Age-dependent variations of intralysosomal enzyme release from human PMN leukocytes under various stimuli.

The intralysosomal beta glucuronidase and elastase release from polymorphonuclear leukocytes (PMNL) of young and aged male subjects were determined after 60-min incubation with 10 micrograms/ml Cytochalasin B (CB), 10(-6) M of Ca ionophore A 23187 and various concentrations of human low density lipoprotein (LDL). The beta glucoronidase secretion was triggered by both A 23187 and LDL; however, no significant differences were found between the enzyme release from PMNLs of young and aged subjects. In contrast, a marked elastase release was triggered in the young group only by LDL, whereas in the aged group, all of the applied drugs induced a significant elastase release. LDL caused the most dramatic enzyme release from PMNLs of aged males. It was concluded that the release of PMNL-elastase after LDL incorporation as well as by CB and Ca ionophore stimulation may be an age-related process.

13C NMR study of actinoidins: carbohydrate moieties and their glycosidic linkages.

The configuration of the glycosidic linkages and the conformation of the carbohydrate moieties in the molecules of the glycopeptide-type antibiotics actinoidins A and B (1a, 1b) have been determined by means of two-dimensional 13C/1H correlation NMR technique and with the application of model compounds 2-4.

Molecular mechanical parameters (MM2 force field) for the N(sp3)-O(sp3) bond.

Based on results of MP2/6-31G* ab initio calculations an MM2 molecular mechanical parameter set has been developed for molecules containing N(sp3)-O(sp3) single bonds, existing parameters concerning the other bonds being retained. The new parameter set was tested for small organic compounds. A simple, generally applicable multilinear regression algorithm has been used and a program written to complement an existing force field (e.g. MM2) with such parameters extracted from quantum chemical computations.

Mechanism of the irreversible inhibition of human cyclooxygenase-1 by aspirin as predicted by QM/MM calculations.

Acetylsalicylic acid (aspirin) suppresses the generation of prostaglandin H2, which is the precursor of thromboxane A2. Aspirin acts as an acetylating agent in which its acetyl group is covalently attached to a serine residue (S530) in the active site of the cyclooxygenase-1 enzyme. The exact reaction mechanism has not been revealed by experimental methods. In this study the putative structure of human cyclooxygenase-1 was constructed from ovine cyclooxygenase-1 by homology modeling, and the acetylsalicylic acid was docked into the arachidonic acid binding cavity of the enzyme. To characterize the shape of the potential energy surface of the acetylating reaction and to determine the relative energies of the stationary points on the surface, a series of ONIOM-type quantum mechanical/molecular mechanical (QM/MM) calculations were carried out at different QM levels of theories applying electronic embedding approximations. The acetylsalicylic acid and the surrounding amino acids were included in these calculations. Frequency analyses were performed to prove the existence of first order saddle points (representing transition states) and local minima on the potential energy surface. It was found that all levels of theories predicted similar transition state geometries. The activation energy values, however, demonstrated significant dependence on the methods that were applied. All the applied "dependable" ab initio and DFT methods predicted that the breakage of the S530 Oγ--Hγ and formation of the Oγ--C(acetylsalicylic acid carbonyl) bonds occur in a single elementary step.

Factor XIII: novel structural and functional aspects.

Factor (F)XIII is a protransglutaminase that, in addition to maintaining hemostasis, has multiple plasmatic and intracellular functions. Its plasmatic form (pFXIII) is a tetramer of two potentially active A (FXIII-A) and two inhibitory/carrier B (FXIII-B) subunits, whereas its cellular form (cFXIII) is a dimer of FXIII-A. FXIII-A belongs to the family of transglutaminases (TGs), which show modest similarity in the primary structure, but a high degree of conservatism in their domain and sub-domain secondary structure. FXIII-A consists of an activation peptide, a ß-sandwich, a catalytic and two ß-barrel domains. FXIII-B is a glycoprotein consisting of 10 repetitive sushi domains each held together by two internal disulfide bonds. The structural elements of FXIII-A involved in the interaction with FXIII-B have not been elucidated; in FXIII-B the first sushi domain seems important for complex formation. In the circulation pFXIII is bound to the fibrinogen γ'-chain through its B subunit. In the process of pFXIII activation first thrombin cleaves off the activation peptide from FXIII-A, then in the presence of Ca(2+) FXIII-B dissociates and FXIII-A becomes transformed into an active transglutaminase (FXIIIa). The activation is highly accelerated by the presence of fibrin(ogen). cFXIII does not require proteolysis for intracellular activation. The three-dimensional structure of FXIIIa has not been resolved. Based on analogies with transglutaminase-2, a three-dimensional structure of FXIIIa was developed by molecular modeling, which shows good agreement with the drastic structural changes demonstrated by biochemical studies. The structural requirements for enzyme-substrate interaction and for transglutaminase activity are also reviewed.

Effects of alpha- and beta-adrenergic blockers on the actions of noradrenaline on body temperature in the newborn guinea-pig.

The effect of NA injected into the lateral cerebral ventricle on Tc was blocked by alpha-adrenergic receptor blockers, but not by beta-receptor blockers, whereas the effect of systemically administered NA was blocked by i.p. administered beta-receptor blockers, but not by alpha-blockers.

The central effect of 5-hydroxytryptamine, gamma-amino-butyric acid and carbachol on thermoregulation in the neonatal guinea pig.

5-Hydroxytryptamine (10 microgram), gamma-amino-butyric acid (20 microgram) and carbachol (1 microgram) were injected through the soft skull into the lateral cerebral ventricle of guinea pigs aged 2 to 12 days, at the slightly subneutral ambient temperature of 30 degrees C. 5-Hydroxytryptamine (5-HT) produced no immediate change in oxygen consumption, whereas colonic temperature fell rapidly. One hour after injecting 5-HT, when colonic temperature had decreased by about 0.5 degrees C, oxygen consumption increased and after a lag of 30--40 min was followed by an increase in colonic temperature. Gamma-amino-butyric acid (GABA) increased oxygen consumption and colonic temperature after a latency of 30--60 min. Carbachol was followed by an immediate decrease in colonic temperature and oxygen consumption. Colonic temperature was below the pre-injection level throughout the five-hour period of observation.

The central effect of noradrenaline on thermoregulation in the neonatal guinea pig.

Noradrenaline (10 microgram) injected into the lateral cerebral ventricle of guinea pigs aged 2 to 12 days produced a rapid increase in oxygen consumption and in colonic temperature at an ambient temperature of 30 degrees C. The increase was most pronounced in the youngest animals and decreased with advancing age, but was still significant at 12 days of age. Species differences and the role of ambient temperature in the responses are discussed.

Alpha- and beta-adrenergic receptor blockers and the effects of noradrenaline on heat production and body temperature.

Noradrenaline (NA) administered systemically or into the lateral cerebral ventricle (ICV) in appropriate doses increased heat production and colonic temperature in 1-12 day-old guinea pigs. The effect of systemically applied NA could be blocked by systemically applied beta-adrenergic receptor blockers, while beta-blockers administered centrally or alpha-adrenergic blockers injected systemically or centrally had no effect on the action of systemically applied NA. Accordingly, the effect of systemically applied NA was mediated by peripheral beta-adrenergic receptors. The effect of centrally applied NA was blocked by alpha-adrenergic receptor antagonists applied into the lateral cerebral ventricle and attenuated by systemically applied phentolamine, but not by phenoxybenzamine or ergotamine. Beta-adrenergic receptor blocking agents applied either ICV or systemically had no effect on the action of centrally applied NA. It is concluded that ICV applied NA acts through central alpha-adrenergic receptors.

Endotoxin and prostaglandin fever of newborn guinea pigs at different ambient temperatures.

At an ambient temperature (Ta) of 30 degrees C, injection of 0.2 micrograms E. coli endotoxin into the lateral cerebral ventricle (icv) of three-day-old or younger guinea pigs was followed by a biphasic febrile rise in body temperature (Tc) and oxygen consumption (VO2), interrupted by a transient fall. At Ta 20 degrees C the change in Tc and VO2 was still biphasic, the first rise was similar as that seen at Ta 30 degrees C, but the subsequent fall was more pronounced. Thus, Tc became lower than before endotoxin and remained below the pre-injection level during and after the second rise. Injection of 10 ng PGE1 icv caused sustained monophasic hyperthermia at both Ta-s. Icv injection of 0.9% NaCl did not affect Tc and VO2 at either Ta. Accordingly, prostaglandins might contribute to, but cannot account for, the whole febrile response to endotoxin.

The mechanism of antibody-dependent cellular cytotoxicity stimulation by somatostatin in rat peritoneal macrophages.

Somatostatin (SS) in 10(-9)-10(-7) M concentrations stimulated the lysis and inhibited the incorporation of IgG2a-coated 51Cr-labeled sheep red blood cell (SRBC) by rat peritoneal macrophages (PM). The intracellular killing capacity of PM remained unchanged. The enhancement of Fc receptor (R) activity and generation of active oxygen species were found to be responsible for the antibody-dependent cellular cytotoxicity (ADCC)-stimulating effect of SS. It was demonstrated that the stimulation of ADCC was abolished by the calmodulin inhibitor trifluoperazine (TFP), whereas it proved to be independent of the Ca2+ uptake. In addition, SS in the ADCC-stimulating concentrations diminished the intracellular cAMP generation and progressively increased the cGMP level. In higher (10(-6)-10(-7) M) concentrations, SS had a controversial effect on PM: it inhibited ADCC through the activation of both the adenylate cyclase and Ca2+ influx.

A possible competition between 5'-monodeiodination of thyroxine and the respiratory burst in human granulocytes.

Thyroxine (T4) pretreatment of A 23187-stimulated human granulocytes in 10(-5)-10(-6) M concentration range inhibited the superoxide anion production of these cells. T4 increased the level of oxidized form of glutathione, whereas the intracellular level of the reduced form decreased. A similar alteration in the ratio of the oxidized to reduced forms of glutathione was detected in granulocytes during yeast cell phagocytosis. In addition, conversion of T4 to triiodothyronine (T3) was also inhibited during phagocytosis. A possible competition between 5'-monodeiodination of T4 and the oxidative burst of human granulocytes is discussed.

Effect of potassium-induced cortical spreading depression on prostaglandin-induced fever in conscious and urethane-anesthetized rats.

Potassium-induced cortical spreading depression (CSD) on prostaglandin E1 (PGE1) induced fever has been investigated in a dose-responsive experimental design in both conscious and urethane-anesthetized adult male Sprague-Dawley rats. While CSD in itself had no effect on nonfebrile body temperature even under cold ambient conditions, CSD significantly suppressed small but not large fevers induced by intracerebroventricular PGE1. The increased oxygen consumption during fever was also reduced. We also explored the possible involvement of the antipyretic peptide arginine vasopressin, in the CSD-induced suppression of fever. Long term castrated rats have significantly reduced ventral septal levels of this peptide, yet CSD was effective in suppressing the initial 40 min of PGE1 fever in these animals. Thus we conclude that increased release of ventral septal arginine vasopressin is probably not involved in the action of CSD on fever.

Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia.

Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.