The relationship between apamin binding and channel block in KCa2 potassium channels.

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are widely distributed throughout the body and are involved in diverse physiological processes including the regulation of neuronal firing and smooth muscle contraction. They are also potential targets in the treatment of cardiac arrhythmia. The KCa2.2 and 2.3 members of the family are blocked by the peptide toxin apamin at low concentrations, however, the mechanism of block by apamin is unclear. In electrophysiological experiments apamin has been reported to block Kca2.2 and 2.3 with IC50 ~100 pM and ~1nM respectively. In contrast, in ligand binding experiments using [125I]-mono-iodoapamin it has been found that apamin does not discriminate between Kca2.2 and 2.3 and that it binds with significantly higher affinity ( ~5pM). This discrepancy has led to the suggestion that, rather than acting as a classical pore blocker, apamin exerts its action by an allosteric mechanism. It is notable that the ligand binding studies reported so far have been conducted with isolated cell membranes in non-physiological solution with low ionic strength. We have investigated this discrepancy between results from ligand binding and electrophysiological studies by comparing binding of [125I]-mono-iodoapamin and inhibition of KCa2 current in intact HEK 293 cells using identical physiological solutions. In these conditions we found that apamin bound to KCa2.1 and KCa 2.3 with KL 60 and 606 pM, close to values of IC50 from electrophysiological experiments. We also compared the ability of some known SK channel blockers, UCL 1848, UCL 1684, gallamine and dequalinium, to displace labelled apamin and inhibit KCa2 current. With these compounds we found a good correlation between K¬i and IC50. These findings suggest that the discrepancy between binding and block might arise from differences in the experimental protocols used. To examine this we examined apamin block of KCa2 current in low ionic strength solutions in which NaCl was iso-osmotically replaced by sucrose. In these conditions 100 pM apamin caused 92 ± 0.1 % block as against 51 ± 5 % block in physiological ionic strength. We conclude that binding data obtained from membrane preparations must be interpreted with care when making comparisons with data from functional experiments and that this has implications for current views on the mechanism of action of apamin as an SK channel blocke

Structure - activity studies with histamine H3 - receptor ligands

Se han sintetizado análogos de tioperamida. Los compuestos han sido ensayados in vitro para explorar los factores que permitan diseñar compuestos derivados de la tioperamida sin grupo tiourea que mejoren la penetración cerebral. Los compuestos más activos como H3-antagonistas contienen un átomo de nitrógeno aromático hetorocíclico sobre la cadena lateral. Estos compuestos se han empleado como cabeza de serie para obtener potentes H3-antagonistas de histarnina con estructura de ariloxietil y ariloxipropilimidazoles. Las relaciones estructura actividad de agonistas se han revisado brevemente. Se han estudiado un grupo de análogos de (S-[2-imidazol-4-il)etil]isotiourea (imetit) con el objeto de explorar la transición entre agonistas y antagonistas. N,N' -dibutil-[S-[3-(imidazol- 4-il)propil]isotiourea es un muy potente antagonistas que tiene Ki=1.5 nM.Analogues of thioperamide have been synthesised and tested in vitro on rat cerebral cortex to explore structure-activity relationships with the intention of designing compounds which do not possess the thiourea group of thioperamide and which may have improved brain penetration. Compounds derived from histamine and having an aromatic nitrogen containing heterocyc1e on the side-chain amino group have been found to act as H3 - antagonists. These have served as leads to provide aryloxyethyl- and aryloxypropylimidazoles which are potent H3 antagonists of histamine. Structure-activity relationships for agonists are brief1y reviewed. Analogues of the very potent and selective agonist, imetit (S-[2-imidazol-4-yl)ethyl]isothiourea) have been studied to explore the transition between agonist, partial agonist and antagonist. The isosteric isourea is also a potent agonist. N,N' -Dibutyl-[S-[3-(imidazol-4-yl)propyl]isothiourea is a very potent antagonist having K¡=1.5 nM

Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny

Molecular Cloning of Rat Mast Cell Protease 1 and Development of Specific Probes for Its Gene Transcript

International audienceRat mast cell protease of type 1 (RMCP1) is a specific marker of connective tissue mast cells selectively occurring in some tissues, e.g., the tongue. Its amino acid sequence is known (Le Trong et al., Biochem. 1987, 26, 6988-6994) but not the corresponding nucleotide sequence. Amplification of mRNAs from rat tongue was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers corresponding to the translated region of rat mast cell protease 2 (RMCP2) gene. The cDNA obtained was subcloned and sequenced, leading to an amino acid sequence which matched the known 227 amino acid sequence. In addition there was, however, two sequences of 11 amino acids at the N-terminus and 13 amino acids at the C-terminus. The amino acid identity was of 74% with RMCP2, and of 76%, 65% and 90% with the mouse proteases MMCP1, MMCP2 and MMCP4, respectively. Based on the sequence of RMCP1 or RMCP2 cDNAs, selective oligoprobes were designed and their specificity established by Northern blot analysis of mRNAs purified from tongue and jejunum, two tissues containing selectively type 1 and 2 protease, respectively. Single 1.2 and 1.0 kb transcripts were evidenced in tongue and jejunum, respectively. In addition, a RT-PCR method was developed to amplify selectively each transcript which may serve as reliable markers in the analysis of mast cell heterogeneity, differentiation and function

Modulation of acetylcholine, capsaicin and substance P effects by histamine H3 receptors in isolated perfused rabbit lungs.

The modulatory role of histamine H3 receptors in pulmonary oedema induced by acetylcholine, capsaicin and by exogenous substance P was investigated in isolated, ventilated rabbit lungs. Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). Acetylcholine (10(-8) to 10(-4) M), substance P (10(-10) to 10(-6) M), capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) induced an increase in the Kf,c. Carboperamide, a novel histamine H3 receptor antagonist, induced a significant leftward shift of the concentration-response curve to acetylcholine and also enhanced the effect of capsaicin on the Kf,c, while it had no significant effect on the response to substance P and 5-HT. Imetit, a new histamine H3 receptor agonist, strongly inhibited the effects of acetylcholine and capsaicin. Imetit also strongly protected the lung against substance P effects but did not prevent the 5-HT-induced increase in the Kf,c. Carboperamide completely blocked the inhibitory effect of Imetit on the acetylcholine response. (R)-alpha-Methylhistamine, an other histamine H3 receptor agonist, had the same protective effect against acetylcholine response as Imetit. We conclude that histamine H3 receptors could protect the lung against acetylcholine- and capsaicin-induced oedema via a prejunctional modulatory effect on the C-fibres. However, since the response to exogenous substance P was also inhibited by histamine H3 receptor stimulation, the presence of such receptors at a postsynaptic level, probably on mast cells, was also suggested

Epidemiological study of infantile rotavirus diarrhoea in Tananarive (Madagascar).

International audienceAn epidemiological study of rotavirus infections was conducted in Tananrive, Madagascar, from November 1988 to October 1990. Rotavirus antigen was detected by ELISA in faecal specimens of 183 of 1,659 children with acute diarrhoea (11%) and in 11 of 631 specimens from children without diarrhoea (1.7%). Rotaviral diarrhoeas were most frequently found in infants aged 6 to 18 months and occurred throughout the year with a definite peak during the first winter months. Analysis of the viral RNA by polyacrylamide gel electrophoresis permitted the characterisation of 170 and 194 strains identified. Nine different electropherotypes (A-I) and one mixed infection were observed. The "short" electropherotypes (A-E) were predominant and represented 140 strains (82.4%), and the "long" electropherotypes (F-I and M) represented 30 strains (17.6%). The "short" electropherotype A (cafb) was the most frequent in our environment (45.3% of cases) and was predominant during the first 14 months of the study. The "long" electropherotype F (bbea) appeared in July 1990 and was predominant during the last three months. Among these children with diarrhoea, the presence of rotavirus was significantly associated with vomiting, fever, and moderate to severe dehydration. However, no significant differences in the occurrence of these symptoms were found between the "short" and "long" electropherotypes