Biological signals最新文献

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by beta-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10(-4) M), a beta-adrenergic antagonist, and was not modified in the presence of prazosin, an alpha-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to beta-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuribenzoate (PCMB, 10(-5) M) and N-ethylmaleimide (NEM, 10(-3) M), indicating the involvement of -SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10(-3) M) and GTP gamma S (10(-4) M), suggesting that the specific alkylated -SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10(-2) M) and fluoroaluminate (AlF4- (F-, 10(-2) M + Al3+, 10(-5) M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.

Animal models were designed to study the changes in immune function, oncogenicity and life span of rats, mice and fruit flies following light-dark (LD) shift manipulations. Alternating the photoperiod of LD 14:10 and DL 10:14 every 3 days in rats disrupted the circadian immune rhythm pattern, decreased the blood leukocyte concentration by 48% and lowered the percentage of lymphocytes in the blood from 71% (control) to 49.2%. In mice, the phagocytosis of neutrophils was only reduced by 7%, but the level of serum hemolysin dropped significantly in the photoperiod-shifted group as compared with animals kept under a constant photoperiod of LD 12:12 or LD 14:10. In Ehrlich-carcinoma- or sarcoma-180-injected mice, a reduction of survival duration, acceleration of tumor growth and depression of the immune system were recorded in the LD-shifted animals. In addition, the life span of fruit flies was shortened by 9.6% by photoperiodic shifting. Melatonin treatment evidently counteracted the deleterious influences of photoperiodic shifting in the above animals. It is suggested that repeated inversion of the LD cycle results in a chronobiological abnormality that, in turn, induces dysfunctions. Reentrainment by exogenous melatonin may inhibit the harmful influences of photoperiodic shifting.

Confocal laser scanning microscopy (CLSM) is extensively used in the study of cellular activities through monitoring the temporal and spatial changes of biologically active molecules such as cAMP and Ca2+ which have been rendered visible by fluorescent labels. During our work with fluo-3 and Ca2+, we noticed two potential sources of artifacts which can make interpretation of the experimental observations difficult. Firstly, the excitation laser light generates heat that enhances the conversion of residual non-fluorescent acetoxymethyl (AM)-esterified indicator to the fluorescent form, thus giving rise to erroneous signals. Secondly, addition of reagents onto the coverslips alters the position of the focal plane, again causing error. In this paper, we present the phenomena and suggest ways to control and eliminate false images.

Male rats were grafted an anterior pituitary within breast muscles on day 5 or under the kidney capsule on day 30 or 60 of life. On the 70th day of life (rats operated on the 5th or 30th day) or on the 100th day of life (rats operated on the 60th day), rats were injected subcutaneously with Freund's complete adjuvant, being killed 2 days later. Rats that had received a pituitary graft on the 30th day showed a greater degree of hyper-prolactinemia than rats grafted on the 5th or 60th day. Analyzed as main factors in a factorial analysis of variance (ANOVA), pituitary transplants augmented splenic natural killer (NK) activity and lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced cell proliferation, and decreased splenic cell number. As indicated by significant interactions between treatment and age of transplantation in a factorial ANOVA, splenic NK activity augmented in rats grafted on the 30th day of life, while LPS and Con A splenic cell proliferation augmented in rats grafted neonatally. Spleen cellularity decreased after pituitary transplants in 30- and 60-day-old rats. In a second study, the effect of cyclosporine on spleen immune responses was tested by administering cyclosporine (5 mg/kg) or vehicle to rats grafted as in experiment 1 for 5 days before sacrifice. Cyclosporine decreased splenic NK activity and LPS- and Con A-induced cell proliferation regardless of the presence of a pituitary graft. In rats grafted on the 30th day of life, cyclosporine reversed the effect of pituitary grafts on splenic NK activity, and ectopic pituitary augmenting NK activity in vehicle-treated rats while decreasing it in cyclosporine-injected rats. Cyclosporine reversed the inhibitory effect of pituitary transplants on spleen cell number. The high circulating prolactin levels found in rats with pituitary grafts were decreased by cyclosporine administration. The results are compatible with age-dependent promoting and inhibitory effects of hyperprolactinemia on the immune responses of the spleen, which were antagonized by cyclosporine immunosuppression.

Ganglioside-stimulated protein kinase, designated PKJ, had been found in several animal brains. These include guinea pig, rabbit, rat, and duck brains. All of the four brain PKJ could be extracted from the particulate fractions using nonionic detergent nonidet P-40 and purified by using identical DEAE-cellulose and phenyl-Sepharose chromatographic methods. These results suggest that PKJ from different animal brains has similar ionic and hydrophobic characteristics. All four of the partially purified PKJ preparations could undergo autophosphorylations in the presence of trisialoganglioside GT1b and 32P-ATP. Analyses of the autophosphorylated proteins by using SDS-polyacrylamide gel electrophoresis and subsequent autoradiography revealed one major radioactive band with apparent M(r) = 68,000. The range of ganglioside-stimulated autophosphorylation was between 6- and 10-fold. The structural similarities of the different animal brain PKJ were further determined by using one-dimensional peptide mapping techniques. Limited proteolytic cleavages of the 32P-labeled 68-kD bands with staphylococcal aureus V-8 protease resulted in four major radioactive fragments with apparent M(r) corresponding to 22, 20, 18 and 15 kD, respectively. By contrast, digestion with chymotrypsin revealed only two major radioactive bands with apparent M(r) of 43 and 26 kD, respectively. These findings indicate that PKJ from guinea pig, rabbit, rat, and duck brains may have similar primary structures.

Melatonin levels were determined in serum and gastrointestinal tract (GIT) tissues of control (C), sham-pinelaectomized (SPx) and pinealectomized (Px) rats sacrificed in mid scotophase. Serum melatonin concentrations of Px rats exhibited the significantly lowest values (8.6 pg/ml), followed by SPx (20.1 pg/ml) and C (37.5 pg/ml) rats. In C, the ileum (542 pg/ml) and jejunum (531 pg/ml) exhibited the highest average GIT concentrations, followed by the colon (362 pg/ml), stomach (359 pg/ml) and cecum (164 pg/ml). However, only jejunum and ileum samples had significantly higher melatonin levels than cecum samples. There were no major differences between GIT melatonin levels in Px and C rats (range: 169-247 pg/ml). Statistically, pinealectomy did not influence melatonin levels in the GIT of rats. The findings support the hypothesis that melatonin concentrations in the tissues of the GIT are independent of pineal production.

Effects of nucleotides and cations on 2-[125I]iodomelatonin binding sites in the goldfish brain were examined. Nucleotides (10(-6)-10(-3) M) dose-dependently inhibited the specific binding with the following order of potency: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) > GTP = GDP > GMP = ATP > cyclic GMP. Cyclic AMP was ineffective. The treatment of membranes with GTP gamma S induced rapid dissociation of 2-[125I]iodomelatonin from membranes when added at the steady state, increased the Kd and decreased the Bmax values as revealed by saturation analysis, and increased the IC50 value of melatonin to inhibit the specific binding. The treatment decreased the specific binding to membrane preparations obtained from six brain regions as well. Inorganic salts (5-200 mM) dose-dependently inhibited the specific binding with the following order of potency: CaCl2 > MgCl2 > LiCl > NaCl > choline chloride > KCl, except for 5 mM MgCl2, which enhanced the specific binding. Saturation experiments demonstrated that 75 mM CaCl2, 100 mM MgCl2 and 200 mM NaCl increased the Kd and decreased the Bmax while 5 mM MgCl2 increased the Bmax value. These results imply that G protein and physiological concentrations of cations are involved in the regulation of melatonin binding sites in the goldfish brain.

Basophils and mast cells play a crucial role in immunological and allergic processes due to the release of inflammatory mediators such as histamine. It has been suggested for a long time that the histamine release (HR) from these cells is closely related to protein kinase (PKC) activity. However, the distinct role of PKC with its large variety of isozymes in different cell types and the actions of these isozymes in HR still remain unclear. Therefore, in the present study, we compared the effects of the two PKC inhibitors 7-O-methyl-UCN-01 (UCN-01-Me) and NPC 15437 as well as two PKC activators, bryostatin 1 and 2, on anti-IgE and Ca(2+)-ionophore-induced HR from human basophils and isolated human skin mast cells (HSMC). In both HSMC and basophils, anti-IgE-induced HR was inhibited by PKC inhibitor UCN-01-Me pre-incubation dose-dependently. In stark contrast, A23187-induced HR was unaffected by UCN-01-Me in both cell types. In our experiments, the inhibitory efficacy of the compound NPC 15437 on HR was much lower than that of UCN-01-Me and showed no statistical significance. Both bryostatins 1 and 2 produced good dose-dependent inhibition of HR from HSMC stimulated with anti-IgE, whereas HR from basophils was potentiated with these compounds. The same effects were observed with basophils stimulated with A23187, where potentiation of HR was up to fourfold of the control at the highest concentrations of bryostatins, while HSMC showed a slight decrease in HR compared to non-bryostatin-treated controls. Basophils and HSMC showed very clear differences in HR when directly stimulated with the bryostatins, since no HR was observed from HSMC while in basophils the HR increased up to 47% of total histamine at the highest concentrations of bryostatins (1 mumol/l). HR from basophils was observed to be strictly dose-dependent. The differences in the cell reactions of the two cell types incubated with these four compounds indicate distinct biochemical roles of PKC in the cascades leading to degranulation of the cells. Furthermore, the experiments with UCN-01-Me support the hypothesis of PKC-beta to play a substantial positive modulatory role for the degranulation of immunologically stimulated basophils.

Activation of D1 dopamine (DA) receptors in the striatum increases the expression of the opioid neuropeptide, dynorphin (DYN). While cAMP is generally accepted as a second messenger in this signal transduction pathway, the role of Fos/FRA proteins and AP-1 binding in mediating D1 receptor-induced changes in DYN expression remains uncertain. In this study, DYN peptide and mRNA levels, as well as Fos/FRA proteins and AP-1 DNA binding activity, were measured in individual animals following acute challenge with the psychostimulant drug methamphetamine (METH). METH caused an initial decrease in striatal levels of DYN A, reflecting increased peptide release. Six hours postinjection, DYN mRNA levels were significantly elevated by METH as compared to vehicle-injected controls. At the same time, these drugs increased the expression of Fos/FRA-ir proteins, in particular the 35- and 40-kDa molecular weight species, and increased binding to the AP-1 DNA element. Analyses of the time course of METH's effects revealed that DYN mRNA levels, Fos/FRA proteins and AP-1 binding activity showed variable increases by 3 h but all were significantly elevated above control levels by 6 h posttreatment. The D1-specific antagonist, SCH 23390, completely blocked the METH-induced changes in DYN peptide and mRNA levels while a D2 receptor antagonist (sulpiride) had little or no effect. These data indicate that stimulant drugs such as METH increase the expression of DYN and AP-1 factors in the striatum via a D1 receptor-mediated mechanism. However, the finding that AP-1 binding merely paralleled, but did not precede, the increase in DYN expression, as would be expected if it were mediating increased gene transcription, suggests these may be correlative, not causally related events.