Alpha-tryptophan synthase of Isatis tinctoria: gene cloning and expression.

Indole producing reaction is a crux in the regulation of metabolite flow through the pathways and the coordination of primary and secondary product biosynthesis in plants. Indole is yielded transiently from indole-3-glycerol phosphate and immediately condensed with serine to give tryptophan, by the enzyme tryptophan synthase (TS). There is evidence that plant TS, like the bacterial complex, functions as an alpha beta heteromer. In few species, e.g. maize, are known enzymes, related with the TS alpha-subunit (TSA), able to catalyse reaction producing indole, which is free to enter the secondary metabolite pathways. In this contest, we searched for TSA and TSA related genes in Isatis tinctoria, a species producing the natural blue dye indigo. The It-TSA cDNA and the full-length exons/introns genomic region were isolated. The phylogenetic analysis indicates that It-TSA is more closely related to Arabidopsis thaliana At-T14E10.210 TSA (95.7% identity at the amino acid level) with respect to A. thaliana At-T10P11.11 TSA1-like (63%), Zea mays indole-3-glycerol phosphate lyase (54%), Z. mays TSA (53%), and Z. mays indole synthase (50%). The It-TSA cDNA was also able to complement an Escherichia coli trpA mutant. To examine the involvement of It-TSA in the biosynthesis of secondary metabolism compounds, It-TSA expression was tested in seedling grown under different light conditions. Semi-quantitative RT-PCR showed an increase in the steady-state level of It-TSA mRNA, paralleled by an increase of indigo and its precursor isatan B. Our results appear to indicate an involvement for It-TSA in indigo precursor synthesis and/or tryptophan biosynthesis.

Increased blocking activity of combined tachykinin NK1- and NK2-receptor antagonists on tachykinergic bronchomotor responses in the guinea-pig.

1. The present study compared the effect of the administration of tachykinin NK1- and NK2-receptor antagonists alone and in combination on exogenous and endogenous tachykinin-induced contractions using three different guinea-pig airway preparations: isolated bronchus, isolated perfused lung and in vivo. 2. In the isolated bronchi, the tachykinin NK1-receptor antagonist CP 99994 (0.01-1 microM) produced concentration-dependent inhibition of contractions induced the tachykinin NK1-receptor agonists substance P (SP) and [Met-OMe11] SP ([Met-OMe11]SP), whereas the tachykinin NK2-receptor antagonist SR 48968 (0.1 microM) had no effect. SR 48968 (0.001-0.01 microM) concentration-dependently inhibited contractions induced by the tachykinin NK2-receptor agonists neurokinin A (NKA) and [beta-Ala8]-neurokinin A (4-10) ([betaAla8]-NKA) whereas CP 99994 (0.1 microM) did not inhibit the contractions. The contractile activity of capsaicin, an agent that releases endogenous tachykinins from sensory C-fibres, was inhibited in a concentration dependent manner by SR 48968 (0.001-0.03 microM) but not by CP 99994 (0.1 microM). Combination of CP 99994 and SR 48968 caused increased inhibitory effects on the concentration-response curves to SP, [Met-OMe1l]SP, NKA, [beta-Ala8]-NKA and capsaicin. 3. In isolated perfused lungs, SR 48968 concentration (0.01-10 microM) dependently inhibited NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction whereas CP 99994 (30 microM) had no effect on SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction. Combination of inactive concentrations of CP 99994 and SR 48968 produced an increased inhibitory effect on all previous stimuli-induced bronchoconstriction. 4. In in vivo guinea-pig studies, intravenous and oral pretreatment with SR 48968 (0.01-1 mg kg(-1) i.v. and 0.1-3 mg kg(-1) p.o., respectively), but not with CP 99994 (1 mg kg(-1) i.v. and 0.3-30 mg kg(-1) p.o., respectively), produced a dose-dependent inhibition of the bronchoconstrictor responses induced by NKA, [beta-Ala8]-NKA and capsaicin. CP 99994 intravenously (0.3 mg kg(-1)) and orally (3-10 mg kg(-1)) inhibited SP-induced bronchoconstriction only. Intravenous and oral low dose combinations of CP 99994 and SR 48968 produced an increased inhibition of SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction, respectively. The present data indicate that combined tachykinin NK1- and NK2-receptor antagonist treatment compared with single antagonist treatment, using CP 99994 and SR 48968, produced an augmented blockade of tachykinin NK1-, NK2- and capsaicin-mediated contractions in guinea pig airways. These findings support the hypothesis that a dual NK1- and NK2-receptor antagonist may provide an advantage over single activity tachykinin NK1- or NK2-receptor antagonists in pulmonary obstructive diseases.

[Estimating the grade of patient satisfaction at the bone marrow transplantation department in Florence hospitals]. / Misurazione del grado di soddisfazione dei pazienti nel reparto di trapianto di midollo osseo di Firenze.

The satisfaction of the patients admitted to the bone marrow transplant unit of Careggi Hospital was evaluated by the nursing team. The aim of the evaluation was to measure the level of satisfaction for the nursing care and services and the areas of improvement. The questionnaire, with 23 questions referring to 5 areas (hotel care, Nurses' reliability, Ability to reassure, to answer to patients' needs and Empathy) derived from the conceptual model of Servqual. Ninety patients were given (or mailed) the questionnaire during a follow-up visit. Patients were asked to answer the questions evaluating each aspect on a scale from 1 (falls short of expectation) to 10 (exceeds all expectations). The answers show a very high satisfaction (> 8) for all the areas except for the food that reported a medium score of 5.2. Further analysis will allow a better understanding of the causes of dissatisfaction.

Preclinical pharmacology of desloratadine, a selective and nonsedating histamine H1 receptor antagonist. 1st communication: receptor selectivity, antihistaminic activity, and antiallergenic effects.

Desloratadine (descarboethoxyloratadine, CAS 100643-71-8) is an active metabolite of loratadine (CAS 79794-75-5) that exhibits qualitatively similar pharmacodynamic activity with a relative oral potency in animals 2.5-4 times greater than loratadine. Its antihistaminic effect lasts 24 h. Desloratadine was shown to be a selective H1 antagonist with more potent antihistaminic activity in vitro than either loratadine or terfenadine (CAS 50679-08-8), as indicated by its displacement of 3H-mepyramine from H1 receptors in rat brain, guinea pig brain, and guinea pig lung, and by its antagonism of histamine-induced contractions of guinea pig ileum. Antihistaminic activity and anitallergic effects also were observed in vivo. After oral administration, desloratadine was 2.5 to 4 times more potent than loratadine in protecting against histamine-induced lethality in the guinea pig and paw edema in the mouse; after topical administration, it was almost 10 times more potent in antagonizing histamine-induced increases in nasal microvascular permeability in the guinea pig. Histamine-induced changes in pulmonary resistance and compliance were also prevented by oral administration of desloratadine and loratadine in the monkey. An oral antiallergic effect was demonstrated by important reductions of acute bronchospasm in the allergic monkey and potent inhibition of allergic cough in the guinea pig. These preclinical studies provide evidence that desloratadine is an antihistaminic agent with a greater potency than loratadine and, together with results from numerous published studies, suggest an antiallergic effect of desloratadine.

Conformational considerations in the design of dual antagonists of platelet-activating factor (PAF) and histamine.

Following the discovery of the first dual antagonist of platelet-activating factor (PAF) and histamine, 1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin- 11-ylidene)piperidine, Sch 37370, 1, a related series of structures, exemplified by (+/-)-1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b] pyridin-11-yl)piperazine, Sch 40338, 2, were prepared. Interestingly, the compounds exhibited a parallel structure antiallergy activity relationship, suggesting that the two series may adopt a common conformation at the PAF receptor. Conformational analysis led to a proposal for this bioactive conformation accessible to both series. The synthesis of novel conformationally constrained analogues that might mimic the proposed bioactive conformation of these compounds, and the evaluation of their in vitro antiallergy activity form the subject matter of this report.

Trans-4-methyl-3-imidazoyl pyrrolidine as a potent, highly selective histamine H3 receptor agonist in vivo.

Extensive structural modification of immepyr (+)-2 led to the discovery of trans-4-methyl-3-imidazoyl pyrrolidine (+/-)-3a as a potent and highly selective H3 agonist. The pyrroline (+/-)-3a was resolved, and its (+) enantiomer, Sch 50971 [(+)-3a], showed a greater separation of H3 and H1 activities in vivo (H3/H1 ratio >> 330) than (R)-alpha-methylhistamine (+)-1 (H3/H1 ratio = 17), the standard H3 agonist.

Pharmacological characterization of histamine H3 receptors in isolated guinea pig pulmonary artery and ileum.

We characterized the histamine H3 receptors involved in the modulation of electrical field stimulated neurogenic contraction of guinea pig pulmonary artery sympathetic, and guinea pig ileum parasympathetic preparations. Simultaneous measures of electrical field stimulation-evoked 3H overflow and tension in [3H]norepinephrine-loaded pulmonary artery were sensitive to tetrodotoxin (300 nM) and insensitive to hexamethonium (100 microM). Only the contractile response was inhibited by prazosin (100 nM). (R)-alpha-Methylhistamine's inhibition of the pulmonary artery contraction and 3H overflow were dose-dependently antagonized by thioperamide (30-100 nM). (R)-alpha-Methylhistamine also inhibited the neurogenic contractions of the isolated ileum (pD2 = 8.2). In the pulmonary artery, the relative potency of the histamine H3 receptor antagonists vs. (R)-alpha-methylhistamine inhibition of neurogenic contractions (pD2 = 7.1) was thioperamide (pA2 = 8.6 +/- 0.1) > burimamide (pA2 = 7.6 +/- 0.2) > impromidine (pA2 = 6.9 +/- 0.02). Similarly, the relative potency of histamine H3 receptor antagonists in the isolated ileum was thioperamide > burimamide > or = impromidine, with pA2 estimates of 8.7 +/- 0.1, 7.3 +/- 0.1 and 7.1 +/- 0.1, respectively. Antagonist potencies suggest a predominant histamine H3A-like receptor population on postganglionic sympathetic neurons innervating the pulmonary artery and parasympathetic neurons innervating the ileum longitudinal muscle.

A novel pyrrolidine analog of histamine as a potent, highly selective histamine H3 receptor agonist.

Employing classical conformational analysis on a known H3 agonist, (R)-alpha-methylhistamine (1), a series of conformationally constrained H3 agonists were proposed and synthesized. Pyrrolidine (+/-)-4a, a compound proposed to mimic the anti-conformation of (R)-alpha-methylhistamine (1), was found to be a potent and selective H3 agonist. The pyrrolidine (+/-)-4a was resolved, and its (+) enantiomer, immepyr [(+)-4a], showed a greater separation of H3 and H1 activities in vivo (H3/H1 ratio >> 550) than (R)-alpha-methylhistamine (1) (H3/H1 ratio = 17), the standard H3 agonist. In fact, no evidence of H1 activity was detected at doses of immepyr [(+)-4a] as high as 100 mg/kg i.v. This pyrrolidine, immepyr [(2R,3S)-(+)-4a], represents, to our knowledge, the first reported cyclic, conformationally restricted analog of histamine to possess selective in vivo H3 agonist activity.

Verongamine, a novel bromotyrosine-derived histamine H3-antagonist from the marine sponge Verongula gigantea.

The novel bromotyrosine derivative verongamine [1] has been isolated from the marine sponge Verongula gigantea. The isolation of verongamine was facilitated by employing histamine-H3 bioassay-guided fractionation. Verongamine is a specific histamine-H3 antagonist at concentrations as low as 1 microgram/ml.

Antibronchoconstrictor activity of the intracellular calcium antagonist HA 1004 in guinea pigs.

HA 1004 is a calcium antagonist vasodilator that inhibits contraction in vascular smooth muscle and lowers arterial blood pressure. The effects of HA 1004 on guinea pig airway smooth muscle contraction were compared to the effects of the calcium antagonists, verapamil and nifedipine and the bronchodilator, albuterol. In vitro, HA 1004, verapamil, nifedipine and albuterol inhibited Ca2+-induced contractions of the depolarized guinea pig trachea. HA 1004 and albuterol also relaxed the basal tracheal tone, whereas verapamil and nifedipine were inactive. The bronchorelaxant activity of HA 1004 was not blocked by propranolol. In vivo, intravenous administration of HA 1004, verapamil, nifedipine and albuterol effectively blocked bronchoconstriction induced by intravenous histamine and methacholine. HA 1004 also reversed a histamine-induced bronchospasm, as did albuterol, verapamil and nifedipine. Intratracheal administration of HA 1004 and albuterol inhibited histamine-induced bronchoconstriction without effecting blood pressure, whereas intratracheal administration of verapamil and nifedipine caused a significant reduction of blood pressure at their pulmonary active doses. These results show that HA 1004 has the ability to relax airway smooth muscle, inhibit contractile responses in the guinea pig airways, and there is separation of its cardiovascular and pulmonary effects when HA 1004 is administered directly to the lungs. The results are discussed in terms of the regulatory enzymes and sources of calcium that are involved in airway smooth muscle contraction.

Bronchodilator and antiallergy activity of forskolin.

Forskolin is a diterpene from the roots of Coleus forskohli which directly activates the adenylate cyclase and raises cyclic AMP levels in a variety of tissues. Forskolin was studied for its effects on the tone of airway smooth muscle and the immunologic release of leukotrienes and histamine. The bronchospasm induced by inhaled antigen in sensitized guinea pigs was prevented in a dose-related fashion by the intravenous (i.v.) or intratracheal administration of forskolin. Forskolin was more potent than aminophylline and less potent than salbutamol. There was no evidence that forskolin would potentiate the in vivo bronchodilator effects of either salbutamol or aminophylline. Forskolin was approximately 100 times more potent than aminophylline by the i.v. and intratracheal routes to reverse an established allergic bronchospasm. Forskolin given intratracheally also inhibited the bronchospasm to i.v. histamine, with a short duration of action. In vitro forskolin (less than 1 microM) inhibited contractions of lung parenchyma provoked by histamine, LTC4 or antigen. Forskolin (1 microM) also inhibited the immunologically stimulated release of LTD4 and histamine from sensitized guinea pig lung. These studies show that forskolin shares with other agents that elevate cyclic AMP levels the ability to relax airway smooth muscle and inhibit mediator release in vitro and elicit a bronchodilation in vivo.

Evaluation of the CNS properties of SCH 29851, a potential non-sedating antihistamine.

SCH 29851 [8-chloro[6,11-dihydro-11-(1-carboethoxy-4-piperidylidene)- 5-H-benzo [5,6]cyclohepta[1,2-b]-pyridine] was discovered as part of a search for a new antihistamine without effects on the central nervous system (CHS). Antihistaminic potency and duration of action of SCH 29851 and other antihistamines were assessed by inhibition of histamine-induced lethality in guinea pigs and histamine-induced paw edema in mice. Evaluation of possible CNS effects included gross observation of mice, rats, dogs and monkeys, prevention of electroshock-induced convulsions, acetic acid-induced writhing and physostigmine-induced lethality in mice and biochemical measures related to sedative liability such as displacement of in vivo 3H-mepyramine binding in mouse brain and in vitro 3H-WB 4101 binding in guinea pig cortex. Comparisons were made to several antihistamines considered to be sedative to varying degrees, including diphenhydramine, promethazine, chlorpheniramine and azatadine and to the newer antihistamines terfenadine and astemizole which are reported to be non-sedating in man at doses that antagonize the effects of histamine peripherally. SCH 29851 had antihistamine activity in the tests used with a potency at least comparable to most standards and was devoid of activity in all the functional and biochemical models used as indices of CNS activity. It is expected that SCH 29851 should be an effective, long acting, antihistamine in man without sedative effects at therapeutic doses.

A method for marking the position of nichrome microelectrode tips in nervous tissue.

A method for revealing nickel deposits from nichrome microelectrodes in the mammalian central nervous system is described. These deposits are stained red by dimethylglyoxime and can be observed directly or in Nissl stained sections. This method allows one to identify the exact position of a nichrome electrode in a microelectrode bundle chronically implanted in the brain.

Benzopyranopyridine derivatives. 1. Aminoalkyl derivatives of the azaxanthenes as bronchodilating agents.

The preparation of the four isomeric azaxanthones 3 and a number of their aromatic ring substituted derivatives is described. These ketones were converted into the title compounds which were examined for their biological properties. The most interesting compound in this series, the 1-methyl-4-piperidylidene derivative of 1-azaxanthene, shows the profile of an orally effective potent bronchodilating agent as well as a moderate antihistamine. Biological properties of this compound were compared to a number of antihistamines as well as known bronchodilating agents. Structure-activity relationships are also discussed.