 1PI interaction is effective also in vivo. Given Kd and the mean plasma levels of the two inhibitors ([alpha 2M] = 2 microM; [alpha 1PI] = 36 microM), it was estimated that > 90% of alpha 2M in human circulation must be complexed to alpha 1PI and lack immediate antiproteinase activity.

Glycosylation pattern of human inter-alpha-inhibitor heavy chains.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan chain: a light chain named bikunin carrying the anti-proteinase activity and two heavy chains, H1 and H2, which exhibit specific properties, e.g. they interact with hyaluronan thus stabilizing the extracellular matrix. In this study, using matrix-assisted laser desorption ionization-time-of-flight MS and amino acid sequencing of tryptic peptides, we provide a detailed analysis of the glycosylation pattern of both heavy chains. H1 carries two complex-type N-glycans of predominantly biantennary structure linked to asparagine residues at positions 256 and 559 respectively. In contrast, the oligosaccharides attached to H2 are a complex-type N-glycan in the N-terminal region of the protein (Asn64) and three to four type-1 core-structure O-glycans mono- or di-sialylated, clustered in the C-terminal region. We propose that these O-glycans might function as a recognition signal for the H2 heavy chain. The biological implications of this hypothesis, notably for the biosynthetic pathway of IalphaI, are discussed.

Disulphide bonds assignment in the inter-alpha-inhibitor heavy chains--structural and functional implications.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan: a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IalphaI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha-trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We mapped the disulphide topology of the IalphaI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IalphaI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IalphaI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated alpha-helicities of 24% and 28%, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment. Inside the IalphaI molecule, the heavy chains are linked to the glycosaminoglycan chain via their C-terminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their C-terminal end and the high susceptibility of IalphaI to these proteinases.

Dendrotoxins: How Does Structure Determine Function?

AbstractDendrotoxins are a family of small proteins isolated from mamba (Dendroaspis) snake venoms. The toxins contain 57–60 amino acid residues cross-linked by three disulphide bridges. They are homologous to Kunitz-type serine proteinase inhibitors, such as aprotinin, (BPTI) although they have little anti-proteinase activity. The dendrotoxins block some subtypes of voltage-dependent potassium channels in neurones. Studies with cloned K+ channels indicate that α-dendrotoxin from green mamba Dendroaspis angusticeps blocks Kvl.1, Kvl.2 and Kv1.6 channels in the nanomolar range. Engineered and modified versions of dendrotoxin are being investigated to define the interactive site and account for differences in K' channel selectivity.

Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine β-trypsin

Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5–Cys55, Cys14–Cys38 and Cys30–Cys51. Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI. Oxidative refolding starts with the formation of single disulfide intermediates. All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli . In each variant the remaining four cysteine residues were replaced by alanine. Four of these variants are shown here to inhibit bovine β-trypsin: three of them contain native and one non-native (Cys5–Cys51) disulfide. All but one (Cys5–Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low. Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI. Remarkably, in some of the variants the bonding constants were found to be higher for the reduced rather than for the oxidized form of the variant. Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value. Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific. In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin. In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra. In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein. This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein. This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster. On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.

Dynamic extracellular matrix remodeling in the heart failure: cardiac hypertrophy, dilatation and fibrosis

Abstract Over 40% of patients awaiting heart transplant surgery suffer from dilated cardiomyopathy, a life-threatening condition of impaired muscle cell metabolism, which forces the walls of the heart to balloon out under pressure. Myocardial infarction, which leads to remodeling, thinning of the ventricle wall, dilatation and heart failure, is one of the leading causes of death. Remodeling of the myocardium following infarction is a poorly understood biological process. Remodeling, by its very nature, implies an alteration in the extracellular matrix (ECM) and in the spatial orientation of cells and intracellular components. Understanding of molecular and cellular mechanisms of remodeling are an extremely worthwhile topic for exploration. Extracellular matrix, in connection with its receptor integrins and cytoskeletal cellular matrix in a 3-D orientation, is responsible for cardiac cell alignment and myocardial structural integrity. Substances that break down the extracellular matrix, specialized proteinases as well as inhibitors of proteinases, appear to be normally balanced in maintaining the integrity of the myocardium. Myocardial infarction leads to an imbalance in proteinase antiproteinase activities allowing alterations in the stability and integrity of the extracellular matrix, leading to tissue remodeling. The latent matrix proteinases are activated following infarct-injury. The degradation of ECM induces contractile and proliferative phenotypes in cardiac fibroblast cells and transforms to myofibroblast cells. The extracellular matrix-derived peptides induce maladaptive response in myofibroblasts and activate MMPs, causing ventricular dilatation and myocardial dysfunction. This review presents new and significant information regarding the role of activated matrix proteinases and their endogenous inhibitors in ischemic heart disease. This will have a significant impact on both our understanding of the tissue remodeling and on basic cell physiology of extracellular matrix turnover, as well as potential avenues for pharmacological approaches for the treatment of ischemic heart disease and heart failure.

Stabilization of lung surfactant particles against conversion by a cycling interface.

The large active particles of pulmonary surfactant are depleted in patients with acute respiratory distress syndrome and in animal models of this disorder. We studied in vitro conversion of large to small particles, separated by differential sedimentation, to determine how factors lavaged from rabbits injured by intravenous oleic acid would affect conversion. In half-filled test tubes rotated end over end, samples from injured animals increased the recovery of large particles from 40 +/- 6% of uncycled samples for controls to 62 +/- 21%. We hypothesized that proteins in the injured samples, and perhaps also the proteinase inhibitors used previously to block conversion (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990), stabilized surfactant particles by limiting access to the cycling interface. Hemoglobin, neutrophil elastase, and alpha1-antiproteinase (alpha1-PI) oxidized to eliminate its antiproteinase activity all stabilized large particles against conversion. Hemoglobin was most effective, increasing recovery from 18 +/- 5% for controls to 86 +/- 5% with 0.4 mg/ml hemoglobin. Native alpha1-PI had no effect on conversion. Our results suggest that acceleration of normal conversion is unlikely to explain the depletion of large particles in injured lungs. They also suggest that conversion of surfactant particles separated by differential sedimentation requires no proteinase susceptible to inhibition by alpha1-PI. They provide an alternate hypothesis related to interfacial effects rather than proteinase inhibition for the previously reported effect of alpha1-PI on conversion of particles separated according to density.

Biology of progesterone-induced uterine serpins.

Progesterone is a pleiotropic regulator of uterine function required for the maintenance of pregnancy in mammals. A major action of progesterone is to induce expression of genes encoding for secretory proteins of the uterine endometrium. These proteins participate in several roles deemed essential for survival of the conceptus and include enzymes, transport proteins, and regulatory proteins. Several serpins have been identified as being part of the milieu of secretory proteins in the progesterone-dominated uterus. Among these are plasminogen activator inhibitor-1, induced by progesterone in cultured human endometrial cells (Miyauchi et al., 1995; Lockwood et al., 1995), and α1-antitrypsin, which is synthesized by human endometrium during pregnancy (Fay et al., 1990) and also can enter uterine fluid as a serum transudate (Bany and McRae, 1992). These proteins likely inhibit serine proteinases secreted by the uterus and placenta and may limit remodeling of the endometrium, participate in local hemostasis and regulate placental invasiveness. In addition to these well-known serpins, the uterus of certain species of ungulates each produce one or more members of a subfamily of serpins that are characterized by endometrial site of synthesis, sequence homology to each other, and weak or no known antiproteinase activity. This chapter will concentrate on the most well-characterized of these uterine-specific serpins — a glycoprotein of the sheep uterus called uterine milk protein (UTMP) — and will compare the properties of this protein to other uterine serpins. Uterine milk protein is the most abundant steroid-induced protein yet described and gram quantities can be obtained from the uterus of a single unilaterally-pregnant ewe (Moffatt et al., 1987a). There is evidence to implicate UTMP in inhibition of lymphocyte funciton and in binding interactions with placental secretory proteins. It is also possible that UTMP acts as a carrier protein to transport maternal products across the placenta.KeywordsUterine EndometriumUterine FluidReactive Center LoopInfer Amino Acid SequencePlacental InvasivenessThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of acrosin‐like activity of lake sturgeon (Acipenser fulvescens) spermatozoa

Acipenserid fish sperm possess trypsin-like activity, resembling acrosin activity of mammalian sperm, which can be measured by hydrolysis of N-α-benzoyl-DL-arginine p-nitroanilide (BAPNA) Ciereszko et al., 1994: J Exp Zool 268:486–491). We found that this activity can be preserved when sperm is frozen on dry ice with 0.6 M sucrose-10% dimethylsulfoxide (DMSO) extender (sperm:extender ratio 1:3), and subsequently stored in liquid nitrogen. However, other methods of freezing (without cryoprotectant at −18°C, −80°C, and −196°C) did not protect this activity. Acrosin-like activity decreased in the course of storage of milt on ice; 88% decline was recorded after 13 days. Acrosin-like activity increased with temperature from 10°C to 30°C, but was inactivated at 40°C to about 40% as compared to the optimum temperature. Triton X-100 inhibited activity by 15% and 72% at 0.01% and 0.1% concentrations, respectively. Activity was not affected by Mg2+ but was inhibited by Zn2+ (30% and 75% in the presence of 0.1 mM and 1 mM, respectively). Maximum velocity of substrate hydrolysis was observed at 2 mM of BAPNA. Acrosin-like activity was effectively inhibited by 4′-acetamidopheny1 4-guanidinobenzoate (AGB), an inhibitor of mammalian acrosin. Sperm acrosin-like activity correlated negatively with anti-proteinase activity of seminal plasma. We conclude that sturgeon acrosin-like activity shares many properties with mammalian acrosin. On the other hand, it has some unique properties which may represent adaptations of this enzyme to the environment of external fertilization. © 1996 Wiley-Liss, Inc.

Characterization of acrosin-like activity of lake sturgeon (Acipenser fulvescens) spermatozoa.

Acipenserid fish sperm possess trypsin-like activity, resembling acrosin activity of mammalian sperm, which can be measured by hydrolysis of N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) (Ciereszko et al., 1994: J Exp Zool 268:486-491). We found that this activity can be preserved when sperm is frozen on dry ice with 0.6 M sucrose-10% dimethylsulfoxide (DMSO) extender (sperm:extender ratio 1:3), and subsequently stored in liquid nitrogen. However, other methods of freezing (without cryoprotectant at -18 degrees C, -80 degrees C, and -196 degrees C) did not protect this activity. Acrosin-like activity decreased in the course of storage of milt on ice; 88% decline was recorded after 13 days. Acrosin-like activity increased with temperature from 10 degrees C to 30 degrees C, but was inactivated at 40 degrees C to about 40% as compared to the optimum temperature. Triton X-100 inhibited activity by 15% and 72% at 0.01% and 0.1% concentrations, respectively. Activity was not affected by Mg2+ but was inhibited by Zn2+ (30% and 75% in the presence of 0.1 mM and 1 mM, respectively). Maximum velocity of substrate hydrolysis was observed at 2 mM of BAPNA. Acrosin-like activity was effectively inhibited by 4'-acetamidophenyl 4-guanidinobenzoate (AGB), an inhibitor of mammalian acrosin. Sperm acrosin-like activity correlated negatively with antiproteinase activity of seminal plasma. We conclude that sturgeon acrosin-like activity shares many properties with mammalian acrosin. On the other hand, it has some unique properties which may represent adaptations of this enzyme to the environment of external fertilization.

Characteristics of Muskellunge Spermatozoa I: Ultrastructure of Spermatozoa and Biochemical Composition of Semen

We investigated the ultrastructure of spermatozoa and some physiological and biochemical parameters of semen in muskellunge Esox masquinongy. Ultrastructure of spermatozoa analyzed with scanning and transmission electron microscopes revealed a spherical head, 1.5 um in diameter. Unlike the spermatozoa of other teleost fishes, an elongated midpiece with abundant mitochondria was found to be characteristic for muskellunge spermatozoa. Three techniques for measuring sperm concentration, counting with a hemacytometer, spermatocrit, and spectrophotometric methods, provided similar results (19.7 ± 6.1 × 109 sperm/mL). The spectrophotometric method is recommended because it is rapid and requires only small amounts of semen. Ionic concentrations and osmolality (289 ± 16.8 milliosmols/kg) of seminal plasma were reported. Sodium (129 ± 6.7 mM) and potassium (27.88 ± 3.27 mM) concentrations were closer to levels found in the seminal plasma of salmonids than cyprinids. Aspartate aminotransferase (AspAT) activity was measured with and without supplement of pyridoxal phosphate (PLP) from sperm extracts. A 96% increase of AspAT was found when PLP was included in the assay. We confirmed the presence of antiproteinase activity (against cod trypsin) in seminal plasma of muskellunge. Using the flow cytometry method, we estimated that the DNA stainability of muskellunge spermatozoa was 0.188 ± 0.008 times that of red blood cells (TRBC) from diploid rainbow trout Oncorhynchus mykiss. The DNA stainability of muskellunge spermatozoa was significantly lower than that of muskellunge embryonic cells, which was 0.474 times that of the TRBC (2.21 pg DNA/cell, based on the internal standard of TRBC of 4.66 pg DNA/cell).

Effects of season and dietary ascorbic acid on some biochemical characteristics of rainbow trout (Oncorhynchus mykiss) semen

During the reproductive season, rainbow trout spermatozoa are stored in the sperm ducts for several months. There is no sperm production at this time since spermatogenesis is completed before spawning. To leam more about characteristics of semen during such a long storage, we analyzed changes in protein concentrations, anti-proteinase activity in seminal plasma and sperm aspartate aminotransferase activity during an extended reproductive period during which fish were fed diets supplemented with various ascorbic acid concentrations. Seminal plasma protein concentration and anti-proteinase activity declined toward the end of the reproductive season. These phenomena may be related to oncoming proteolytic events leading to degradation of the sperm. Protein concentrations and anti-proteinase activities were strongly correlated within groups of different ascorbic acid supplementations and several sampling dates (r=0.6-0.9 in most cases, p<0.05). Ascorbic acid deficiency resulted in a decrease in both parameter levels as compared to levels in groups with vitamin C supplement (p<0.08). Deficiency also resulted in lower stimulation of aspartate aminotransferase by an exogenous pyridoxal 5'-phosphate in comparison to fish fed vitamin C-supplemented diets (p<0.05). These results support earlier studies suggesting a protective role of ascorbic acid toward maintaining sperm quality.

Anti-inflammatory effect of a PAF receptor antagonist and a new molecule with antiproteinase activity in an experimental model of acute urate crystal arthritis

Platelet activating factor (PAF) is a potent mediator of allergic and inflammatory reactions in different pathological conditions. During recent years there has been increasing evidence that PAF can play an important role in the pathogenesis of arthritis. The PMN proteinases make an important contribution to the final tissue joint destruction in arthritis. In a rabbit model of acute crystal arthritis, we have compared the anti-inflammatory effect of two new molecules: BN 50727 with anti-PAF activity, and BN 50548 an inhibitor of PMN proteinases. These molecules were administered dissolved in DMSO at doses of 6 mg/kg three times daily i.p., beginning 24 h before the induction of arthritis. Compared with the untreated animals those receiving the drugs, presented a significant diminution in: (1) the synovial fluid volume; (2) the amount of cells infiltrating the joint cavity and the synovial membrane; and (3) the PGE 2 concentration. Furthermore, in both groups of treated rabbits there was a significant decrease in synovial IL-6 concentration and in C-reactive protein serum levels and an important decline of histopathological score. The treatment with BN 50548 induced a significant reduction of TNF levels in the synovial fluid vs DMSO-treated and untreated rabbits. These results further strengthen that in an acute experimental arthritis model, molecules with capacity to antagonize the in vivo action of PAF have an anti-inflammatory effect reflecting an important role for this mediator in the pathogenesis of arthritis. We have also seen that an inhibitor of proteinases is capable of improving the joint inflammation apparently through a decrease in tumor necrosis factor (TNF) and interleukin-6 (IL-6) synovial levels. Furthermore, the proteinase inhibitor treatment preserves the loss of articular proteoglycan content, in an acute arthritis model. In conclusion, BN 50727 and BN 50548, two compounds with PAF antagonist and antiproteinase activity, respectively exert an anti-inflammatory effect in an experimental model of acute urate crystal arthritis, probably due to a decrease in TNFa and IL-6 synthesis.