Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels.

Hydrogen sulphide (H2S) has been recently hypothesized to be an endogenous adipocyte-derived relaxing factor, evoking vasorelaxation of conductance and resistance vessels. Although the activation of ATP-sensitive potassium channels is known to play a central role in H2S-induced vasorelaxation, activation of vascular Kv7 voltage-gated potassium channels has also been suggested. To investigate this possibility, the ability of selective activators and blockers of distinct classes of potassium channels to affect vasodilation induced by the H2S-donor NaHS, as well as NaHS-induced Rb(+) efflux in endothelium-denuded rat aortic rings, was investigated. NaHS-induced changes of membrane potential were fluorimetrically assessed on human vascular smooth muscle (VSM) cells. Modulation of Kv7.4 channels by NaHS was assessed by electrophysiological studies, upon their heterologous expression in CHO cells. In isolated aortic rings, NaHS evoked vasorelaxing responses associated with an increase of Rb(+)-efflux. NaHS promoted membrane hyperpolarization of human VSM cells. These effects were antagonized by selective blockers of Kv7 channels. The H2S-donor caused a left-shift of current activation threshold of Kv7.4 channels expressed in CHO cells. Altogether, these results suggest that the activation of Kv7.4 channels is a key mechanism in the vascular effects of H2S. Given the relevant roles played by Kv7.4 channels in VSM contractility and by H2S in circulatory homeostasis regulation, these findings provide interesting insights to improve our understanding of H2S pathophysiology and to focus on Kv7.4 channels as novel targets for therapeutic approaches via the "H2S-system".

The bacterial community in 'taberna' a traditional beverage of Southern Mexico.

AIMS: To characterize the bacterial community of taberna, an alcoholic traditional beverage from the Southern part of Mexico produced by the fermentation of the coyol palm sap (Acrocomia aculeate). METHODS AND RESULTS: Bacterial 16S rDNA libraries were constructed from metagenomic DNA extracted during the fermentation process at 0, 60 and 108 h. A total of 154 clones were sequenced, and 13, 10 and nine unique sequences were found at each sampling time. At the onset of the fermentation, Zymomonas mobilis, Fructobacillus spp., Pantoea agglomerans and other Gammaproteobacteria were detected. After 60 h, lactic acid bacteria were found and 30% of clones in the library were related to Lactobacillus nagelii, L. sucicola and L. sp. By the end of the experiment, i.e. after 108 h, the bacterial community included Z. mobilis, Lact. nagelii and Acetobacter pasteurianus. CONCLUSIONS: Our results suggest that Z. mobilis population represented an important proportion of the bacterial community (60-80%), as well as the lactobacilli during the fermentation process. The bacterial diversity was low and decreased as the fermentation progressed. SIGNIFICANCE AND IMPACT OF THE STUDY: This culture-independent study suggests that Z. mobilis and lactobacilli play an important role in the alcoholic fermentation of the taberna beverage.

An in vivo comparison of working length determination of two frequency-based electronic apex locators.

AIM: To compare in vivo the accuracy of two electronic apex locators (EALs) by means of digital radiographic imaging system. METHODOLOGY: Electronic working lengths of 831 canals were determined with the DentaPort ZX and Raypex 5 apex locators and confirmed radiographically. The radiographic images acquired with the aid of a digital radiographic imaging system (VisualiX eHD; Gendex Dental Systems, Des Plaines, IL, USA) were blindly analysed by two independent evaluators. The distance between the file tip and the radiographic apex was measured using dedicated software (VixWin Pro, Gendex Dental Systems, Des Plaines, IL, USA) and the mean distance achieved between different tooth type and EALs were compared statistically. Statistical analyses were performed using the t-test for independent samples and one-way anova with the null hypothesis set as 5%. Positive or negative values were recorded when the file tip was detected beyond or short of the radiographic apex, respectively. RESULTS: The mean distance between file tip and radiographic apex were -1.08 +/- 0.73 and -1.0 +/- 0.67 mm considering DentaPort ZX and Raypex 5 groups, respectively, with no significant differences (P > 0.05). No statistically significant differences were found amongst the same tooth type when comparing both groups (P > 0.05) or amongst different teeth type in the same group (P > 0.05). CONCLUSIONS: Within the limitations of this in vivo study, the DentaPort ZX and Raypex 5 were similar in terms of accuracy.

Sheep manure vermicompost supplemented with a native diazotrophic bacteria and mycorrhizas for maize cultivation.

An orthogonal experimental design L9 (3(4)) with 10 repetitions was used to investigate the effect of Glomus claroideum (0, 1 or 2g(-1) plant), G. fasciculatum (0, 1 or 2g plant(-1)), native diazotrophic bacteria (0, 10(3) and 10(5) UFC ml(-1)) and sheep manure vermicompost (0%, 5% and 10% v/v) on maize plant growth, N and P in leaves and mycorrhization percent. Vermicompost explained most of the variation found for leaf number, wet weight, stem height, and diameter. Both mycorrhizas increased the plant wet weight but G. fasciculatum the most. Mycorrhization increased the P content, but not the N content. Mycorrhizal colonization increased when diazotrophic bacteria and vermicompost were added. It was found that weight of maize plants cultivated in peat moss amended with vermicompost increased when supplemented with G. fasciculatum and diazotrophic bacteria.

Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas.

Preeclampsia is a mainly vascular disease of pregnancy, probably caused by an imbalance between vasodilator and vasoconstrictor agents that results in generalized vasospasm and poor perfusion in many organs. Among these factors, endothelin-1 (ET-1), a potent vasoconstrictor, is highly increased in preeclamptic women, while nitric oxide (NO), a vasodilator of human utero-placental arteries, is reduced in the same patients. The present study was designed to investigate the interactions between ET-1 and the NO system in the feto-placental unit; to this purpose we also examined the messenger ribonucleic acid (mRNA) expression of ET-1, inducible NO synthase (iNOS), and endothelial NOS (eNOS) in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive (NT) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblastic cells. Interestingly, by Northern blot analysis we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Furthermore, exogenous ET-1 (10(-7) mol/L) was able to up-regulate its own mRNA expression in both NT and PE trophoblastic cells. iNOS and eNOS mRNA expression was then detected, by semiquantitative PCR, in both NT and PE trophoblastic cells. PE trophoblastic cells expressed lower iNOS mRNA levels compared with NT pregnancies. On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells. Moreover, in the presence of ET-1 we observed a decrease in iNOS and an increase in eNOS mRNA expression levels in both NT and PE trophoblastic cells compared with the respective untreated cells. In conclusion, we demonstrate that ET-1 expression is increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, is decreased; conversely, eNOS expression is increased. Finally, ET-1 is able to influence its own as well as NOS isoform expression in normal and PE trophoblastic cultured cells. These findings suggest the existence of a functional relationships between ET(s) and NOS isoforms that could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.

Evidence for a functional link between the heme oxygenase-carbon monoxide pathway and corticotropin-releasing hormone release from primary cultures of human trophoblast cells.

The gene expression and synthesis of both constitutive and inducible heme oxygenase (HO) isoforms have been recently described in human placental cells, but the functional role(s) of this biochemical pathway in placental physiology and pathology is still unclear. In the present study, we have investigated whether HO activity is involved in the control of CRH secretion from trophoblast cells. Fluctuations in HO activity were induced in primary cultures of human trophoblast cells using well-known activators and inhibitors of HO, and the subsequent changes in CRH secretion were monitored measuring CRH immunoreactivity released into the incubation medium. It was found that the increase in HO activity induced by hemin or cobalt chloride (CoCl(2)) was associated with parallel significant increases in CRH release. This effect was probably caused by the gaseous HO end-product, carbon monoxide (CO), because it was blocked by the HO inhibitor tin-mesoporphyrin-9, but it was not mimicked by stable HO end-products, biliverdin and bilirubin. We have also investigated whether stimulation of CRH release induced by HO was mediated by the cyclooxygenase (COX) pathway. Indeed, hemin also caused significant increases in PGE2 release in this experimental paradigm. However, CoCl(2), which also enhances CRH release, had no stimulatory effect and actually inhibited PG secretion; moreover, a nonselective COX inhibitor, indomethacin, failed to counteract hemininduced CRH release. Taken collectively, these findings suggested that modulation of CRH secretion by the HO-CO system occurs through a mechanism independent of COX activity.

Endothelins enhance prostaglandin (PGE(2) and PGF(2alpha)) biosynthesis and release by human luteal cells: evidence of a new paracrine/autocrine regulation of luteal function.

We have previously shown that endothelin-1 (ET-1) is normally found in human luteal cells, where it is able to significantly inhibit both basal and hCG-induced progesterone production. To further expand our comprehension of the possible roles of endothelins (ETs) in luteal physiology, in this study we used primary cultures of luteal cells exposed to graded doses of ET-1 and ET-3; PGF(2alpha) and PGE(2) were assayed in the culture medium to investigate whether ETs also influence cyclooxygenase activity in these cells. We found that both ETs are able to significantly stimulate PGF(2alpha) and PGE(2) release in a dose- and time-dependent manner. ET-1 was always more effective than ET-3. Experiments with two endothelin receptor antagonists (the BQ485 and BQ788 compounds, which block the ET-A and ET-B receptors, respectively) showed that the two endothelins induce PG production through different receptors and signaling pathways. In conclusion, here we demonstrate the ability of ETs to influence PG synthesis and release from human luteal cells. As PGs are deeply involved in corpus luteum activity, and ETs were also able to influence progesterone production, the present new data suggest an interesting interplay among progesterone, PGs, and ETs in the control of corpus luteum physiology.

Paracrine regulation of insulin-like growth factor I (IGF-I) an IGF-II on prostaglandins F2alpha and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects.

The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.

Human umbilical vein endothelial cells: a new source and potential target for corticotropin-releasing factor.

Corticotropin-releasing factor (CRF) plays a key role in the modulation of fetal-placental unit function during human pregnancy. CRF has a potent vasoactive action on fetal-placental circulation. As products secreted from endothelial cells affect vascular wall reactivity, we investigated whether cultured human umbilical vein endothelial cells (HUVEC) may represent a source and a target for CRF. With RT-PCR we showed that HUVEC express CRF and CRF receptor type 2 messenger ribonucleic acids. Cultured HUVEC also released CRF peptide in a time-dependent way, and the CRF release was differently regulated by various molecules. Dexamethasone decreased CRF release, whereas progesterone and 17beta-estradiol markedly increased it. Forskolin and PGF2alpha were potent stimulators of CRF release from HUVEC. Among the peptides, CRF secretion was stimulated by interleukin-1beta and by endothelin-1. Our study shows for the first time that HUVEC express CRF messenger ribonucleic acid and peptide as well as the CRF R2 gene, and that CRF release is differentially regulated by several distinct molecules. We here propose that CRF has a role in the regulation of the fetal-placental circulation.

Growth hormone induces in vitro maturation of follicle- and cumulus-enclosed rat oocytes.

The aim of this study was to assess the possible role of growth hormone (GH) on rat oocyte maturation. This effect was analyzed in follicle-enclosed, cumulus-enclosed and denuded oocytes obtained from immature pregnant mare's serum gonadotropin (PMSG)-treated rats. The addition of GH to the cultures significantly accelerated maturation in both follicle- and cumulus-enclosed oocytes while no effect was seen on denuded oocytes maturation. Also, GH accelerated meiotic maturation in follicle-enclosed oocytes from immature untreated rats. The GH action was not mediated by lactogenic receptors since prolactin (Prl) did not affect the maturation process while it was mediated by insulin growth factor-I (IGF-I) as suggested by the block of GH action observed in the presence of antibodies anti-IGF-I. Finally, no GH effect was found when dbcAMP was added to the cultures. Our results demonstrate that GH is capable of inducing maturation in oocytes from both primed and unprimed rats. Since the presence of physiological levels of GH in the ovary is now well established, the present data strongly suggest a potential relevance of GH in the reproductive biology.

Growth hormone-releasing factor stimulates meiotic maturation in follicle- and cumulus-enclosed rat oocyte.

This study was designed in order to assess the possible role of growth hormone-releasing factor (GRF) on oocyte maturation. This effect was analyzed in follicle-enclosed, cumulus-enclosed and denuded oocytes obtained from immature pregnant mare's serum gonadotropin-treated rats. The addition of GRF to the cultures significantly accelerated maturation in follicle- and cumulus-enclosed oocytes while no effect was seen on denuded oocytes. Also, the neuropeptide was able to induce maturation in follicle-enclosed oocytes obtained from immature untreated rats. The GRF action was probably not mediated by the vasoactive intestinal peptide (VIP) receptors since the two hormones had different effects on oocyte maturation and on cAMP production by granulosa cells. In addition the disappearance of the GRF effect observed in the presence of antibodies anti-GH suggested that GRF required the intermediacy of GH to accomplish its effect on oocyte maturation. Finally, GRF did not affect meiotic maturation when dbcAMP was added to the cultures. Our results demonstrate the ability of GRF to accelerate maturation in oocytes from both primed and unprimed rats. Since the presence and the involvement of GRF at the ovarian levels is now well established, the present data strongly suggest an important potential role of GRF in the ovarian physiology.

Growth hormone induction of rat granulosa cell tissue-plasminogen activator expression and progesterone synthesis.

The plasminogen activator (PA) system is present in the ovary and appears to be involved both in follicular growth and ovulation. Similarly, the growth hormone (GH) has been demonstrated to positively affect some ovarian activities. Interestingly, GH appears not only as a mediator of gonadotropin effects, but also as having an independent action of its own on the ovary. In the present study we wanted to investigate if GH could affect ovarian plasminogen activator (PA) activity and steroidogenesis. Granulosa cells from immature rats, injected with pregnant mare serum gonadotropin (PMSG) for inducing follicular growth, were cultured for 24 h with increasing concentrations of GH. A significant dose-dependent increase in tPA activity was observed in the GH-treated cells. This effect was exerted at the mRNA level and the use of cycloheximide, a protein synthesis inhibitor, suggested that GH did not require any other intermediary protein for inducing tPA-mRNA. Furthermore, cAMP levels were not affected by GH treatment. Finally, GH was found to increase progesterone (P) synthesis by granulosa cells. The correlation between the PA system and ovulation and the importance of a normal steroidogenesis for the ovarian physiology claim for a key role of GH in the ovarian activities.

Growth hormone stimulates androsterone synthesis by rat theca-interstitial cells.

The aim of this study was to assess the possible role of growth hormone (GH) on androsterone synthesis. This effect was analyzed in theca-interstitial cells obtained from immature female rats. The addition of GH to the cultures significantly stimulated androsterone (A) synthesis in a dose- and time-dependent way and this effect was not due to a cellular number increase. When added to the hCG cultures, GH significantly enhanced androgen production even though it did not synergyze with the chorionic gonadotropin. The addition of antibodies anti-IGF-I to the GH cultures did not modify the growth hormone effect suggesting that GH probably does not require IGF-I to achieve its effect on A production. Finally, no effect of GH on cAMP levels were observed in the cultures at the end of the treatment. Our results demonstrate that GH is able to significantly induce A synthesis by rat theca-interstitial cells. Since the presence of GH and its receptors in the ovary is now well established the present data strongly suggest a potential relevance of GH in reproductive biology.

Control of human luteal steroidogenesis: role of growth hormone-releasing hormone, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide.

OBJECTIVE: To examine the possible effect of growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide on basal and hCG-stimulated P production by human luteal cells. DESIGN: Cultures of human luteal cells from the early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Università Cattolica, a public care center. PATIENT(S): Ten nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders, such as leiomyomatosis. INTERVENTION(S): Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURE(S): Luteal cells were incubated with GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide with or without hCG at different concentrations. RESULT(S): Pituitary adenylate cyclase-activating peptide stimulated P production in a dose- and time-dependent manner, whereas GHRH and vasoactive intestinal peptide did not affect luteal steroidogenesis. None of the three peptides were found to synergize with hCG. CONCLUSION(S): Pituitary adenylate cyclase-activating peptide can influence human luteal steroidogenesis.

Insulin-like growth factor (IGF)-I and IGF-II stimulate progesterone production by human luteal cells: role of IGF-I as mediator of growth hormone action.

OBJECTIVE: To examine the possible direct effect of insulin-like growth factor (IGF)-I and IGF-II on basal and hCG-stimulated P production by cultured human luteal cells. The possible role of IGF-I as mediator of GH action on luteal steroidogenesis also was investigated. DESIGN: Cultures of human luteal cells from early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Universita Cattolica, a public care center. PATIENTS: Eight nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders such as leiomyomatosis. INTERVENTIONS: Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURES: Luteal cells were incubated with IGF-I or IGF-II with or without hCG at different concentrations. Growth hormone also was used alone and with an anti-IGF-I-antibody. RESULTS: We found that IGF-I and IGF-II were able to stimulate directly the P production at all used concentrations and that both of them significantly amplified the steroidogenic hCG effect. Finally, IGF-I was shown to mediate the positive GH action on P synthesis.

Maternal plasma calcitonin gene-related peptide levels do not change during labor and are not influenced by delivery route.

OBJECTIVE: Calcitonin gene-related peptide (CGRP) circulates in maternal circulation throughout pregnancy, and specific receptors for CGRP (CGRPrs) are expressed by human myometrium. Because CGRP induces a dose-dependent relaxation of human myometrium, we examined a role for CGRP in modulation of myometrial smooth muscle contractility during pregnancy and labor. The aim of the present study was to evaluate the changes of maternal serum CGRP levels during parturition, according to the mode of delivery and in relation to cervical dilatation. METHODS: Circulating CGRP levels were measured in the following groups of healthy women: nonpregnant women, during the follicular phase of the menstrual cycle (n = 19); at term pregnancy (39-40 weeks; n = 24); after elective cesarean delivery (39-40 weeks; n = 20); and at spontaneous vaginal delivery (39-40 weeks; n = 16). In a subgroup of women, blood samples were collected longitudinally throughout labor at various cervical dilatations in the progress of labor (n = 8). RESULTS: Pregnant women at term not in labor had significantly higher CGRP levels than nonpregnant women (P =.021). No significant difference was found between women who delivered vaginally and those who had elective cesarean, and there were no correlations between CGRP plasma levels and cervical dilatation. CONCLUSIONS: Parturition is characterized by no significant changes in maternal serum CGRP levels, and no significant correlation exists between plasma CGRP levels and cervical dilatation during labor.

[Effect of 3,4,5-trimethoxy-benzoyl-aminocaproic acid on the cytolytic process in myocardial infarct. Working hypothesis]. / Influenza dell'acido 3,4,5-trimetossi-benzoil-aminocaproico sul processo citolitico in corso di infarto del miocardio. Ipotesi di lavoro

The equilibrium of the ion pump is seriously disturbed at the cell level in acute myocardial infarct. The damaged cells are unable to take up potassium and this remains in the intercellular spaces, bound to acid substances released by the necrotic site. This results in self-perpetuating damage to surrounding tissue, together with extension and aggravation of the infarct. Early treatment with 3,4,5-trimethoxy-benzoyl-aminocaproic acid enables this secondary type of lesion to be prevented, since this substance is capable of fixing potassium ions and returning them to the cell.

[Agenesis of the gallbladder. Description of 2 cases in 2 siblings]. / L'agenesia della colecisti. Descrizione di due casi in due fratelli.

Gallbladder and cystic duct agenesis is a rare malformation with frequent familial occurrence. We report a case in a girl, diagnosed by nonoperative means, and a second case in her brother, discovered with a screening in the same family, operated with ultrasonography.