Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

Role of heparin in the formation of food lipemia

Influence of endogenic and exogenic heparin in vivo on the basic forms of serum lipids content: cholesterol ethers, triacylglycerols, free fatty acids; as well as that glycosaminoglycan effect in vivo and in vitro on total lipoproteine lipase (LPL) activity and lecithin-cholesterol acyltransferase (LCAT) activity of human blood serum were investigated on food lipidemia model. The decrease of intercell reserve heparin content and increase of the background and post-heparin levels of blood serum LPL activity were indicated after two hours food load. The role of two factors, endogenic heparin being one of them, in the increase of postprandial LPL activity of blood serum were discussed. At the same time, some inhibition of blood serum LCAT activity two hours after food reception (evidently, as a result of endogenic heparin action) and to a considerable extent inhibition of cholesterol etherification under the action of exogenic heparin in vivo were ascertain. Heparin in vitro (50 U/ml of blood serum) did not influence LCAT and total LPL activities. It was summarised that endogenic heparin is a factor, taking part in lipolysis processes regulation.     

The effect of anticoagulants on Blennius pholis L. Leucocytes

The effects of ethylenediamine-tetraacetic acid (EDTA), heparin and tri-sodium citrate (TSC) on various haematological parameters in the blenny, Blennius pholis, were investigated. EDTA and heparin, at the concentrations tested, proved adequate in preventing coagulation although leucocyte viability was reduced when the higher levels were used. TSC failed to prevent coagulation at all concentrations tested. Short (5 min) and long (20 min) term contact of these anticoagulants with blood did not produce any significant changes in leucocyte proportions. Heparin is regarded as the most suitable anticoagulant for use with B. pholis blood.     

Effects of heparin and benzyl alcohol on lymphocyte-mediated cytotoxicity in vitro

The effect of heparin on the in vitro lysis of EL4 tumor cells by immune BALB/C lymphocytes was investigated by using a ⁵¹Cr-release cytotoxicity. assay. Powdered heparin did not inhibit lymphocyte-mediated cytotoxicity (LMC) at concentrations up to 500 units/ml. Heparin containing 9 mg/ml of benzyl alcohol (BA), as preservative, significantly reduced the LMC. BA alone at 1 mg/ml inhibited LMC without any apparent toxic effect on the target cells or on the immune lymphocytes. The inhibitory effect of three different heparin preparations was found to be related to the BA concentration rather than the amount of heparin. However, low concentrations of heparin (0.5 or 1 unit/ml) significantly enhanced the LMC. Our findings are in contrast to previous reports suggesting a depressive effect of heparin on LMC.     

Effect of anticoagulants in vitro on the viability of lymphocytes and content of free fatty acids in plasma

Summary These authors attempted to test the effect of anticoagulants on lymphocytes viability by reproducing the procedure used for lymphocyte isolation for various immunologic tests in which blood specimens are allowed to stay at room temperature for 2 h before lymphocytes are isolated. Blood was obtained with three different anticoagulants i.e. heparin, citrate, and CPDA (citrate, phosphate, dextrose, and adenine). Plasma was lyophilized and extracted with ethanol. Dried ethanol extracts were suspended in medium (RPMI 1640+10% fetal bovine serum) and incubated with a lymphocyte cell line (MOLT-4). After 24 h of incubation the viability of cells was examined. The following death rates of the cells were observed: heparin −63±4.6% (mean±SEM), citrate −27±6.7%, and CPDA 6.2±0.6% (P<0.0005). A significant correlation was found between these results and changes in the concentrations of free fatty acids in the extracts. These results emphasize the importance of choosing the right anticoagulant when the viability of lymphocytes is obligatory.     

Influence of heparin on the removal of serum lipoprotein lipase by the perfused liver of the rat

Isolated rat livers were perfused with whole rat blood containing postheparin lipoprotein lipase (LPL) activity. LPL activity disappeared rapidly from the perfusate; the extraction ratio (portal vein-hepatic vein difference) was 0.70 for all time periods studied. Control experiments established that the disappearance of LPL was not due to non-specific inactivation in the apparatus or to the release of an inhibitory by the liver. The addition of heparin to the perfusate in suitable concentration (4 units/ml) almost completely blocked the disappearance of LPL activity from the perfusate. In addition to the perfusion experiments, we studied the effect of heparin on LPL activity when added to the LPL assay system. When heparin was added to the assay system containing fresh postheparin serum from rats, it stimulated LPL activity by about 70%. When heparin was added to postheparin serum which had been perfused through the liver, it stimulated LPL activity over 200%, but it did not restore LPL to its preperfusion value. These observations are compatible with a two-step inactivation system for LPL by the liver. The first step may involve a dissociation of a heparin-apoenzyme complex followed by destruction of the heparin. The second step may involve the removal of the apoenzyme of LPL.     

Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 microl ml(-1) or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 degrees C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8.0 microl ml(-1) (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 10 degrees C. Aeromonas salmonicida strain MT004 (As MT004) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of 0.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity.     

The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.)

Hematological parameters (Ht, Hb, RBC, WBC, PLT), erythrocyte size, and osmotic fragility, differential leukocyte count, ROS production in common carp blood collected on three anticoagulants: heparin (10 IU/mL, Na2EDTA (0.1, 0.5, and 1 mg/mL), and sodium citrate (0.3 mg/mL) were compared. Na2EDTA caused partial blood hemolysis in Ht tubes which made Ht measurement impossible, and resulted in high variability of the results. Both, citrate and Na2EDTA increased sensitivity of red blood cells to hemolysis. Na2EDTA also induced erythrocyte anisocytosis and anisonucleosis. Na2EDTA significantly increased ROS production but no effect of anticoagulants on WBC, PLT or differential leukocyte count was observed. The obtained results show that Na2EDTA should not be used for evaluation of red blood cell parameters and erythrocyte morphology, and for ROS production measurement in common carp. Heparin proved to be the most appropriate anticoagulant to use for this species, although Na2EDTA and sodium citrate may be used for WBC and leukocyte differential count evaluations.     

The influence of preparative techniques on glycolytic metabolism in resting leucocytes

Literature values for ‘resting’ glycolytic and respiratory rates of guinea pig exudate polymorphonuclear leucocytes as reported by various authors were calculated to the same unit basis to determine what differences might exist. Comparison experiments investigated preparative techniques to determine the significance with respect to the variations discovered. Procedures using 12 or 0.1% casein in saline as peritoneal irritant, 0 or 37 °C collection temperatures, siliconed or non-siliconed glassware, and hemolysis treatment to remove red blood cells, did not affect glycolytic rate in leucocytes. Three different anticoagulants used, heparin, citrate, and ethylenediamine tetracetic acid (EDTA) did account for the variability in the reported glycolytic rates by increasing the lactate production as compared with cells not exposed to anticoagulant. Citrate and EDTA increased the lactate production approx. 65%, heparin 22%, with the increase apparently related to the calcium chelating ability. Calcium ions were found to depress the glycolytic lactate production proportionately as the concentration increased up to 0.0026 M in the incubation medium. The removal or absence of calcium ions from the medium increased the lactate formation.     

Immunosuppressive properties of electrophoretically "slow" and "fast" form alpha 2-macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation

Lymphokine activated killer cell lysis of K562 cells was inhibited by alpha 2-macroglobulin (alpha 2M), soybean trypsin inhibitor, and alpha 1-proteinase inhibitor. In serum free medium 2 mg/ml alpha 2M suppressed target cell lysis in a 4-h cytotoxic assay with about 40%. Suppression was dose and time dependent. Cytotoxicity was unaffected by alpha 2M concentrations less than 0.25 mg/ml, and by alpha 2M added later than 1.5 h from start of assay. Pre-treatment of effector (but not of target) cells with alpha 2M was even more suppressive than the presence of alpha 2M during assay. Cell-mediated cytotoxicity was not inhibited by alpha 2M treated with methylamine or by various alpha 2M-proteinase complexes. In contrast, alpha 2M-proteinase complex as well as native alpha 2M suppressed the proliferation of Ag-activated T cells. However, methylamine-treated alpha 2M did not inhibit T cell proliferation, and suppression by alpha 2M-proteinase complex was significantly reduced after inhibition of the alpha 2M-bound proteinase. On incubation at 4 degrees C with lymphokine-activated killer cells, alpha 2M reacted with cell associated proteinases and changed from electrophoretically "slow" to "fast" form. Cell associated proteinases bound by alpha 2M showed chymotrypsin- and trypsin-like specificities and their activity surpassed activity caused by cellular leakage and secretion. The present results strongly indicate that alpha 2M mediates immunosuppression in its capacity as a proteinase inhibitor and suggest inhibition of (T)cell surface-associated proteinases as a possible mode of suppression.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets.

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.     

Recovery of functional capacity of human granulocytes after freeze-preservation with dimethyl sulphoxide.

Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose (ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at ?196 °C and reconstituted in a solution containing dextran polymer 70.A battery of tests including nitroblue tetrazolium (NBT) reduction, Candida phagocytic and candidacidal capacity was used to compare anticoagulants and reconstitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period.Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstitution method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. Lignocaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional protective effect on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields.Using these findings to optimize conditions reconstituted neutrophils retained 31.7% of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.     

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.     

Effect of thyroid hormones on activity dynamics of enzymes of intestinal mucosa of juvenile roach <Emphasis Type="Italic">Rutilus rutilus</Emphasis>

The effect of thyroid hormones on activity dynamics of enzymes (proteinases and glycosidases) of intestinal mucosa of juvenile roach Rutilius rutilus was investigated. Application of substances increasing and decreasing the level of thyroid hormones in blood plasma significantly influences the growth rate and the activity of proteinases and glycosidases functioning in the intestinal mucosa. In most cases, the activity level of trypsin-like proteinases and the activity of glycosidases in the fish exposed to triiodothyronine were significantly higher than in the control. The activity level of chymotrypsin-like proteinases in fish form the group with exposure of exogenous triiodothyronine only in the end of the experiment surpassed the values of this parameter in the control fish. In the fish developing at deficiency of thyroid hormones, the growth rate and proteinases activity were significantly lower in comparison with the control.     

Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).     

杠柳新苷P和E对东方粘虫和小地老虎幼虫中肠蛋白酶活性的影响

从杀虫植物杠柳Periploca sepium Bunge根皮中分离得到的杠柳新苷P具有很高的杀虫活性, 为了探索其杀虫机理, 采用经典的昆虫蛋白酶活性测定方法, 比较研究了杠柳新苷P和无杀虫活性的杠柳新苷E对东方粘虫Mythimna separata与小地老虎Agrotis ypsilon 6龄幼虫中肠类胰蛋白酶和类胰凝乳蛋白酶活性的影响。结果表明： 对东方粘虫弱碱性类胰蛋白酶, 杠柳新苷P表现出强激活作用（酶活性为对照的3.43倍）, 激活时间可长达8 h, 而杠柳新苷E则无明显激活作用。杠柳新苷P和E对东方粘虫弱碱性类胰蛋白酶活性的影响二者差异显著（P=0.01）, 杠柳新苷P药后2, 4和8 h, 东方粘虫中肠弱碱性类胰蛋白酶的活性分别是杠柳新苷E药后的15.4, 106.8和242.7倍。酶活性测定结果还表明, 与东方粘虫相比, 小地老虎中肠类胰蛋白酶活性相对较低, 且杠柳新苷P的激活作用也较弱, 这可能是杠柳新苷P对东方粘虫具杀虫活性, 而小地老虎对其不敏感的原因之一; 另外, 杠柳新苷P和E对试虫中肠类凝乳胰蛋白酶活性均无明显影响。据此推测, 杠柳活性成分新苷P对东方粘虫中肠弱碱性类胰蛋白酶的激活作用可能是造成试虫中毒的机理之一。     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Effect of heparin on thrombin inactivation by antithrombin-III.

The inactivation of thrombin by heat and by its physiological inhibitor, antithrombin-III, shows quite different dependence on heparin concentration. Heparin at 250 microgram/ml protects thrombin against heat inactivation, and thrombin behaves heterogeneously in this reaction. In the absence of heparin, the thermodynamic activation parameters change with temperature (deltaH+ = 733 kJ/mol and 210 kJ/mol at 50 and 58 degrees C respectively). When heparin is present, heat inactivation of the protected thrombin species proceeds with deltaH+ = 88 kJ/mol and is independent of temperature in the same range. On the other hand, heparin at 0.125-2.5 microgram/ml accelerates the thrombin-antithrombin-III reaction. Thrombin does not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0 and 37 degrees C appear to follow first-order kinetics. Activation enthalpy is independent of heparin concentration or temperature, deltaH+ = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin specific activity. Heparin seems to exert its effect by increasing activation entropy. On the basis of these data we suggest a mechanism of action of heparin in the thrombin-antithrombin-III reaction which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.