Search

Objective: To analyze and compare the expression profile of TAC3, TACR3, KISS1, and KISS1R in mural granulosa and cumulus cells from healthy oocyte donors and patients with different infertility etiologies, including advanced maternal age, endometriosis, and low ovarian response. Design: Genetic association study. Setting: Private fertility clinic and public research laboratory. Patient(s): Healthy oocyte donors and infertile women undergoing in vitro fertilization (IVF) treatment. Intervention(s): IVF. Main Outcome Measure(s): Gene expression levels of KISS1, KISS1R, TAC3, and TACR3 in human mural granulosa and cumulus cells. Result(s): Infertile women showed statistically significantly altered expression levels of KISS1 (-2.57 +/- 2.30 vs. -1.37 +/- 2.11), TAC3 (-1.21 +/- 1.40 vs. -1.49 +/- 1.98), and TACR3 (-0.77 +/- 1.36 vs. -0.03 +/- 0.56) when compared with healthy oocyte donors. Advanced maternal age patients, endometriosis patients, and low responders showed specific and altered expression profiles in comparison with oocyte donors. Conclusion(s): Abnormal expression levels of KISS1/KISS1R and TAC3/TACR3 systems in granulosa cells might be involved in the decreased fertility associated to advanced maternal age, endometriosis, and low ovarian response. ((C) 2020 by American Society for Reproductive Medicine.)

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

Infertility is a global disease affecting one out of six couples of reproductive age in the world, with a male factor involved in half the cases. There is still much to know about the regulation of human male fertility and thus we decided to focus on two peptide families that seem to play a key role in this function: tachykinins and kisspeptins. With this aim, we conducted an exhaustive review in order to describe the role of tachykinins and kisspeptins in human fertility and their possible implications in infertility etiopathogenesis. Many advances have been made to elucidate the roles of these two families in infertility, and multiple animal species have been studied, including humans. All of this knowledge could lead to new advances in male infertility diagnosis and treatment, but further research is needed to clarify all the implications of tachykinins and kisspeptins in fertility.

The portal of the forecourt of the old monastery of San Agustin in Seville (Spain) has been preserved in fragments. The notarial document specifying the conditions for its construction has been preserved, although the drawing that accompanied it has been lost. To a certain extent, this loss is remedied by other designs created by Hernan Ruiz. We also have archive photographs that bear witness to its state of repair some years before it was dismantled. Armed with all of this material, we attempted to carry out a virtual anastylosis of the portal using new technologies such as digital photogrammetry, reverse engineering, BIM and visual programming. The creation of the 3D digital model was accompanied by the interpretation of the data obtained from documentary sources. The end result is a digital graphical model of heritage information (HBIM) which we hope will provide a useful tool for the analysis, promotion and anastylosis.

[Objective] To explore the relationship between iron and infertility by investigating iron-related gene expression in granulosa and uterine cervical cells., [Design] Case-control study., [Setting] Two tertiary hospitals., [Patient(s)] Two independent cohorts of fertile (n = 18 and n = 17) and infertile (n = 31 and n = 35) women., [Intervention(s)] In vitro fertilization., [Main Outcome Measure(s)] Gene expression levels of ferritin light chain (FTL), ferritin heavy chain (FTH), transferrin receptor (TFRC), and ferroportin (SLC40A1) mRNA were analyzed in granulosa and cervical cells., [Result(s)] In the first cohort, fertile and infertile women were similar in body mass index. Ferroportin mRNA levels were decreased in granulosa cells from infertile women in parallel with increased serum hepcidin levels. A positive association between ferroportin and TFRC mRNA, a gene associated with intracellular iron deficiency, was observed only in granulosa cells from fertile women. The major findings were replicated in a second independent cohort., [Conclusion(s)] Ferroportin mRNAs and circulating hepcidin identify a subset of infertile women and may constitute a target for therapy.

PURPOSE: The neurokinin B (NKB)/NK3 receptor (NK3R) and kisspeptin (KISS1)/kisspeptin receptor (KISS1R), two systems essential for reproduction, are present in human granulosa cells (GCs) of healthy women and contribute to the control of fertility, at least partially, by acting on the gonads. However, little is known about the expression of these systems in GCs of women with polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of NKB/NK3R and KISS1/KISS1R in mural granulosa (MGCs) and cumulus cells (CCs) of PCOS women. METHODS: A cross-sectional study was performed in 46 healthy women and 43 PCOS women undergoing controlled ovarian stimulation. MGCs and CCs were collected from pre-ovulatory follicles after transvaginal ultrasound-guided oocyte retrieval and the expression of the genes encoding NKB (TAC3), NK3R (TACR3), KISS1, and its receptor (KISS1R) was analyzed using real-time quantitative RT-PCR. RESULTS: TAC3, TACR3, and KISS1 mRNA levels were decreased in MGCs and CCs of PCOS women. TAC3 positively correlated with KISS1 in MGCs of healthy women and TACR3 was positively associated with KISS1R in CCs from healthy women. These associations were not observed in PCOS women. CONCLUSION: The NKB/NK3R and KISS1/KISS1R systems are dysregulated in MGCs and CCs of PCOS women. The lower expression of these systems in GCs could contribute to the abnormal follicle development and defective ovulation that characterize the pathogenesis of PCOS.

Aims The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca2 +, Na+, K+ and H+ ions in the sperm cytoplasm. Ion channels are key regulators of the sperm membrane potential, cytoplasmic Ca2 + and intracellular pH (pHi), which leads to regulate motility, capacitation, acrosome reaction and other physiological processes crucial for successful fertilization. Expression of epithelial sodium channels (ENaC) and voltage-gated sodium channels (Nav) in human spermatozoa has been reported, but the role of Na+ fluxes sodium channels in the regulation of sperm cell function remains poorly understood. In this context, we aimed to analyze the physiological role of Nav channels in human sperm. Main methods Motility and hyperactivation analysis was conducted by CASA analysis. Flow cytometry and spectrophotometry approaches were carried out to measure Capacitation, Acrosome reaction, immunohistochemistry for Tyr-residues phosporylation, [Ca2 +]i levels and membrane potential. Key findings Functional studies showed that veratridine, a voltage-gated sodium channel activator, increased sperm progressive motility without producing hyperactivation while the Nav antagonist lidocaine did induce hyperactivated motility. Veratridine increased protein tyrosine phosphorylation, an event occurring during capacitation, and its effects were inhibited in the presence of lidocaine and tetrodotoxin. Veratridine had no effect on the acrosome reaction by itself, but was able to block the progesterone-induced acrosome reaction. Moreover, veratridine caused a membrane depolarization and modified the effect of progesterone on [Ca2 +]i and sperm membrane potential. Significance Our results suggest that veratridine-sensitive Nav channels are involved on human sperm fertility acquisition regulating motility, capacitation and the progesterone-induced acrosome reaction in human sperm.

Today’s society is sensitive to the architectural heritage conservation. This implies to perform works to maintain these buildings and to assure their structural security. In the last years, the structural analysis of historical masonry constructions has experienced a great progress, thanks to the use of techniques based on the study of the dynamic properties of building structures. In this context, changes on the structural health state of a building are one of the elements that can be assessed considering changes on their dynamic properties. This is useful to evaluate the effectiveness of structural interventions on this kind of buildings by analysing these properties before and after it. This paper focuses on the Jura Chapel, in Jerez de la Frontera (Spain). This chapel is part of San Juan de los Caballeros Church and is dated from the 15th century. In 2015 and after the identification of some structural damages in the chapel vault, an intervention was initiated to improve its structural behaviour and to recover its original appearance. The present work reports the evaluation of the effects that this intervention has on the structural health state of the building, using nondestructive techniques based on ambient vibration tests (AVT) and Operational Modal Analysis (OMA). The AVT were performed for both prerestored and restored states and under environmental loads. A discussion about the validity of doing AVT from extrados when a vault presents disconnection between ribs and web is included in the paper. As a result, the first five natural frequency values have increased while the corresponding mode shapes have not changed significantly. This proves a structural health improvement caused by the repairing process without changing the original behaviour of the structure. This work shows OMA capabilities for evaluating the effectiveness of intervention works on the health state of a historical masonry structure.

© 2015 Ivyspring International Publisher. Background and Objective: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. Methods: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), ami-nopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). Results: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall suvival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. Conclusions: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

Study question Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? Summary answer The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. What is known already The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown. Study design, size, duration This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. Participants/materials, setting, methods The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. Main outcome and the role of chance NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB. Limitations, reasons for caution The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells. Wider implications of the findings Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells. Study funding/ competing interests This work was supported by grants from Ministerio de Economia y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucia (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.

Objective To analyze the mRNA and protein expression of cannabinoid receptors CB1 and CB2 in chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Design and methods Fresh and formalin-fixed tissue samples of ChRCC and RO were analyzed by using real-time quantitative RT-PCR and immunohistochemical techniques (n = 40). Results Quantitative RT-PCR analysis showed that CB1 mRNA was underexpressed by 12-fold in ChRCC and had a variable expression in RO. CB1 protein showed intense positive immunostaining in both neoplasms. Both CB2 mRNA and protein were not expressed in tumor and non tumor renal tissue. Conclusion This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of renal tumors.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca2+ levels ([Ca2+]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca2+]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

[Objective] To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium., [Design] Laboratory study., [Setting] University research laboratories and academic hospital., [Patient(s)] Women undergoing hysterectomy for symptomatic leiomyomas., [Intervention(s)] Quantitative polymerase chain reaction, immunohistochemistry and Western blot., [Main Outcome Measure(s)] Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium., [Result(s)] TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells., [Conclusion(s)] These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors., Supported in part by research grants from the Agencia Canaria de Innovación y Sociedad de la Información, ACIISI, (PI 2007/001), and from Ministerio de Economía y Competitividad (CTQ2011-25564 and RTC-2014-1431-1), Spain, in both cases with joint financing by FEDER funds from the European Union.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

Objective To investigate the presence of neurokinin B (NKB)/NK 3 receptor (NK 3 R) and kisspeptin/KISS1 receptor (KISS1R) messenger RNA (mRNA) and proteins throughout the human female genital tract. Design In vitro study. Setting Academic research laboratories and academic hospitals. Patient(s) Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. Intervention(s) Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. Main Outcome Measure(s) Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK 3 R and kisspeptin/KISS1R in target tissues. Result(s) Expression of the genes encoding NKB ( TAC3 ) and NK 3 R ( TACR3 ), and kisspeptin ( KISS1 ) and its receptor ( KISS1R ) was found in the uterus, ovary, and oviduct. Both NKB and NK 3 R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK 3 R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. Conclusion(s) NKB/NK 3 R and kisspeptin/KISS1R are present in female peripheral reproductive tissues with colocalization of both systems in some non-neuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues.

Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration, [Ca(2+)]i, sperm motility, sperm hyperactivation and the acrosome reaction. Changes in [Ca(2+)]i were monitored using Fura-2, sperm kinematic parameters were measured using computer-assisted sperm analysis (CASA), and the acrosome reaction was measured using fluorescein isothiocyanate-coupled Pisum sativum agglutinin lectin (FITC-PSA method). We found that kisspeptin and its receptor are present in sperm cells, where both are mainly localized in the sperm head, around the neck and in the flagellum midpiece. Exposure to kisspeptin caused a slow, progressive increase in [Ca(2+)]i, which reached a plateau about 3-6 min after kisspeptin exposure. In addition, kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation. The effects of kisspeptin on sperm motility and hyperactivation were inhibited by the antagonist of KISS1R, peptide 234. Kisspeptin did not induce the acrosome reaction in human spermatozoa. These data show for the first time that kisspeptin and its receptor are present in human spermatozoa and modulate key parameters of sperm function. This may represent an additional mechanism for their crucial function in the control of male fertility.

Substance P (SP) and neurokinin A (NKA) are members of the tachykinin peptides family. SP causes endothelial-dependant relaxation but the contractile response to tachykinins in human vessels remains unknown. The objective was to assess the expression and the contractile effects of tachykinins and their receptors in human saphenous veins (SV). Tachykinin expression was assessed with RT-PCR, tachykinin receptors expression with RT-PCR and immunohistochemistry, and functional studies were performed in organ bath. Transcripts of all tachykinin and tachykinin receptor genes were found in SV. NK 1-receptors were localized in both endothelial and smooth muscle layers of undistended SV, whereas they were only found in smooth muscle layers of varicose SV. The expression of NK2- and NK3-receptors was limited to the smooth muscle in both preparations. NKA induced concentration-dependent contractions in about half the varicose SV. Maximum effect reached 27.6 ± 5.5% of 90 mM KCl and the pD2 value was 7.3 ± 0.2. NKA also induced the contraction of undistended veins from bypass and did not cause the relaxation of these vessels after precontraction. The NK2-receptor antagonist SR48968 abolished the contraction induced by NKA, and a rapid desensitization of the NK2-receptor was observed. In varicose SV, the agonists specific to NK1- or NK 3-receptors did not cause either contraction or relaxation. The stimulation of smooth muscle NK2-receptors can induce the contraction of human SV. As SV is richly innervated, tachykinins may participate in the regulation of the tone in this portion of the low pressure vascular system. © 2011 Elsevier Ltd., C. Clement, Department of Vascular Surgery, University Hospital, Reims, France. The work by FMP and LC was supported by a grant from the Ministerio de Ciencia e Innovación (CTQ2007-61024/BQU), Spain.

6 páginas, 3 figuras, 3 tablas. The angiotensin-converting enzymes (ACE and ACE2) are highly expressed in renal tubules and play an important role in the regulation of renal function by the intrarenal renin-angiotensin system (iRAS). Dysregulation of these cell-surface peptidases has been associated with renal injury. Most of these studies, however, have focused on non-neoplastic kidney diseases. In the present study, ACE and ACE2 activity and protein and mRNA expression were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Enzyme activity was measured by spectrofluorometric (ACE2) and spectrophotometric assays (ACE), and protein and mRNA expression were determined by immunohistochemistry and qRT-PCR assays, respectively. The enzyme activities and immunohistochemistry showed that both enzymes are mainly downregulated in these neoplasms. qRT-PCR studies in CCRCC showed no positive correlation between ACE and ACE2 activity/protein expression and mRNA levels, whereas downregulation of ACE2 mRNA levels was observed in tumors from the distal nephron (ChRCC and RO). These findings suggest a metabolic imbalance in iRAS and a role of this system in renal neoplastic diseases, and point to ACE and ACE2 as potential prognostic/diagnostic markers. This work was supported by grants from the Jesús Gangoiti-Barrera Foundation, Gobierno Vasco (GIC07/84), MEC (CTQ2007-61024/BQU) and SAIOTEK (SA-2008/00046).

7 páginas, 3 figuras, 1 tabla. Several studies in cell cultures and in animal models have demonstrated that cannabinoids have important antitumoral properties. Because many of these effects are mediated through cannabinoid (CB) receptors CB1 and CB2, the study of their expression in human neoplasms has become of great interest in recent years. Fresh and formalin-fixed tissue samples of 20 consecutive clear cell renal cell carcinomas (CCRCCs) were collected prospectively and analyzed for the expression of both CB receptors by using RT-PCR, Western blot (WB), and immunohistochemical techniques. RT-PCR assays demonstrated the expression of mRNA encoding the CB1 in tumor tissue and in adjacent non-neoplastic kidney.Conversely, WB and IHC revealed a marked downregulation of CB1 protein in tumor tissue; CB2 was not expressed. The obtained data suggest a possible implication of the endocannabinoid system in renal carcinogenesis. A posttranscriptional downregulation of CB1 and the absence of expression of CB2 characterize CCRCC. This work was supported by grants from the Jesús Gangoiti-Barrera Foundation, Gobierno Vasco (GIC07/84), Ministerio de Educación y Ciencia (CTQ2007-61024/BQU), and SAIOTEK (SA-2008/00046).

6 páginas, 1 figura, 4 tablas. Since tachykinins appear to be involved in the pathogenesis of allergic asthma, we investigated a possible association between 28 single nucleotide polymorphisms of the tachykinin genes TAC1, TAC3 and TAC4, and asthma susceptibility. A case–control study was conducted on 102 patients and 100 healthy subjects from the Canary Islands (Spain). A significant association with asthma was observed for two SNPs: rs2291855 in the TAC3 gene conferring asthma protection (Odds ratio [OR]: 0.46; 95% Confidence Interval [CI]: 0.22–0.97; P = 0.038), and rs4794068 in the TAC4 gene associated with an increased risk for asthma (OR: 1.94; 95% CI: 1.06–3.54; P = 0.03). The present study represents a preliminary step in elucidating the association between tachykinin gene polymorphisms and asthma susceptibility. This work was funded by BFU2005-04495-C02-01 and 02 from Ministerio de Educación y Ciencia (Spain) and the FPU grant AP2005-4921 to T.E.K.

The phylogenetic relationships of 15 species of the obscura group of Drosophila were analysed by use of one- and two-dimensional electrophoresis. Genetic distances based on two-dimensional data are five times smaller than those based on native proteins. From the data, it is proposed that the species radiation of the obscura group happened in two evolutionary bursts, the first one giving rise to at least four palearctic proto-lineages (bifasciata, obscura (including D. subsilvestris), subobscura, and microlabis) and one or two proto-nearctic lineages (affinis, pseudoobscura), and the second, more recent burst giving rise to the current speciation within lineages. Zusammenfassung Phylogenie der Arten der Drosophila obscura-Gruppe abgeleitet von ein- und zweidimensionaler Protein-Elektrophorese Die phylogenetischen Verwandtschaftsbeziehungen von 15 Arten der obscura-Gruppe der Gattung Drosophila wurden mit Hilfe von ein-und zweidimensionaler Elektrophorese von Proteinen untersucht. Die genetische Distanzen, die aus den Ergebnissen der zweidimensionalen Elektrophoresen ermittelt wurden, waren funfmal kleiner als solche, die von nativen Proteinen kommen. Aufgrund der Untersuchungsergebnisse wird angenommen, das die Radiation der Arten der obscura-Gruppe in zwei evolutiven Schuben erfolgt sei; der erste Schub hatte zu zumindest vier palaerktischen (bifasciata, obscura mit D. subsilvestris, subobscura und microlabis) und zwei proto-ne arktischen Linien (affinis, pseudoobscura) gefuhrt. In einem zweiten Schub waren dann die endgultigen rezenten Arten entstanden.

We investigated the function and expression of voltage-gated Na+ channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca2+] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50–60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca2+]i, which were abolished by treatment with 0.1 μM TTX. Using end-point and real-time quantitative RT-PCR,...

The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat.

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK1 receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RT-PCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin NK1 receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK2 receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK3 receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK(2) receptor splice variant named beta isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the alpha isoform. Moreover, neurokinin A was unable to inhibit the [(3)H]SR48968 binding to the beta isoform up to microM concentrations. In cells expressing the human tachykinin NK(2) receptor beta isoform, contrary to those expressing the alpha isoform, natural or selective tachykinin receptor agonists (1 microM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca(2+)](i). The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca(2+)](i) increase in cells expressing the alpha or beta isoform of the human tachykinin NK(2) receptor. The present data indicate that the human tachykinin NK(2) receptor beta isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK(2) receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands.

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as excitatory neurotransmitters. Currently, the concept that tachykinins act exclusively as neuropeptides is being challenged, since the best known members of the family, substance P, neurokinin A and neurokinin B, are also present in non-neuronal cells and in non-innervated tissues. Moreover, the recently cloned mammalian tachykinins hemokinin-1 and endokinins are primarily expressed in non-neuronal cells, suggesting a widespread distribution and important role for these peptides as intercellular signaling molecules. The biological actions of tachykinins are mediated through three types of receptors denoted NK(1), NK(2) and NK(3) that belong to the family of G protein-coupled receptors. The identification of additional tachykinins has reopened the debate of whether more tachykinin receptors exist. In this review, we summarize the current knowledge of tachykinins and their receptors.

Tachykinins may be involved in reproduction. A reverse transcription-polymerase chain reaction assay was used to analyze the expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. The preprotachykinin (PPT) genes, PPT-A, PPT-B and PPT-C, that encode substance P/ neurokinin A, neurokinin B, and hemokinin-1, respectively, and the genes that encode the tachykinin NK1 ,N K 2, and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of neprilysin (NEP), the main enzyme involved in tachykinin metabolism, was also expressed in the uterus. Isolated cumulus granulosa cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of the tachykinin NK1 and NK2 receptors. Mouse oocytes expressed PPT-A and -B mRNA transcripts. A low expression of the three tachykinin receptors was observed but PPT-C and NEP were undetectable. Two- and 8- to 16-cell mouse embryos expressed only a low-abundance transcript corresponding to the NK1 receptor. However, the mRNAs of PPT-B, PPT-C and NEP appeared in blastocyst-stage embryos. A low-abundance transcript corresponding to the NK2 receptor was the only target gene detected in mice sperm. Female mice or rats treated neonatally with capsaicin showed a reduced fertility. A reduction in litter size was observed in female rats treated in vivo with the tachykinin NK3 receptor antagonist SR 142801. These data show that tachykinins of both neuronal and nonneuronal origin are differentially expressed in various types of reproductive cells and may play a role in female reproductive function. embryo, female reproductive tract, fertilization, neuropeptides, polypeptide receptors

Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell-surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. The neurokinin B (NKB) precursor PPT-B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT-A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT-C, the gene encoding the novel tachykinin peptide hemokinin-1 (HK-1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. Tachykinin NK2 receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK1 receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK3 receptor was present only in tissues from nonpregnant women. The mRNA expression of the tachykinin-degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA≫SP≥NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. The tachykinin NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–l0) was approximately equipotent with NKA, but the tachykinin NK1 and NK3 receptor-selective agonists [Sar9Met(O2)11]SP and [MePhe7]NKB were ineffective in the myometrium from nonpregnant women. The uterotonic effects of [Lys5MeLeu9Nle10]NKA(4–10) were antagonized by the tachykinin NK2 receptor-selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys5MeLeu9Nle10]NKA(4–10). These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK2 receptors in the regulation of myometrial contraction. Keywords: Human myometrium, [Lys5MeLeu9Nle10]NKA(4–10), NK1 and NK2 receptors, neprilysin, neurokinin A, neurokinin B, substance P, tachykinins, uterine contractions, SR48968 Introduction Tachykinins are members of a family of neuropeptides that includes substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). Their biological actions are mediated through three receptors belonging to the family of G protein-coupled receptors (GPCR), denoted NK1, NK2 and NK3, that have highest affinity for SP, NKA and NKB, respectively (Henry, 1986; Regoli et al., 1994a,1994b; Lecci et al., 2000). SP and NKA, but not NKB, are present within capsaicin-sensitive sensory nerves in the periphery and are locally released by a number of physical and chemical stimuli (Holzer, 1988; Maggi & Meli, 1988). SP and a new member of the tachykinin family, hemokinin-1 (HK-1), that has recently been cloned in the mouse (Zhang et al., 2000), rat and human (Kurtz et al., 2002) are also produced in non-neuronal cells (Joos & Pauwels, 2000; Zhang et al., 2000). This distribution suggests an important role for tachykinins in intercellular communication. Previous studies from other laboratories and from ours indicate a role of tachykinins in the regulation of uterine function. Early immunohistochemical studies showed the presence of tachykinin-immunoreactive nerves supplying the uteri of several species including the mouse, rat, guinea-pig and human (Alm et al., 1978; Huang et al., 1984; Traurig et al., 1984; Papka et al., 1985; Samuelson et al., 1985; Heinrich et al., 1986; Alm & Lundberg, 1988; Reinecke et al., 1989; Traurig et al., 1991; Papka & Shew, 1994). The association of these nerves with smooth muscle indicates that tachykinins released from their peripheral terminals may influence uterine contractility. Preprotachykinin-A (PPT-A), the precursor of SP and NKA, is also expressed in non-neuronal cells such as monocytes and macrophages (Ho et al., 1997); these can be associated with the mammalian uterus (Cocchiara et al., 1997). SP has also recently been reported to participate in stress-induced abortion in the mouse and possibly in the human (Arck et al., 1995; Markert et al., 1997; Marx et al., 1999; Joachim et al., 2001). NKB has recently been reported to be expressed in the human placenta (Page et al., 2000) and the gene that encodes it, preprotachykinin-B, is expressed in the uterus of the rat and the human (Pinto et al., 2001;2002). NKB has been implicated in the symptoms associated with pre-eclampsia (Page et al., 2000). The major tachykinin-degrading enzyme neprilysin (NEP) (Matsas et al., 1983;1984; Hooper et al., 1985; Hooper & Turner, 1985) is also present in the uterus (Ottlecz et al., 1991; Head et al., 1993; Riley et al., 1995). This intrauterine distribution supports the view that tachykinins may be important intercellular signalling molecules within the female reproductive tract (Pinto et al., 2001; 2002). To date, the majority of molecular (Pinto et al., 1999; Candenas et al., 2001), immunohistochemical (Traurig et al., 1984;1991; Papka et al., 1985) and functional (Fisher et al., 1993; Pennefather et al., 1993b; Fisher & Pennefather, 1997; Magraner et al., 1998; Patak et al., 2000a) studies of the uterine distribution of tachykinins, tachykinin receptors and/or degradation of tachykinins have been conducted using the rat. It is now emerging, however, that there are species (Patak et al., 2000a,2000b;2002a) as well as hormonal- and pregnancy-related differences in tachykinin expression and actions in the mammalian myometrium (Pinto et al., 1999;2001;2002; Candenas et al., 2001). We have previously described the uterotonic effects of tachykinins on myometrium of late-pregnant women (Patak et al., 2000b). However, no systematic molecular studies of the expression of tachykinin precursor genes, neither of tachykinin receptors nor of NEP in either pregnant or nonpregnant human uterus, have been undertaken to date. Moreover, despite early reports that SP and eledoisin elicit contractions of the nonpregnant human uterus (Molina & Zappia, 1976; Ottesen et al., 1983), there have been no systematic functional studies of the actions of tachykinin peptides on myometrium from nonpregnant women. For these reasons, the aim of the present study was to examine further the tachykinins, their receptors and NEP on the human myometrium. We have therefore performed molecular studies using myometrium from both nonpregnant and pregnant women, and functional studies to characterize the effects of tachykinins on uterine contraction of tissue from nonpregnant women. A preliminary account of some of the results of this study has been presented previously (Patak et al., 2002b; Pinto et al., 2002).

We investigated whether fenoterol was able to enhance contractile responsiveness to neurokinin A (NKA) on the guinea-pig isolated trachea. We then studied the effects of two inhibitors of nuclear factor kappa B (NFkappaB), gliotoxin and pyrrolidine dithiocarbamate, and of the tachykinin NK(1), NK(2) and NK(3) receptor antagonists, SR 140333, SR 48968 and SR 142801 and determined whether tachykinin receptor gene expression was up-regulated in the trachea after exposure to fenoterol. Fenoterol (0.1 microM, 15 h, 21 degrees C) induced an increased contractile response to NKA (mean of difference in maximal tension between control and fenoterol +/- S.E.M; +0.47 +/- 0.14 g, n = 26, P0.01). This hyperresponsiveness was strongly reduced by co-incubation with gliotoxin (0.1 microg/ml) or pyrrolidine dithiocarbamate (0.1 mM) and abolished by SR 140333 (0.1 microM) and SR 142801 (0.1 microM). SR 48968 (0.1 microM) diminished the tracheal contractility to NKA but failed to reduce the hyperreactivity induced by fenoterol. Tachykinin NK(1) receptor (NK(1)R), NK(2) receptor (NK(2)R) and NK(3) receptor (NK(3)R) gene expression was analyzed by semiquantitative RT-PCR. Compared to control tissues, NK(1)R and NK(2)R mRNA expression was increased by about 1.6-fold and 1.4-fold, respectively, in tissues treated with fenoterol. We were unable to detect the presence of NK(3)R mRNA in the guinea-pig trachea. In conclusion, fenoterol induces tracheal hyperresponsiveness to NKA and an up-regulation of NK(1)R and NK(2)R gene expression. The hyperresponsiveness implicates the NFkappaB pathway and is abolished by tachykinin NK(1) (SR 140333) and NK(3) (SR 142801) receptor antagonists.

The complete A + T-rich region of mitochondrial DNA (mtDNA) has been cloned and sequenced in the species of the Drosophila subobscura subgroup D. subobscura, D. madeirensis and D. guanche. Comparative analysis of these sequences with others already published has identified new sequence motifs that are conserved in Drosophila and other insects. A putative bi-directional promoter and a stop signal are proposed to be involved in the primary mtDNA strand replication of Drosophila. This region strongly resolves relationships of the species included in a phylogenetic analysis, both for closely related species and also at deeper phylogenetic levels when only the left and central domains are taken into account.

In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK(1) receptor (NK(1)R), the tachykinin NK(2) receptor (NK(2)R), and the tachykinin NK(3) receptor (NK(3)R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK(1)R, the NK(2)R, or the NK(3)R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK(1)R and NK(3)R varied along the course of pregnancy and at postpartum. Uterine NK(2)R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK(2) receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 microM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.

Although the Canary Islands were settled by humans, possibly of Berber origin, as late as 2500 years ago, the precise course and numbers of early migrations to the archipelago remain controversial. We have therefore analysed mtDNA variation (HVS-I as well as selected RFLP sites) in 300 individuals from the seven Canary Islands. The distribution and variation across the islands in a specific mtDNA clade of Northwest African ancestry suggest that there was one dominant initial settlement process that affected all the islands, from east to west. This indicates that a certain genetic affinity of present-day Canary Islanders to Northwest African Berbers mainly stems from the autochthonous population rather than slaves captured on the neighbouring African coast. The slave trade after the European conquest left measurable, though minor, traces in the mtDNA pool of the Canary Islands, which in its majority testifies to the European immigration.

Tachykinin neuropeptides, such as substance P, are localized to a population of sensory fibers that innervate the mammalian female reproductive tract. In the present study, we have characterized tachykinin NK1 receptor (NK1R), NK2 receptor (NK2R), and NK3 receptor (NK3R) gene expression by semiquantitative RT-PCR in uteri from ovariectomized rats and studied their regulation in response to 17beta-estradiol (E2), progesterone (P4), or a combination of both. In addition, we analyzed the expression and regulation of the neutral endopeptidase 24.11 (NEP), the most important enzyme involved in tachykinin degradation in the rat uterus. In uteri from control (olive oil-treated) rats, RT-PCR assays revealed single bands corresponding to the expected product sizes encoding complementary DNA for NK1R (232 bp), NK2R (491 bp), NK3R (325 bp), and NEP (221 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. Compared with control rats, NK1R messenger RNA (mRNA) was increased by 2-fold in uteri from rats treated with E2, was decreased by 3.3-fold in rats treated with P4, and was decreased by 1.8-fold in rats treated with both E2 and P4. Uterine NK2R mRNA levels were not altered by any steroid treatment. E2 treatment decreased by 15-fold NK3R mRNA. P4 was without effect if administered alone and did not influence the E2-induced decrease in NK3R mRNA. NEP mRNA levels were about 4-fold lower in E2-treated than in P4-treated rats. Functional studies were carried out in uteri from E2- or P4-treated ovariectomized rats to characterize the contractile response evoked by the selective tachykinin receptor agonists [Sar9Met(O2)11]substance P (NK1R selective), [Nle10]NKA-(4-10) (NK2R selective), and [MePhe7]NKB (NK3R selective) in the presence of the NEP inhibitor phosphoramidon (1 microM). A marked correlation was observed between the magnitude of the contractile response to each agonist and the level of expression determined by RT-PCR for each tachykinin receptor. The present findings show that tachykinin NK1R, NK2R, NK3R, and NEP are expressed in the rat uterus and that ovarian steroids differentially regulate their expression.

The aim of our study was to characterize the tachykinin receptor population in the oestrogen-primed rat uterus. For this purpose, we investigated the receptor type(s) responsible for tachykinin-induced contraction of longitudinally-arranged smooth muscle layer. The effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and several of their analogues with well-defined selectivities for tachykinin NK1, NK2 and NK3 receptors were studied and their inhibition by the selective nonpeptide tachykinin receptor antagonists (S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR 140333, NK1-selective), (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968, NK2-selective) and (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)- 4-phenylpiperidin- 4-yl)-N- methylacetamide (SR 142801, NK3-selective) was evaluated. Additionally, expression of tachykinin receptor mRNA was examined by using the reverse transcription-polymerase chain reaction (RT-PCR). SP, NKA, [Nle10]-NKA(4-10), the analogue with selectivity at the tachykinin NK2 receptor type, and NKB elicited concentration-dependent contractions of the rat uterus. The pD2 values were 5.95±0.19; 6.73±0.21; 7.53±0.12 and 5.76±0.21, respectively. The selective agonist for the tachykinin NK1 receptor [Sar9Met(O2)11]-SP produced a small phasic response in the nanomolar concentration range. The selective tachykinin NK3 receptor agonist [MePhe7]-NKB failed to induce any significant contraction. In the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM), the log concentration-response curves to exogenous tachykinins and their analogues were shifted significantly leftwards. The pD2 values were 6.12±0.10, 8.04±0.07, 7.89±0.03 and 6.59±0.07 for SP, NKA, [Nle10]-NKA(4-10) and NKB, respectively. In the presence of phosphoramidon (1 μM), [Sar9Met(O2)11]-SP (1 nM–0.3 μM) induced concentration-dependent contractions of increasing amplitude when only one concentration of drug was applied to each uterine strip and the pD2 value was 7.61±0.89. [MePhe7]-NKB induced small, inconsistent contractions and, therefore, a pD2 value could not be calculated. In experiments performed in the presence of phosphoramidon (1 μM), SR 48968 (3 nM–0.1 μM) caused parallel and rightward shifts in the log concentration-response curves of NKA. The calculated pKB value was 9.16±0.08 and the slope of the Schild regression was 1.28±0.24. SR 48968 (0.1 μM) also antagonized responses to SP with an apparent pKB value of 7.63±0.13. SR 48968 (0.1 μM) inhibited contractions elicited by NKB (1 nM–3 μM) and [Nle10]-NKA(4–10) (0.1 nM–3 μM) but had no effect on the response evoked by [Sar9Met(O2)11]-SP (0.1 μM). SR 140333 (0.1 μM) inhibited responses to SP with an apparent pKB value of 7.19±0.22. This compound did not significantly affect responses to NKA, [Nle10]-NKA(4-10) and NKB, but suppressed [Sar9Met(O2)11]-SP (0.1 μM)-induced contraction. SR 142801 (0.1 μM) had no effect on responses to natural tachykinins or their analogues. Total RNA was extracted from some of the uteri used in functional studies. RT-PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for tachykinin NK1 (587 base pairs) and NK2 receptors (491 base pairs) (n=6 different animals). A very low abundance transcript corresponding to the 325 base pairs product expected for the tachykinin NK3 receptor was detected. The present data show that functionally active tachykinin NK1 and NK2 receptors are expressed in the oestrogen-primed rat uterus. The NK2 receptor type seems to be the most important one involved in the contractile responses elicited by tachykinins. NK3 receptors are present in trace amounts and seem not to be involved in tachykinin-induced contractions. British Journal of Pharmacology (1998) 123, 259–268; doi:10.1038/sj.bjp.0701613

[Background] Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development., [Methods] In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival., [Results] GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs., [Conclusions] This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms., This work was supported by grants from the Basque Government (IT8-11/13), the University of the Basque Country UPV/EHU (UFI 11/44, EHUA 12/15) and the Gangoiti Barrera Foundation.

Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium?In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated.The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression.Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro.RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples.Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349).This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells.The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice.

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum and N. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical to N. tabacum, two almost identical to N. plumbaginifolia, and a fourth similar to this parental species, but with some N. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37% N. tabacum DNA contribution, respectively.

The genetic polymorphism of eight red cell enzymes was analyzed in population samples from the Northwest African Continent and from the South of Spain in order to study their genetic relationships with the Canarian population. The Moroccan, Berber and Spanish populations, although geographically more distant from the Canary Islands than the Saharan and Mauritanian ones, are genetically more closely related to the Canarian population. The glucose-6-phosphate dehydrogenase Gc allele earlier found only in the Canary Islands was detected in the Berber sample. The Spanish, Berber and African Black contributions to the Canarian hybrid population was estimated to 70, 20 and 10%, respectively.

Introduction Lung inflation may have deleterious effects on the alveoli during mechanical ventilation. However, the consequences of stretch during excessive lung inflation on basal tone and responsiveness of human bronchi are unknown. This study was undertaken to devise an experimental model of acute mechanical stretch in isolated human bronchi and to investigate its effect on airway tone and responsiveness. Methods Bronchi were removed from 48 thoracic surgery patients. After preparation and equilibration in an organ bath, bronchial rings were stretched for 5 min using a force (2.5 × basal tone) that corresponded to airway-inflation pressure > 30 cm H2O. The consequences of stretch were examined by using functional experiments, analysis of organ-bath fluid, and ribonucleic acid (RNA) isolation from tissue samples. Results Following removal of the applied force the airways immediately developed an increase in basal tone (P < 0.0001 vs. paired controls) that was sustained and it did so without significantly increasing responsiveness to acetylcholine. The spontaneous tone was abolished with a Rho-kinase inhibitor and epithelium removal, a leukotriene antagonist or nitric oxide synthase inhibitors reduced it, whereas indomethacin, sensory nerve inhibitors or antagonists for muscarinic, endothelin and histamine receptors had no effect. Stretch enhanced leukotriene-E4 production during the immediate spontaneous contraction of human bronchi (P < 0.05). Moreover, stretch up-regulated the early mRNA expression of genes involved in wingless-type mouse mammary tumor virus integration-site family (WNT)-signaling and Rho-kinase pathways. Conclusions Stretching human bronchi for only 5 min induces epithelial leukotriene release via nitric oxide synthase activation and provokes a myogenic response dependent on Rho-kinase and WNT-signaling pathways. From a clinical perspective, these findings highlight the response of human airway to acute mechanical stress during excessive pulmonary inflation., This research was supported by a grant (CTQ2007-61024) from Ministerio de Educación y Ciencia (Spain). A 70% of this grant is contributed by FEDER funds.

SUMMARY Nuclear hybrids have been obtained by fusion of mesophyll protoplasts of Nicotiana plumbaginifolia and x-irradiated or iodoacetate-treated mesophyll protoplasts of a kanamycin-resistant line of N. tabacum. The effect of irradiation on the recovery of asymmetric hybrids was evaluated by analysis of their morphology, fertility, chromosome number, isozyme patterns, restriction patterns in their organelle DNAs, and presence of the kanamycin-resistance gene. The results presented in this paper show that x-ray irradiation leads to a significant reduction in the amount of N. tabacum genome present in the hybrids and demonstrates, once more, the power of this technique to induce directional loss of genomic traits of the irradiated parent.

Several studies in cell cultures and in animal models have demonstrated that cannabinoids have important antitumoral properties. Because many of these effects are mediated through cannabinoid (CB) receptors CB1 and CB2, the study of their expression in human neoplasms has become of great interest in recent years. Fresh and formalin-fixed tissue samples of 20 consecutive clear cell renal cell carcinomas (CCRCCs) were collected prospectively and analyzed for the expression of both CB receptors by using RT-PCR, Western blot (WB), and immunohistochemical techniques. RT-PCR assays demonstrated the expression of mRNA encoding the CB1 in tumor tissue and in adjacent non-neoplastic kidney. Conversely, WB and IHC revealed a marked downregulation of CB1 protein in tumor tissue; CB2 was not expressed. The obtained data suggest a possible implication of the endocannabinoid system in renal carcinogenesis. A posttranscriptional downregulation of CB1 and the absence of expression of CB2 characterize CCRCC. (J Histochem Cytochem 58:1129–1134, 2010)

Aminopeptidase N (APN/CD13) and neutral endopeptidase (NEP/CD10) are enzymes present in human sperm cells and involved in regulation of sperm motility of noncapacitated spermatozoa. We investigated the involvement of APN/CD13 and NEP/CD10 in motility and in kinematic parameters of human capacitated spermatozoa. Sperm cells isolated by a discontinuous Percoll gradient (40%–80%) followed up by swim-up techniques were incubated with the APN/CD13-specific inhibitor, leuhistin (100 μmol L−1), and the NEP/CD10-specific inhibitor, thiorphan (1 μmol L−1). The complete inhibition of both APN/CD13 and NEP/CD10 improved sperm motility. Spermatozoa incubated with the APN/CD13-specific inhibitor leuhistin showed asymmetrical trajectories, whereas sperm trajectories were more regular after treatment with the NEP/CD10-specific inhibitor thiorphan. In conclusion, APN/CD13 and NEP/CD10 modulate the motility of capacitated spermatozoa, although each of the enzymes seems to participate in the control of different aspects of sperm motility. Therefore, both inhibitors may be useful for sperm activation at different functional stages of spermatozoa.

The presence of CB1 and CB2 cannabinoid receptors and their physiological role in the kidney has been described in animal models but not in humans. Our aim in this study was to evaluate the presence of these receptors in human kidney, adult and fetal. For this purpose, RT-PCR, western-blot and immunohisto-chemical assays were performed. RT-PCR confirmed the presence of CB1 receptor mRNA receptor and the absence of the CB2 receptor mRNA in adult and fetal kidney. Western-blot and immunohistochemical assays revealed the presence of the CB1 cannabinoid receptor protein, which displayed a similar distribution in fetal and adult kidneys. Proximal and distal convoluted tubule cells and intercalated cells in the collecting ducts showed marked positivity. Conversely, the CB2 cannabinoid receptor protein was consistently negative in all cases. Our data suggest a possible implication of the endocannabinoid system in the physiology and development of the human kidney.

Background Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO). Methods Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining. Results The activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors). Conclusions These results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.