Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of rat brain (mu-, delta-, kappa 2-receptors) and guinea pig cerebellum (kappa-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively). The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations (relative potencies of 0.8 and 8.8% on the GPI and MVD) while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative spinal analgesic action of dynorphin1-8, dynorphin1-13, and a kappa-receptor agonist U50,488

The effect of intrathecal injections of dynorphin1-8 (DYN1-8), dynorphin1-13 (DYN1-13), and a putative kappa agonist, U50,488 was tested in the rat tail-flick test. DYN1-8 and DYN1-13 (5, 10, 20 micrograms) produced a dose-related biphasic antinociceptive response consisting of an initial and a delayed response. Injection of U50,488 (20, 40 60 micrograms) produced a monophasic response. The antinociceptive effect of DYN1-8 (5, 10, 20 micrograms) and DYN1-13 (20 micrograms), was present 24 h postintrathecal injection. Pretreatment with systemic naloxone (2 mg/kg s.c.) attenuated the delayed response, but not the initial response induced by DYN1-8 and DYN1-13. The initial response was attenuated by pretreatment with intrathecal naloxone at a dose of 0.5 and 2.0 micrograms. The antinociceptive effect of U50,488 (20, 60 micrograms) was not affected by pretreatment with 2.0 micrograms intrathecal naloxone, but was significantly reduced by 4 micrograms of the antagonist. Both DYN1-8 and DYN1-13 (5 micrograms) augmented the antinociceptive effect of intrathecally administered morphine (5, 10 micrograms). Intrathecal injection of DYN1-8 (5, 10, 20 micrograms), DYN1-13 (5 micrograms), and morphine (10 micrograms) reduced the spontaneous output of urine measured at 2 and 24 h postintrathecal injection. A similar injection of U50,488 (20 micrograms) had no significant action on the urinary output. The results show that long and short dynorphin fragments have a comparable activity and the spinal antinociceptive actions of dynorphin are sensitive to low doses of intrathecal naloxone. The activity profile of spinally administered dynorphins differs from that of the kappa agonist U50,488.     

14-beta-Methyl-8-oxacyclorphan (BC-3016), a morphinan derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(1-13)

14-beta-Methyl-8-oxacyclorphan (BC-3016) was tested for its ability to depress the electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD) and to compete with the binding of prototype ligands selective for kappa-, mu-, or delta-opioid receptors in membrane preparations of rat brain and guinea pig cerebellum. BC-3016 was a very potent agonist in the GPI and MVD preparations, with ID50 of 0.7 and 31 nM, respectively. The activity of levorphanol, a standard alkaloid related to BC-3016, was much lower in both assays with ID50 values of 44 and 86 nM, respectively. Conversely, the activity of BC-3016 was quite comparable to that of dynorphin-A(1-13) in both preparations. In the GPI assay, a putative kappa-receptor antagonist, MR-2266, was 6.6 and 5.5 times more potent than naloxone in blocking the activity of BC-3016 and dynorphin-A(1-13), respectively. BC-3016 was also very potent in displacing bound [3H]ethylketocyclazocine ([3H]EKC) to membrane preparations of the guinea pig cerebellum, a brain component containing predominantly kappa-opioid receptors (Ki of 0.58 nM). Its potency in the displacement of the bound mu-ligand, 3H-labelled (D-Ala2,MePhe4,Gly-OH5)-enkephalin ([3H]DAGO), to rat brain homogenates was somewhat lower (Ki of 0.8 nM) but still high when compared with its ability to displace the delta-ligand, 3H-labelled (D-Ser2, Thr6)-Leu-enkephalin ([3H]DSLET) to rat brain homogenates (Ki of 4.45 nM). The affinity of BC-3016 for the opioid receptor was 2.1-fold higher than that of U-50488H, a selective kappa-opioid ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperprolactinemia and sexual function in men

Male hyperprolactinemia (HPRL) is known to induce different types of sexual dysfunctions. In order to determine the incidence of HPRL among patients referred for sexual dysfunction, serum prolactin (PRL) was assayed in 1053 clinically idiopathic cases. Among 850 cases complaining of erectile impotence, 10 with marked HPRL (1.1%, PRL above 35 ng/ml) were found, of whom 6 cases were associated with a pituitary adenoma. 17 mild HPRL (2%, PRL 20-35 ng/ml) were also found. Among 124 cases with premature ejaculation, 13 (10%) mild HPRL were found. Serum PRL was normal in 51 cases complaining of an ejaculation without orgasm, and 27 patients exclusively complaining of reduced sexual desire. Our results lay stress on the fact that serum PRL must be assayed in every case of clinically idiopathic erectile impotence. Indeed, 5 of the 10 marked HPRL patients would have been misdiagnosed if we had only assayed this hormone when plasma testosterone was below the normal range. Moreover, in order to shed some light on the mechanisms by which HPRL disturbs male sexual function, the sexual behaviour of 17 markedly HPRL males was compared to their serum levels of PRL and testosterone, first before treatment, then at regular intervals during treatment. Our main conclusion is that impotence cannot be totally explained by a decrease in plasma testosterone, because this steroid hormone was within the normal range 7 of the 16 impotent patients. Moreover, when serum PRL was lowered by bromocriptine, 6 patients recovered their potency before plasma testosterone clearly increased, and in 3 of those patients before it reached the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical conformational analysis of phospholipids II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.     

A reduction in the hyperventilation threshold by intravenous infusion of adrenaline in the treadmill exercise

Seven healthy male subjects underwent a treadmill incremental work test in control conditions and during an intravenous epinephrine infusion (10 micrograms/min). At all exercise intensities, epinephrine increased heart rate, ventilation, respiratory quotient and plasma lactate levels without significant changes in oxygen consumption. Under epinephrine infusion, the "anaerobic threshold", considered as the critical intensity at which ventilation began to increase non linearly with oxygen consumption, appeared at a lower intensity and for a higher plasma lactate level than in control conditions. We conclude that the hyperventilation threshold does not necessarily reflect a muscular hypoxia. It could be due to an effect of catecholamines on peripheral chemoreceptors, maybe by alpha-adrenergic vasoconstriction in the carotid bodies.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA.

The binding specificity of the duplicated segments borne by Clostridium thermocellum endoglucanase CelD and by the cellulosome-integrating protein CipA was investigated. The fusion protein CelC-DSCelD, in which the duplicated segment of CelD was fused to the COOH terminus of endoglucanase CelC, bound with an affinity of 4.7 x 10(7) M-1 to the fusion protein MalE-RDCipA, in which the seventh receptor domain of CipA was grafted onto the COOH terminus of the Escherichia coli maltose-binding protein MalE. The affinity of CelC-DSCelD for the homologous chimeric protein MalE-RDORF3p, carrying the receptor of the surface protein ORF3p, was 6.9 x 10(6) M-1. The fusion protein CelC-DSCipA, in which the duplicated segment of CipA was grafted onto the COOH terminus of CelC, did not bind detectably to MalE-RDCipA or MalE-RDORF3p. However, Western blotting (immunoblotting) experiments indicated that the duplicated segment of CipA was able to bind to a set of C. thermocellum proteins which are different from those recognized by the duplicated segment of CelD. These results argue against the hypothesis that ORF3p interacts with the duplicated segment of CipA. More probably, ORF3p binds to individual cellulases and hemicellulases harboring duplicated segments.     

Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG.

The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined. The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C. thermocellum endoglucanase CelB (51.5% identical residues). The N terminus displayed a typical signal peptide, followed by a catalytic domain. The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism. Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector. A low- and a high-M(r) form of the protein were purified. The two forms displayed identical enzymatic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus. An antiserum raised against the forms purified from E. coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C. thermocellum known as the cellulosome.