Lebetase, an alpha(beta)-fibrin(ogen)olytic metalloproteinase of Vipera lebetina snake venom, is inhibited by human alpha-macroglobulins.

The effects of the plasma proteinase inhibitors alpha(2)-macroglobulin (alpha(2)M) and the alpha(2)M-related pregnancy zone protein (PZP) were evaluated towards the metalloproteinase lebetase, isolated from Vipera lebetina venom. We demonstrate that lebetase interacts with both inhibitors. Cleavage of alpha(2)M by lebetase resulted in the formation of 90-kDa fragments, and covalent complexes of alpha(2)M with lebetase were observed. The proteolytic activity of lebetase against fibrinogen and azocasein could be inhibited by alpha(2)M. Cleavage of PZP also resulted in the formation of 90-kDa fragments, and complexes of both dimer and tetramer forms of PZP with lebetase were detected. The amino acid sequence identification of the sites of specific proteolysis of alpha(2)M and PZP demonstrate that the cleavage sites are within the bait regions of both proteins. Lebetase I cleaves between Arg(696)-Leu(697), which is one of the most common cleavage sites in alpha(2)M by proteinases. The other two cleavage sites in alpha(2)M by lebetase are Gly(679)-Leu(680) and His(694)-Ala(695). The cleavage between Pro(689)-Gln(690) is the only cleavage site identified in PZP. In that lebetase is an anticoagulation agent in vivo, we propose that the interaction of lebetase with alpha(2)M may suggest a reduced fibrin(ogen)olytic activity of lebetase in human.

Isolation, partial characterization and complete amino acid sequence of the toxic phospholipase A2 from the venom of the common viper, Vipera berus berus.

A basic, toxic phospholipase A2 was purified from the venom of Vipera berus berus (Vbb) by a single purification step, using hydrophobic chromatography. The primary structure of isolated protein was established from peptides generated by Gly-specific papaya proteinase IV, beta-trypsin, CNBr and mild acid hydrolysis. The enzyme consists of a single chain of 122 amino acid residues with 14 Cys in positions characteristic for the phospholipase A2 subgroup IIA. As far as we know, this is the first complete Vipera berus phospholipase A2 amino acid sequence reported.

Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom.

A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vgamma (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70 degrees C does not alter the arginine esterase activity of the enzyme.

Factor X activator from Vipera lebetina snake venom, molecular characterization and substrate specificity.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6-9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly - factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly - factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg(52)-Ile(53) bond in the heavy chain of human factor X and the Arg(226)-Val(227) bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.

Biochemical characterization of lebetase, a direct-acting fibrinolytic enzyme from Vipera lebetina snake venom.

Lebetase, the fibrinolytic enzyme isolated from Vipera lebetina (Levantine viper) snake venom is a metalloenzyme that contains one mole of Zn2+ and one mole of Ca2+ per mole of protein. Lebetase is inhibited by dithiothreitol, suggesting that disulfide bonds are necessary for holding the structure. Vipera lebetina venom contains several isoforms of lebetase in the interval of pI 4.6-5.4. Two lebetase fractions I (pI of the main component 5.0) and II (pI of the main component 5.3) degrade fibrin and fibrinogen by hydrolysis of the alpha and beta chains. The molecular weights of the cleavage products produced by the two different lebetase fractions are identical. The metal ions, Cd2+, Cu2+, Co2+, inhibit fibrinolytic and caseinolytic activity of lebetase I and II. Using mass spectrometry we characterized differences in molecular masses of lebetase I and II (22719 Da and 22912 Da). Vipera lebetina venom from a single snake contains mainly one form of lebetase. Lebetase I is more stable at low pH than lebetase II. The lebetases I and II inhibit platelet aggregation induced by ADP in a dose-dependent manner.

Medium molecular weight factor X activating enzyme from Vipera berus berus venom.

Vipera berus berus venom contains several factor X activating enzymes. One of them (VBFXAE) was separated by gel-filtration on Sephadex G-100 superfine and on a bacitracin-agarose column. The enzyme is a single-chain glycoprotein with mol. wt 38,000. The enzyme has several molecular forms with pI 3.5-4.5. After neuraminidase treatment the enzyme has pI 4.5. VBFXAE contains 2 Ca per mole. The activator is inactive on synthetic substrates, on casein, prothrombin, and fibrinogen, and appears to act specifically on factor X. The activator also weakly hydrolyses the insulin B-chain at the positions Ala14-Leu15 and Tyr16-Leu17. The cleavage of the insulin B-chain is inhibited by EDTA, suggesting the metalloproteinase nature of the enzyme.

Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom.

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.

Surgical, infectious and thromboembolic complications of central venous catheterization.

The authors have analysed the incidence of specific complications in a series of 420 intracaval catheters placed in 388 patients, using six transcutaneous puncture techniques: supraclavicular and infraclavicular subclavian, external and internal jugular, antecubital and brachiocephalic approaches. Strict and moderate criteria were used to evaluate the frequency of complications. Using strict criteria, the lowest rate of surgical complications (5%) was found with the antecubital and external jugular approach, followed by infraclavicular (6.7%) and supraclavicular (9.3%) subclavian techniques; the highest rate was seen with internal jugular (10%) and brachiocephalic (15%) routes. As to inflammatory and infectious complications, the sequence was as follows: brachiocephalic (2.5%), infraclavicular (4.4%) and supraclavicular (5.3%) subclavian, and internal jugular (7%) veins; a 10% incidence was associated with external jugular and antecubital techniques. Manifest thromboembolic complications were observed only in the brachiocephalic and antecubital groups (2.5% and 10%, respectively), the overall incidence of pulmonary embolism being 0.2%. None of the approaches used can be recommended as an exclusive method of choice. The risks of central venous catheterization should be minimalized by adherence to strict principles of placing as well as care of the indwelling intravenous catheters.

Venepuncture, catheterization and failure to position correctly during central venous cannulation.

The authors analyse the incidence of puncture, catheterization and failures of positioning in a series of 420 central venous catheterizations performed in 388 patients, using six transcutaneous approaches: supraclavicular and infraclavicular subclavian, external and internal transjugular, antecubital and brachiocephalic. Puncture failures were recorded at 7.9% per 36 failures from 456 attempts, catheterization failures at 5.8% per 26 failures from 446 attempts, and misplacements at a rate of 5.7% per 23 misplacements during 420 catheterizations. The results show that, in respect of puncture, catheterization and success of positioning, the supraclavicular subclavian approach may be recommended preferentially. The internal jugular, brachiocephalic and infraclavicular subclavian approaches are also advantageous. A high rate of failures and misplacements was recorded in the antecubital and external jugular groups. The antecubital veins, however, should be reserved for orthopnoeic sitting patients, the success rate being significantly increased if the patients are sitting.

The effects of open heart surgery on growth hormone, cortisol and insulin levels in man. Hormone levels during open heart surgery.

Plasma growth hormone, cortisol, insulin and blood glucose concentrations were measured intra- and postoperatively in ten patients who underwent open heart surgery with moderate hypothermia. Diazepam-ketamine anaesthesia for 10-20 min failed to precipitate any significant alterations in the levels of measured hormones and blood glucose. In the pre-bypass period of surgery, an increase in cortisol and a slight elevation in growth hormone levels was observed; insulin level showed no change in spite of marked hyperglycaemia. The bypass period, including hypothermia and haemodilution, was accompanied by unchanged cortisol and elevated growth hormone levels, while insulin demonstrated a slight rise which did not correspond with the degree of hyperglycaemia. The post-bypass period with rewarming the restoring spontaneous circulation was characterized by further marked increase in cortisol and growth hormone levels and, in spite of decreasing levels of blood glucose, by a paradoxical elevation in plasma insulin. It is suggested that hypothermia, haemodilution, reduced tissue perfusion affecting endocrine glands, as well as denaturation of some hormones in the oxygenator, participate in the moderate endocrine response, disproportionate to the stress of cardiopulmonary bypass surgery. The rise in hormone levels on terminating bypass seems to be dependent on the improved blood flow to endocrine glands due to recovered spontaneous circulation, rewarming and, as for insulin, presumably even on the reduced inhibitory effect of catecholamines.

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom.

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.

[Molecular medicine]. / Molekulárna medicína.

The introduction of new methods into medicine, such as recombinant DNA, production of monoclonal antibodies, and particularly polymerase chain reactions, yielded much knowledge and information in fields like preventive medicine (prenatal screening), reproduction strategies (preimplantation diagnosis of genetic defects), molecular diagnosis (oncological and infectious diseases), gene therapy, etc. In many areas the practical use of these sophisticated approaches has become technically feasible yet due to the high cost involved it is still limited. The introduction of some screening programs will be associated with ethical problems of serious impact. To date we can already establish presymptomatic diagnosis of some progressive diseases for which however no therapy is available. Potential abuse of confidential genetic information as well as the possibilities of using gene therapy for "corrective eugenics" are loaded with serious ethical problems. Prospective doctors have to be prepared for this situation in the course of undergraduate studies of medicine. (Ref. 11.)

The development of lipid metabolism. Changes in the level of fatty acids in serum during postnatal ontogenesis in the rat.

In young rats of 5 different age groups the spectrum (per cent) of 6 FFA was determined. The results show, that palmitic acid (C16:0) represents the main FFA in all age groups; at the end of the first week forms nearly 50 per cent. In adult animals (8-week-old) values decreased to 26 per cent. Similarly, the myristic acid (C14:0) was found highest in suckling animals (10 per cent) and decreased with age. The ratio of saturated to unsaturated FA (S/U) decreased with age. In other animals of 7 different age groups the concentration of total and of individual FFA was investigated. The results were expressed in nmol.l-1. Results showed, that the total concentration of FFA during the first postnatal month is nearly 2-times higher than in adults. The ratio of S/U FFA decreased from 1.81 to 0.85. The concentrations of individual FFA showed different age-dependent trend. Myristic and stearic acids (C14:0 and 18:0) increased with age till the 4th week. The concentrations of palmitic, oleic and linoleic acids were high during the first 3 weeks of age and decreased after weaning. The concentration of palmitate-oleic acid (C16:1) was lowest in all age groups as well as that of palmitic (C 16:0) was always the highest. These results show, that changes in concentration of individual FFA with age are not dependent only on the intake of the milk. (Tab. 3, Fig. 1, Ref. 8).

[The effect of age and thyroxine on lipogenesis during ontogeny. Activity of malic enzyme]. / Vplyv veku a tyroxínu na lipogenézu pocas ontogenézy. Aktivita "malic" enzýmu.

In young rats of different ages of postnatal development the effect of short-term administration of thyroxine (T4) on the activity of malic enzyme (ME) was studied in brown adipose tissue (BAT), liver, heart and kidney. Thyroxine was injected in a dose sufficient to saturate all receptor sites, i.e. 1 microgram per 1 gram of B.W. during 3 consecutive days. The highest activities of ME were found in BAT. An increase in enzyme activities with age was observed in all tissues studied in control animals. Injections of T4 increased the activity od ME in the liver; in BAT a significant increase was found only the younger age group (6 days). In older animals (40 days) with high values in BAT of controls no further increase induced by T4 was observed. In the kidney as well as in the heart no significant changes were found after T4 administration. It is concluded that in developing animals BAT may play a more important role in lipid synthesis than considered so far. (Fig. 1, Ref. 7.)

[Kinetics of thyroxine in ontogenesis studies using the metabolism of radiothyroxine]. / Kinetika tyroxínu pocas ontogenézy studovaná pomocou metabolizmu rádiotyroxínu.

Radiothyroxine turnover rate was studied postnatally in 9, 18, and 35 day old rats in various intervals during 24 hours after intrajugular injection. Value of fractional turnover rate indicated higher thyroid activity in 18 day old rats than in those aged 9 or 35 days. These results support our previous conclusions on the maturation of thyroidal regulatory mechanisms in this period of development which were based on the rate of radioiodine uptake and changes in serum thyroxine concentrations. In another series of animals, the distribution of radiothyroxine in comparison with distribution of radioiodide was studied 4 hours after intrajugular injection on days 8, 12, 16, 20, 24, and 30 of postnatal development. In case of hormone distribution, all values in the brain, liver, kidney, intestine, and serum were much higher in young animals than in adult 60 day old rats. Particularly, in the brain up to 16th day, the concentration of T4 was six times higher than in adult animals. The highest percentage of the injected radiothyroxine dose was found in the liver, intestine and stomach, total values in these organs exhibited a progressive increase from 20% on day 8 to 50% on day 24. When compared with litter-mates injected with inorganic radioiodide, the radioactivity in animals treated with hormone was in most organs markedly higher. The opposite results were found only in the stomach, i.e. the uptake of iodide exceeded the uptake of hormone. These results suggests, that differences between age groups are not due by differences in vascularity and although indirectly they may be considered as reflecting changes in size of thyroxine binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relation between levels of essential fatty acids in maternal milk, in maternal blood and in the blood of neonates 1 and 5 days after birth]. / Vztah medzi koncentráciou esenciálnych mastných kyselín v materskom mlieku a ich obsahom v krvi matiek a v krvi ich novorodencov 1. a 5. postnatálny den.

BACKGROUND: Linolenic, linoleic, and arachidonic acids as well as other polyunsaturated fatty acids are necessary for health as the precursors of eicosanoids and for the structure of developing membranes. OBJECTIVES: The aim of the present study was the determination of the level of 11 individual free fatty acids (FFA) in the milk and in the blood of mothers and newborns during the perinatal period. METHODS: In 21 women the FFA was determined in their colostrum as well as in the venous blood at the delivery in the hospital, and then again 5 days later at leaving the hospital. Simultaneously, the blood of newborns was collected as umbilical samples at birth and as venous blood on the 5th day. The study was performed on health term infants and mothers with normal gestational age. RESULTS: The results show a marked increase in total milk FFA as well as in most, but not all, individual FFAs during the followed period of the first 5 days. The values in the milk were always remarkably higher (the increase more than 2 orders) than in the blood. We have found no significant statistical correlation between values in the blood and those in the milk. The concentrations of all very important omega-3 FFAs (which are present in fish oil and in foods of marine fish origin) were always lower in all blood and milk samples in comparison with the levels of omega-6 FFAs (which are prevailing in lipids of our usual nutrition as are margarines and most of commercial oils). CONCLUSIONS: Our findings seem to be very important for preventive medicine and need to study further the relationship of low intake of O3FA to increased incidence of various allergies and other pathological syndromes in children. The very large range of a variance in the values of FFAs in the milk suggests the need of more profound study of the role of the food composition probably during the whole period of pregnancy, mainly as to the type of lipid composition. (Tab. 1, Fig. 6, Ref. 24.)

[Ethical principles in working with live animals and human fetal tissue in biomedical research]. / Etické zásady pri práci so zivými zvieratami a pri práci s fetálnymi tkanivami cloveka v biomedicínskom výskume.

The regulations for the protection of experimental animals which are suggested are based on internationally recognized ethical principles. The research institutions should establish and maintain appropriate policies and procedures to ensure the human care and use of live vertebrate animals. Recent developments in the use of human fetal tissue for therapeutical transplantations represent some serious ethical problems. Proposals for the ethical use of human fetal tissues are suggested. (Ref. 8).

The development of lipid metabolism. Effect of short-term application of thyroxine on the level of fatty acids and albumin in serum during ontogenesis in rat.

In young rats of different age the effect of short-term administration of thyroxine (T4) on the level of 6 FFA was determined. The concentration of FFA was expressed in mmol/l as well as the molar ratio to serum albumin. The results show that the level of the total FFA and the ratio of saturated to unsaturated (S/U) FFA are high in control animals till the age of 21 days in comparison with adult animals. Administration of T4 caused marked increase in the total FFA in the youngest age group (8-day-old) and the effect increased with age. Differences in the trend of changes in individual FFA were found. In the concentration of myristic acid (C 14:0) a significant increase was found only till the age of 16 days; in the case of palmit-oleic acid the increase was significant in all age groups. T4 caused no significant changes in the values of stearic acid (18:0). When the data were expressed in terms of the molar ratio FFA/albumin, the changes were more pronounced. The peak values found in 16-day-old animals were 33.9 +/- 0.75; they represent a 2-times higher supply of FFA to tissues in comparison with control animals. In this study it was also found that the concentration of serum albumin in newborn animals is low and represents just 57% of adult values. The short-term application of T4 caused no changes in the concentration of serum albumin. The mechanism of T4 action on FFA during ontogenesis was not explained in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of brown adipose tissue in the rat and thyroxine administration influence.

The role of the thyroid hormone in the activity of brown adipose tissue (BAT) is in most papers underestimated and it is the noradrenaline which is considered as the main regulator. The aim of the presented experiments was the study of the effect of 2 or 3 administrations of thyroxine (T4) applied to young rats of different age on the weight of BAT. T4 was injected in relatively large doses, i.e. 1 microgram/gram BW/day dissolved in 0.005 mmol NaOH intraperitoneally. Changes in T4 injected animals were compared with their litter-mate control, which were injected with solvents. In control animals the relative weight in mg/100 g BW was significantly higher during the entire first postnatal month in comparison with adult animals. Three administrations of T4 caused significant increase in relative as well as in absolute weight in all age groups. The difference was significant in dry tissue also. When the change was expressed in percentage the greatest effect of T4 was found in 15- and 19-day-old animals. Another experiment followed the effect of only two injections of T4 on BAT to animals of 7 different litters but of the same age, i.e. on day 5 and 6 and euthanized as 7-day-old. In all litters the increase after T4 treatment was significant; the increase in % ranged from 20 to 40% and was influenced by the level in controls. The relative weight in control animals ranged from 620 to 920 mg/100 g BW. This between-litter variance is greater than the effect of T4.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes in cholesterol and triacylglycerol serum levels in rats during postnatal ontogenesis]. / Zmeny v koncentrácii cholesterolu a triacylglycerolov v sére potkanov pocas postnatálnej ontogenézy.

In the serum of young normal rats during the first two months of life the concentrations of triacylglycerols (TG) and of cholesterol (CH) were determined. Low values in both studied parameters found in newborn animals were followed by a marked increase during the first week of life. In comparison with adult animals high values remained during the first five weeks of life. The results are discussed with regard to the high intake of lipids by milk during the period of sucking, as well as from the point of possible mobilization of tissue lipid stores because of the similar trend in the ontogenetic expression of the gene coding the hormone-sensitive lipase (HSL). On the basis of evaluation of the analytical as well as biological variance it was found, that for the level of 50% of confidentiality the serum concentrations of cholesterol should be in the range X +/- 0.26 mmol/l. For the level of 95% the range of values is X +/- 0.67 mmol/l. It is suggested that these results could be used as reference values for serum lipids in the rat during ontogenesis. (Fig. 1, Ref. 21.)