EPI-peptide designer: a tool for designing peptide ligand libraries based on epitope-paratope interactions.

MOTIVATION: Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. RESULTS: To identify the interacting residues in a given antibody-antigen (Ab-Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab-Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. AVAILABILITY AND IMPLEMENTATION: All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ CONTACT: liza@icb.ufmg.br SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Immunosuppressive PAS-1 is an excretory/secretory protein released by larval and adult worms of the ascarid nematode Ascaris suum.

Helminths use several strategies to evade and/or modify the host immune response, including suppression or inactivation of the host antigen-specific response. Several helminth immunomodulatory molecules have been identified. Our studies have focused on immunosuppression induced by the roundworm Ascaris suum and an A. suum-derived protein named protein 1 from A. suum (PAS-1). Here we assessed whether PAS-1 is an excretory/secretory (E/S) protein and whether it can suppress lipopolysaccharide-induced inflammation. Larvae from infective eggs were cultured in unsupplemented Dulbecco's modified Eagle medium (DMEM) for 2 weeks. PAS-1 was then measured in the culture supernatants and in adult A. suum body fluid at different time points by enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody MAIP-1. Secreted PAS-1 was detected in both larval culture supernatant and adult body fluid. It suppressed lipopolysaccharide (LPS)-induced leucocyte migration and pro-inflammatory cytokine production, and stimulated interleukin (IL)-10 secretion, indicating that larval and adult secreted PAS-1 suppresses inflammation in this model. Moreover, the anti-inflammatory activity of PAS-1 was abolished by treatment with MAIP-1, a PAS-1-specific monoclonal antibody, confirming the crucial role of PAS-1 in suppressing LPS-induced inflammation. These findings demonstrate that PAS-1 is an E/S protein with anti-inflammatory properties likely to be attributable to IL-10 production.

Growth and production of cowpea beans under potassium doses in soil of cerrado in Amapá, Brazil.

The cowpea bean [Vigna unguiculata (L.) Walp.], a legume of great socioeconomic importance, it was previously cultivated exclusively for subsistence and commercial purposes, especially in the North and Northeast regions. This crop has a low production cost and high nutritional value, in addition to a high potential for productivity growth and expansion to other regions. The objective of this work was to evaluate parameters of growth and production in cowpea culture, as a function of potassium fertilization in soil of the cerrado of Amapá. The parameters of growth and production of the cowpea culture were evaluated, as a function of potassium fertilization in the soil of the cerrado of Amapá. The experiment was conducted in a greenhouse, using a completely randomized experimental design, with four replications, in a 5x2 factorial scheme, totaling 40 experimental units, which were composed of plastic pots containing 7 dm3 of soil collected from the arable layer (0-20 cm ) of a typical Hyperdystrophic Yellow Argisol, with a sandy clay loam texture, in a cerrado area in the municipality of Porto Grande-AP. The factors consisted of the control treatment (without K), four doses of K (45, 90, 135 and 180 kg ha-1) in the form of potassium chloride, and two cowpea cultivars (Pretinho and BRS Tumucumaque). The cultivar BRS Tumucumaque shows better growth and production of cowpea plants. Doses of 90 kg ha-1 provided greater height (98.75 cm) and stem diameter (10.0 mm). As for production, the dose of 135 kg ha-1 caused greater grain weight gain (5.25 g) and dry mass of pods (13.92 g), and the doses of 90 and 180 kg ha-1 induced greater number and length of pods (3.16 pods) respectively. These results show better responsiveness of the BRS Tumucumaque cultivar at doses of 90, 135 and 180 kg ha-1 in the type of soil where the study was conducted.

Mast cell degranulation contributes to susceptibility to Leishmania major.

Leishmaniasis causes high morbidity and mortality in tropical and subtropical areas. Mast cells can be activated by Leishmania or Leishmania products in vitro and in vivo. Several innate immunity mediators, including some released by mast cells, play roles in the outcome of the disease. In this study, we examined whether pharmacological inactivation of mast cells before infection with L. major interferes with the progressive disease in BALB/c mice. The results show that, when mast cells are degranulated before challenge with L. major, susceptible mice become more resistant to infection, as measured by decrease of lesion size and lower parasite loads. Mast cell degranulation reduced IL-4 production. Moreover, mast cells degranulation enhanced mRNA expression for IFN-gamma, inducible nitric oxide, CCL2 and CCL5 in response to infection. Mast cell degranulation also decreased parasite loads in IL-4 KO animals, indicating that mediators other than IL-4 are involved in susceptibility in vivo. Taken together, our results disclose a role for mast cells in the induction of susceptibility to infection. This work contributes to a better understanding of the role of mast cells in Leishmania infection, and suggests a new field of study for strategies to contain the parasite, restricting its dissemination.

Trans-enamel and trans-dentinal cytotoxic effects of a 35% H2O2 bleaching gel on cultured odontoblast cell lines after consecutive applications.

AIM: To evaluate the trans-enamel and trans-dentinal cytotoxic effects of a 35% H(2)O(2) bleaching gel on an odontoblast-like cell lines (MDPC-23) after consecutive applications. METHODOLOGY: Fifteen enamel/dentine discs were obtained from bovine central incisor teeth and placed individually in artificial pulp chambers. Three groups (n = 5 discs) were formed according to the following enamel treatments: G1: 35% H(2)O(2) bleaching gel (15 min); G2: 35% H(2)O(2) bleaching gel (15 min) + halogen light (20 s); G3: control (no treatment). After repeating the treatments three consecutive times, the extracts (culture medium + gel components that had diffused through enamel/dentine discs) in contact with the dentine were collected and applied to previously cultured MDPC-23 cells (50 000 cells cm(-2)) for 24 h. Cell metabolism was evaluated by the MTT assay and data were analysed statistically (alpha = 5%; Kruskal-Wallis and Mann-Whitney U-test). Cell morphology was analysed by scanning electron microscopy. RESULTS: Cell metabolism decreased by 92.03% and 82.47% in G1 and G2 respectively. G1 and G2 differed significantly (P < 0.05) from G3. Regardless of halogen light activation, the application of the bleaching gel on the cultured odontoblast-like cells caused significantly more severe cytotoxic effects than those observed in the nontreated control group. In addition, significant morphological cell alterations were observed in G1 and G2. CONCLUSION: After three consecutive applications of a 35% H(2)O(2) bleaching agent, the diffusion of the gel components through enamel and dentine caused severe toxic effects to cultured pulp cells.

Chemotherapy and the future: microdialysis as a local administration technique.

Chemotherapy has an important role in cancer treatment. Although there have been developments and good results, chemotherapy still has many limitations mainly due to its toxicity and to the resistance mechanisms of tumour cells. Therefore, besides other important improvements in chemotherapy agents and indications, recent researches have focused on the development of locoregional administration techniques, with which therapeutic weapons can reach the tumour with a higher concentration and fewer side-effects. At present, local chemotherapy includes delivery systems or prodrug strategies, arterial infusions, intraperitoneal administration and aerosolised agents. We will describe a new local cancer chemotherapy method, using microdyalisis procedures, which may revolutionise the actual tumour management because of the higher effectiveness and the absence of side-effects. Finally, the applications and limitations of this technique will be considered.

Breast cancer patients with micrometastases in sentinel lymph nodes: differences considering additional metastatic lymph nodes.

AIMS: Characterization of breast cancer patients with micrometastases in sentinel lymph node (SLN) and establish differences between micrometastatic breast cancers with additional metastatic lymph nodes (LNS) versus no other lymph node invasion. METHODS: Analysis of 30 breast cancers, N1mi or pN0(i+), diagnosed and treated in our department from July 2000 to July 2008. RESULTS: Micrometastases in SLNs were found in 30 patients. Complete axillary dissection revealed other metastatic LNs in 24%. Concerning breast cancers with additional LN invasion versus no other LN invasion, tumors located in the superior-external quadrant were more frequent in the former group. Other characteristics as clinical presentation, histological subtype, focality, cytonuclear grade, hormone receptors and Her2 expression were not significantly different in either group. Regarding SLN invasion, the presence of at least two micrometastatic foci were significantly more relevant in patients with other metastatic LN invasion (p < 0.01). Micrometastases diagnosed only after immunohistochemistry (IHC) were exclusively found in patients without other LN invasion, reaching statistical significance (p < 0.05). CONCLUSIONS: Complete axillary dissection revealed additional LN invasion in 24% of patients with micrometastases in the SLN. Tumors with additional LN invasion were more frequently found in the superior external quadrant and SLNs harbored at least two micrometastatic foci. Micrometastases diagnosed exclusively by IHC techniques were more relevant in cases without additional lymph node invasion.

What is the diagnostic value of nipple discharge cytology and galactography in detecting duct pathology?

The present study aimed to evaluate the diagnostic value of nipple discharge (ND) cytology and galactography. Ninety-four patients submitted to duct excision, representing a total of 98 duct excisions, were retrospectively analyzed from January 1997 to May 2007. Histology of ducts excised revealed 35% duct ectasia (DE), 31% duct papilloma (DP), 20% potential malignant transforming lesions (PMTL), 6% breast cancer (BC), 1% adenoma and 6% normal breast tissue. Cytology had a sensibility and specificity in detecting duct pathology of, respectively, 40% and 61.3%, a positive predictive value (PPV) of 53.8% and a negative predictive value (NPV) of 47.5%. Concerning malignant and PMTL, cytology had a sensibility and specificity of, respectively, 46.2% and 62.3%, a PPV of 25% and a NPV of 82.5%. Breast cancer was never suggested by positive cytology. Galactography had a sensibility and specificity in detecting duct pathology of, respectively, 77.4% and 29.2%, a PPV of 58.5% and a NPV of 50%. For malignant and PMTL, galactography had a sensibility and specificity of, respectively, 80% and 26.7%, a PPV of 19.5% and a NPV of 85.7%. However, galactography never suggested a diagnosis of cancer. Cytology and galactography performed together in 49% showed a low Kappa Index (KI < 1), allowing us to conclude that cytology and galactography detect different pathologies. Cytology showed a poor performance in predicting histological diagnosis in face of ND. Galactography had a good sensibility in excluding malignant lesions and PMTL. Galactography was significantly more sensitive for duct pathology but cytology was more specific for malignant lesions and PMTL.

Parameters for assessing the aquatic environmental impact of cosmetic products.

The cosmetic industry's growing concern about the impact of its supply chain on the environment, sustainability of raw materials, and biodiversity increases the need to ensure that the final product has a lower environmental impact. The objective of this review is to summarize and compare the information available from international organizations and legislation regarding the main criteria used to assess raw materials for aquatic toxicity, as well as the most suitable alternative methods for obtaining assessment parameters. Using the literature available in databases, a review of the scientific literature and international legislation, this work discusses and compares the parameters established by international organizations such as the Environmental Protection Agency (EPA) and Cradle to Cradle (C2C), as well as European legislation, namely, European Regulation 1272/2008, for assessing environmental impact. Defining the ecotoxicity parameters of the main classes of raw materials in rinse-off cosmetic products can enable the development of products that are more environmentally sustainable, prioritizing substances with less environmental impact.

Sinonasal Melanoma: A Case Report and Literature Review.

Purpose. Sinonasal malignant mucosal melanoma is a rare, aggressive tumour. Nasal obstruction and epistaxis are the most commonly reported symptoms, although symptomatology may develop late and be nonspecific, which tends to delay diagnosis, resulting in a poorer prognosis. Case Report. This report describes a 64-year-old male patient with nasal obstruction and epistaxis. Computed tomography of the facial sinuses revealed a large lesion in the right nasal cavity, with infiltration into the left cavity, ethmoidal cells, and erosion of the cribriform plate. Initial incisional biopsy revealed an undifferentiated carcinoma of the right maxillary sinus, staged as T4aN0M0. Induction chemotherapy was initiated with cisplatin and etoposide. Response to treatment was complete. The patient was then submitted to radiotherapy with concomitant cisplatin. Immunochemical analysis revealed positivity for vimentin, S100, and HMB-45 (human melanoma black 45), a result compatible with a diagnosis of malignant melanoma. Discussion. Due to the rarity of the tumour and the patient's complete response to chemotherapy and since no blackened lesion had been found at the previous exam, treatment was continued as planned. The patient remains healthy, with no metastasis or recurrence. He is currently being monitored by the clinical oncology team.

Soluble VCAM-1 and E-selectin in breast cancer: relationship with staging and with the detection of circulating cancer cells.

In breast cancer, the correct evaluation of cancer dissemination is essential to establish prognosis and treatment choices. This study analyses the relationship between circulating levels of soluble VCAM-1 and E-selectin and the presence of circulating cancer cells in breast cancer patients. Plasma levels of VCAM-1 and E-selectin were measured by enzyme-linked immunosorbent assay (ELISA). The presence of circulating cancer cells was diagnosed using a RT nested-PCR assay detecting the cancer specific transcript, epidermal growth factor receptor variant III (EGFRvIII) mRNA. Blood samples were collected from 64 patients divided in three groups: group A of 11 women selected for neoadjuvant chemotherapy; group B of 13 women with metastatic disease and group C, with 40 women having completed their treatment at least one year ago and with no evidence of relapse. The mutant transcript was detected in 45.5% of patients from group A, in 61.5% of patients from group B and in none of the group C patients. For both VCAM-1 and E-selectin, plasma levels increased with disease staging and with the presence of EGFRvIII mRNAin peripheral blood. The differences were statistically significant (p < 0.025) when group C was compared with all patients from group B, with patients from group B with EGFRvIII positive results or with all patients with EGFRvIII positive results. Increased plasma levels of VCAM-1 and E-selectin are associated with advanced stage of breast cancer and with the presence of circulating cancer cells. The combined analysis of these parameters may contribute to a more accurate evaluation of cancer dissemination.

Endometrial adenocarcinoma after endometrial ablation. A case report.

The authors present a case of endometrial adenocarcinoma after endometrial ablation, emphasizing the importance of close surveillance of these patients, patient selection and education. Even patients with none of the risk factors for endometrial cancer or contraindications to endometrial ablation should be checked carefully.

Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10µg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

Primary breast lymphoma.

OBJECTIVES: Retrospective evaluation of the clinical behavior, treatment and prognosis in five cases of primary breast lymphoma. METHODS: From 1999 to 2003, five patients with primary breast lymphoma were diagnosed in our department. RESULTS: Primary breast lymphoma (PBL) was diagnosed in five patients, whose median age was 63.4 (41-79) years. In four out of five patients, a diagnosis of lymphoma was made after the evaluation of a palpable breast mass measuring 1.5 to 6 cm. All of them were classified as non-Hodgkin's B cell lymphomas and three of five cases were diffuse large cell lymphomas. All patients were submitted to chemotherapy; in only one patient was surgery performed. CONCLUSIONS: A relatively high rate of PBL was observed in our department compared with other oncology centers. Beyond its scarce appearance, PBL is very difficult to distinguish from primary breast carcinoma. Histology remains the major diagnostic tool.

Transdentinal cell photobiomodulation using different wavelengths.

OBJECTIVE: The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23). METHODS AND MATERIALS: Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level. RESULTS: Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2). CONCLUSION: The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Modulation of coronary flow and cardiomyocyte size by sensory fibers.

Cardiac tissue is densely innervated by sensory neurons that are believed to play important modulatory roles in cardiac functions. In this study, pretreatment of neonate rats with capsaicin was performed. In adult rats, cardiomyocyte size and amount of fibrous tissue in left ventricles as well as in vitro coronary flow were evaluated. The chronotropic and inotropic responses to beta-adrenoceptor agonists (norepinephrine and isoproterenol), muscarinic agonists (carbachol and pilocarpine), and calcitonin gene-related peptide (CGRP) were also investigated with the use of the isolated right atria preparation. Capsaicin pretreatment significantly (P<0.05) reduced both basal coronary flow (18% reduction) and cardiomyocyte size (34% reduction) without affecting the amount of fibrous tissues in the left ventricles. The positive inotropic and chronotropic effects in response to norepinephrine in the isolated rat heart did not significantly differ between control and capsaicin-treated rats. Similarly, the positive chronotropic effects in response to norepinephrine, isoproterenol, and CGRP as well as the negative chronotropic responses to carbachol and pilocarpine in the isolated right atria were not affected by capsaicin pretreatment. Our data are consistent with the suggestion that reductions of both basal coronary flow and cardiomyocyte size seen in hearts from capsaicin-pretreated rats may be consequences of CGRP depletion. The cardiomyocyte size reduction produced by capsaicin treatment may be related to a modulatory role of CGRP as a growth factor.

Phase II study of high-dose megestrol acetate in patients with advanced ovarian carcinoma.

The EORTC Gynaecological Cancer Cooperative Group conducted a phase II study of high dose oral megestrol acetate: 800 mg/day for 1 month followed by 400 mg/day as maintenance treatment, in heavily pretreated patients with ovarian cancer. Of 72 patients included in this study, 54 were fully evaluable for response and toxicity. The response rate was low with only 1 patient having a partial response, 9 patients with stable disease and 44 patients with progressive disease. The toxicity profile was low. However, 1 patient died after 2 months of treatment, and in 3 patients thrombo-embolic events occurred. Weight gain varied in 20 of the 61 patients from 0.5 to 16 kg. This study does not suggest that the overall 10% benefit from hormonal therapy for chemotherapy refractory ovarian cancer will improve by increasing the dose.

Phase II study of carboplatin in patients with advanced or recurrent endometrial carcinoma. A trial of the EORTC Gynaecological Cancer Group.

The aim of this study was to investigate the efficacy and toxicity of carboplatin given as monotherapy in endometrial adenocarcinoma. Cisplatin is one of the most active drugs in gynaecological cancer types, but at the cost of an associated high toxicity. In this high-risk population of endometrial cancer patients, it is necessary to have chemotherapy regimens with a low toxicity. Patients eligible for this study were those with histologically-confirmed endometrial adenocarcinoma with evidence of recurrent and/or metastatic disease. Carboplatin was administered every 4 weeks as a first- (dose: 400 mg/m(2)) or second- (dose: 300 mg/m(2)) line chemotherapy. Of the 64 patients who entered the trial, 60 were eligible, 53 patients were evaluable for toxicity and 47 for efficacy. A total of 169 cycles of carboplatin was given with a median of 2 cycles per patient (range 1-11 cycles) to a median cumulative dose of 798 mg/m(2) (range 290-3879 mg/m(2)). No grade 4 toxicity or toxic deaths occurred. White Blood Cell (WBC) toxicity grade 3 was noted five times, mainly in the radiotherapy pre-treated patients. Grade 3 non-haematological toxicity consisted mainly of nausea and vomiting (21%). There was a total of eight responses (3 Complete Responses (CR) and 5 Partial Responses (PR) with an overall response rate (ORR) of 13% (95% Confidence Interval (CI) 6-25). No responses occurred in patients treated with prior chemotherapy. In evaluable patients, the ORR in all patients (n=47) and in those receiving first-line chemotherapy (n=33) were, 17% (95% CI 8-31) and 24% (95% CI 11-42), respectively. After a median follow-up of 379 days, the median duration of response was 488 days (range 141-5303 days) with two very long responses in patients with a CR. Carboplatin has a low toxicity and is active in chemotherapy-naive advanced endometrial carcinoma patients. These results lead us to propose its use in association in first-line chemotherapy in recurrent or advanced endometrial carcinoma patients. The choice of the initial dose can be determined according to whether the patients have received prior radiotherapy treatment.

Correolide, a nor-triterpenoid blocker of Shaker-type Kv1 channels elicits twitches in guinea-pig ileum by stimulating the enteric nervous system and enhancing neurotransmitter release.

Correolide (1 - 10 microM), a nortriterpene purified from Spachea correae and a selective blocker of Kv1 potassium channels, elicits repetitive twitching in guinea-pig ileum. This effect is not seen in guinea-pig duodenum, portal vein, urinary bladder or uterine strips, nor in rat or mouse ileum. The time course and amplitude of the correolide-induced twitches in guinea-pig ileum are similar to those elicited by electrical stimulation of the enteric nervous system. The correolide-induced twitching is not affected by pre-treatment with capsaicin (1 microM), but is facilitated by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl esther (L-NAME, 200 microM). The correolide-induced twitching is abolished by tetrodotoxin (1 microM) or hexamethonium (100 microM), and is markedly inhibited by nifedipine (0.3 microM) or atropine (0.2 microM). The atropine-resistant component is inhibited by selective antagonists of NK1 and NK2 tachykinin receptors, namely GR 82334 and GR 94800 (1 microM each). The former compound is more effective in inhibiting the correolide-induced, atropine-resistant activity. Correolide intensified the twitching of ileum segments exposed to saturating concentrations of margatoxin (MgTX), which suggests that Kv1 sub-types other than Kv1.1 (Kv1.4 or Kv1.5) are involved in the relatively greater degree of stimulation of the enteric nervous system by correolide, as compared to MgTX. We propose that blockade of Kv1 channels by correolide increases the excitability of intramural nerve plexuses promoting release of acetylcholine and tachykinins from excitatory motor neurons. This, in turn, leads to Ca(2+)-dependent action potentials and twitching of the muscle fibres.