Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays.

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity. This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays. In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor alpha, with a lower potency but comparable efficacy to that of 17 beta-estradiol. Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F(2alpha,) and bradykinin than tissues obtained from estradiol valerate treated rats. The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F(2alpha,) and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 beta-estradiol. Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.

The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare.

Cyclo-oxygenase- and capsaicin-sensitive afferent fibres affect Beta-adrenoceptor-evoked response in the rat urinary bladder.

As reported previously, the beta-adrenoceptor-evoked response in the in vitro rabbit detrusor is inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). Prostaglandins can restore this response. This study was designed to better define physiological influences on beta-adrenoceptors in the urinary bladder and to ascertain whether the observations made in the rabbit urinary bladder in vitro were valid in vivo, as well as in a different animal species, i.e. in the rat. The NSAID indometacin partially inhibited the isoproterenol-evoked response in the rat detrusor muscle. When the neurokinin A (NKA) antagonist MEN-10376 was administered after indometacin, the beta-adrenoceptor-evoked response was completely abolished. The isoproterenol-evoked beta response was dose dependently reduced following treatment with the capsaicin antagonist ruthenium red. Taken together, these data indicate an involvement of prostanoids, NKA- and capsaicin-sensitive fibres in the beta-adrenoceptor-evoked response in the rat urinary bladder.

Effects of natural tachykinins on ovine lower urinary tract smooth muscle.

1. Numerous studies have demonstrated that the urinary bladder is particularly sensitive to tachykinins; rat, rabbit and guinea pig bladders, besides human detrusor, have been the most extensively studied, whereas very little is known about most large animal detrusors. The aim of this work was to study natural tachykinin activity on the lower urinary tract of ovine to make a comparison with data obtained in laboratory animals. 2. As in other animal species, tachykinins are also able to contract ovine bladder smooth muscle. 3. The results reported in this study indicate that in ovine bladder, neurokinin 2 (NK2) receptors are expressed most. In fact, on lamb and sheep bladder neurokinin A (NKA), a NK2- almost selective peptide, was shown to be > 100% more active than the natural tachykinins kassinin (KASS) and eledoisin (ELED). Eledoisin was shown to be 50% less active than KASS, which is typical behaviour for an almost exclusively NK2 receptor population. Moreover, NK1- preferential peptides, namely substance P (SP) and physalaemin (PHYS), showed a lack of activity even when applied at high concentrations. 4. The results reported in this study show that lamb and sheep detrusor represent a good alternative model for the characterization of NK2-selective tachykinins.

Interactions between A-9THC and capsaicin on isolated lamb bladder detrusor.

A number of studies have demonstrated that capsaicin, a capsicum alkaloid, can affect isolated bladder tissue with either a relaxation or a contraction, depending on the species, by acting on VR1 receptors. In a previous work on isolated lamb detrusor, we demonstrated that capsaicin generally produces a relaxation of the tissue; this relaxation seems to be mediated by CGRP. Endogenous cannabinoids, such as anandamide, produce some of their actions by stimulating VR1 receptors and this seems to cause the release of peptides, e.g. CGRP. The aim of this work was to ascertain whether a cannabinoid, delta-9-tetrahydrocannabinol (delta-9THC), was able to interfere with the response of the isolated lamb detrusor to capsaicin. A-9THC, at concentrations between 1.6 x 10(-7) and 1.3 x 10(-6) M, displayed no activity on tissues. Instead, following delta-9THC, most of the tissues responded to capsaicin with a contraction that was abolished by atropine (9.0 x 10(-7) M). It has been reported that cannabinoids can inhibit the release of CGRP by stimulation of CB1 and CB2 cannabinoid receptors. Delta-9THC could act stimulating these receptors and thus inhibiting CGRP release and vesical relaxation. The muscle relaxing component removal could favour the contracting component, usually not active.

Pharmaco-toxicological screening of commercially available Italian natural mineral waters.

The consumption of natural mineral water has increased enormously during the past few years, yet doubts are arising about the real utility of using such water instead of ordinary drinking water (tap water). Mineral water's 'special' mineral composition might have properties favourable to health, which should be assessed by clinical and pharmacological analyses. A comparative pharmaco-toxicological study was performed on 14 commercially available Italian natural mineral waters with a wide range of mineral content. The waters were microbiologically analysed and the Allium cepa test done; in addition, Irwin, charcoal meal and diuresis tests were also performed on laboratory animals. No toxicological signs were recorded at the Allium cepa assay for any but the water with the highest mineral content. The diuretic effect was comparable to that of control tap water. Waters with high magnesium content significantly enhanced intestinal motility while at the Irwin test none of the water samples evoked behavioural changes. All the tested samples were microbiologically pure. In conclusion, mineral water can be an alternative to aqueduct water in places where the latter comes from superficial water and has to be subjected to hard potabilisation techniques; water from the 'Acqua Marcia' aqueduct (control) was found to have characteristics comparable to the mineral waters studied.

Toxicological evaluation of commercial mineral water bottled in polyethylene terephthalate: a cytogenetic approach with Allium cepa.

The aim of this study was to ascertain the possible toxicological effects of chemicals released into mineral water packaged in polyethylene terephthalate (PET) bottles. Two commercial mineral waters, bottled both in PET and glass and stored under different conditions, were examined using the Allium cepa test. The influence of the water samples on macroscopic (root length, colour and form) and microscopic (root tip mitotic index, chromosome aberrations) parameters was examined. The water samples were analysed after: (A) controlled-condition storage (no direct light exposure and 18 +/- 2 degrees C), (B) storage at 40 degrees C for 10 days, in the dark (migration test in accordance with 82/711/EEC), and (C) exposure to sunlight and varying temperatures (18-38 degrees C, mean temperature 25 +/- 3 degrees C). The two water samples bottled in PET induced cytogenetic aberrations regardless of the storage conditions. These signs of toxicity were evident even only 8 weeks after bottling, which is well within the recommended expiry date. Storage conditions were very important, as is suggested by the finding that chromosomal aberrations were particularly apparent after exposure to direct sunlight. However, as plant systems are not considered as primary screening tools by current international guidelines for mammalian systems, extrapolation of the results from this test system to other systems and, eventually, to human beings should be based on results from a battery of assays covering various metabolic pathways.

Effects of natural tachykinins on porcine lower urinary tract smooth muscle.

1. The aim of our study was to ascertain the possible differences and/or similarities in natural tachykinin activity in vitro on lower urinary tract of large-sized animals as compared with data obtained in laboratory animals. 2. Besides tachykinins normally present in mammals, namely substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), we tested non-mammalian tachykinins, such as eledoisin (ELED), physalaemin (PHYS), kassinin (KASS) and PG-kassinin II (PG-KASS II). 3. NKA, KASS and ELED were found to be the most potent peptides in contracting detrusor strips from porcine bladder. In particular, NKA showed a pD2 of 7.14, whereas KASS and ELED showed pD2 values of 7.20 and 7.22, respectively. The activity of NKB and PG-KASS II corresponded to 72.4 and 55.0% respectively of that of NKA. SP and PHYS activity corresponded to only 2% of that of NKA. 4. NKA (pD2 7.92) was the most active peptide in contracting bladder neck tissues as well. ELED and KASS were found to have lower, similar pD2 values (7.62 and 7.70, respectively), whereas NKB and PG-KASS II were much less active (pD2 7.12 and 6.74, respectively). Moreover, SP and PHYS showed an activity range lower than 2% of that of NKA. 5. The reported results confirm that, on pig vesical neck and detrusor, NK1 receptors represent a minority as compared with NK2 and NK3 receptors. By contrast, the presence of NK2 receptors is demonstrated by a greater potency of NKA. The presence of NK3 receptors both on detrusor and neck is evidenced by NKB activity and by results achieved with PG-KASS II.

Response to isoprenaline of rabbit detrusor muscle following exposure to 5, 8, 11, 14 eicosatetraynoic acid. Role of prostanoids on beta-adrenergic-evoked response.

1. Incubation with indomethacin or ibuprofen causes a fall in basal tone and inhibits isoprenaline-evoked response in rabbit detrusor muscle strips. To ascertain whether this is due to cyclo-oxygenase inhibition, we investigated the influence on beta-adrenoceptor response of 5, 8, 11, 14 eicosatetraynoic acid (ETYA) which, unlike nonsteroidal anti-inflammatory drugs, has no action in vitro other than cyclo-oxygenase inhibition. 2. Incubation of detrusor strips with ETYA (7 or 14 microM) for 60 min significantly reduced tissue response to isoprenaline by an extent similar to that induced by indomethacin, but only slightly affected basal tone. 3. In tissues preincubated with indomethacin, the addition of PGE2 or PGE1 to restore basal tone, completely re-established isoprenaline-evoked responses. 4. Our findings in rabbit detrusor strips show that inhibition of cyclo-oxygenase and of prostanoid production is the main mechanism involved in lowered beta-adrenoceptor evoked response. 5. In addition, these findings strengthen the conviction that ibuprofen and indomethacin act largely through mechanisms other than inhibition of prostanoid production.

Response to isoproterenol of rabbit detrusor strips following exposure to NSAIDs.

To ascertain whether NSAIDs affect the physiological mechanisms involved in bladder detrusor muscle motility we investigated the influence of indomethacin, ibuprofen and acetyl salicylic acid on the beta-adrenergic-evoked response in isolated male-rabbit detrusor strips. Incubation with indomethacin for 60 min significantly reduced the tissue response to isoproterenol (up to 10.8+/-4.9%), entirely abolished spontaneous contractions and caused a fall in basal tone. Ibuprofen significantly inhibited the tissue response to isoproterenol (up to 25.5+/-5.8%), abolished spontaneous contractions and caused a fall in basal tone. After the spasmogen KCl had been added to restore basal tone, the highest indomethacin and ibuprofen concentrations still significantly inhibited the isoproterenol-evoked response, indomethacin being the most active compound. Acetyl salicylic acid, at the doses used, neither inhibited spontaneous contractions nor changed basal tone whereas it significantly inhibited tissue response to isoproterenol (up to 69. 6+/-4.6%). Our findings in rabbit detrusor strips show that NSAIDs affect the beta-adrenergic-evoked response. They probably do so mainly through mechanisms other then inhibition of prostaglandin production.

Sequence analysis of a 40.2 kb DNA fragment located near the left telomere of yeast chromosome X.

We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae. The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long. Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y' repeat region of 65.4 and 12.8 KDa, the cytochrome B pre-mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein. Of the 16 remaining ORFs, eight show highest homologies with the S. cerevisiae hexose transporters family (two ORFs), the yeast alpha-glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O-acetyltransferase, the S. cerevisiae 137.7 KDa protein located in the Y' region and a protein of unknown function of Schizosaccharomyces pombe. Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks. DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends. Disruption studies have been performed on two ORFs encoding putative proteins of unknown function.

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Sequencing and analysis of a 20.5 kb DNA segment located on the left arm of yeast chromosome XI.

A 20.5 kb DNA fragment from the left arm of chromosome XI of Saccharomyces cerevisiae has been sequenced and analysed. Thirteen open reading frames (ORFs) for proteins longer than 100 amino acids were discovered. Among them, two are the known genes MRP49 and TPK3; two others encode proteins which show strong similarity with a yeast putative protein kinase and a yeast choline transport protein; one other shows weaker similarity with a yeast Ca2+/calmodulin-dependent protein kinase. Moreover, two putative proteins encoded by ORFs located in the sequenced fragment are closely similar to non-yeast proteins: the Caenorhabditis elegans elongation factor 2 and a glutamic acid-rich protein of Plasmodium falciparum.

DNA sequencing of a 36.2 kb fragment located between the FAS1 and LAP loci of chromosome XI of Saccharomyces cerevisiae.

We have completely sequenced on both strands a continuous DNA segment of 36.2 kb located on the left arm of Saccharomyces cerevisiae chromosome XI. Sequence analysis reveals the presence of 20 open reading frames (ORFs) at least 100 amino acids long. Five of these ORFs correspond to known genes; five others show homology with known proteins; the ten remaining ORFs identified show no detectable homology with other protein sequences contained in data banks and may represent new biological functions.

Pumiliotoxin B-like alkaloid in extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea: effects on blood pressure and heart of the rabbit.

Extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea (PS) displayed striking, reversible and, in part, dose-dependent effects on the systemic blood pressure and the heart of the rabbit. Similarly to the results obtained in the rat, the blood pressure response in the rabbit consisted in an initial short-lasting fall, followed by a significant and persistent rise. The initial hypotensive effect was inhibited by atropine, indicating a cholinergic mechanism. The inhibition of the pressure rise by prazosin or guanethidine, but not by surrenalectomy or hexamethonium, suggests a catecholamine release from adrenergic nerve terminals of the vasculature. PS produced on the heart a variety of rhythm disorders, caused both by a release of acetylcholine and a direct effect on the myocardium. It is worth mentioning that tetrodotoxin, a typical sodium channel blocker, reduced or abolished the effects of PS both on the heart and the blood pressure, suggesting that sodium channels may directly or indirectly participate in the mechanism of action of PS.

The complete sequence of K3B, a 7.9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames.

We have determined the nucleotide sequence of a segment from chromosome III of Saccharomyces cerevisiae extending over 7.9 kb between the PGK1 and CRY1 loci. The fragment contains seven open reading frames, YCR241, YCR242, YCR243, YCR244, YCR245, YCR246 and YCR247, of more than 70 codons. The study of the effects of a global disruption of YCR242, YCR243, YCR244, YCR245 and YCR247 shows that they are not essential for growth and division.

The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae.

The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.

A comparative study of behavioural and autonomic effects of atropine and Atropa belladonna.

Atropa belladonna L. (Solanaceae) tincture was compared with atropine for its anticholinergic activity, both in vivo and in vitro. In all tests, the biological activity of A. belladonna resulted greater than that suggested by its alkaloid content. The results suggest the presence in A. belladonna leaves of unknown compounds with a significant biological activity.

A comparative study of Atropa belladonna and atropine on an animal model of urinary retention.

The ability of Atropa belladonna L. and atropine to produce urinary retention has been studied in the rat. Our results suggest that A. belladonna is more effective than expected on the basis of its alkaloidic content.