A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Effect of GLG-V-13, a class III antiarrhythmic agent, on potassium currents in rabbit ventricular myocytes

The effects of a new Class III antiarrhythmic drug, GLG-V-13, on the 4-aminopyridine sensitive transient outward current, on the inward rectifier potassium current, on the ATP sensitive potassium current and on the rapid and slow components of the delayed rectifier potassium current were studied in single rabbit ventricular myocytes using the whole-cell voltage-clamp technique. GLG-V-13 blocked the rapid component of the delayed rectifier potassium current in a dose-dependent manner, with an estimated EC50 value of 0.36 microM. At high concentration, the slow component of the delayed rectifier potassium current was also depressed by the drug (40% effect at 10 microM concentration). The transient outward current, the inward rectifier potassium current and the ATP sensitive potassium current were not influenced by GLG-V-13, even at 10 microM concentration. Thus, GLG-V-13 blocks predominantly the rapid component of the delayed rectifier potassium current which may play a significant role in the prolongation of repolarization by the drug in ventricular tissue.     

Variability of Hypericins and Hyperforin in Hypericum Species from the Sicilian Flora

Within Sicilian flora, the genus Hypericum (Guttiferae) includes 10 native species, the most popular of which is H. perforatum. Hypericum’s most investigated active compounds belong to naphtodianthrones (hypericin, pseudohypericin) and phloroglucinols (hyperforin, adhyperforin), and the commercial value of the drug is graded according to its total hypericin content. Ethnobotanical sources attribute the therapeutic properties recognized for H. perforatum, also to other Hypericum species. However, their smaller distribution inside the territory suggests that an industrial use of such species, when collected from the wild, would result in an unacceptable depletion of their natural stands. This study investigated about the potential pharmacological properties of 48 accessions from six native species of Hypericum, including H. perforatum and five ‘minor’ species, also comparing, when possible, wild and cultivated sources. The variability in the content of active metabolites was remarkably high, and the differences within the species were often comparable to the differences among species. No difference was enlightened between wild and cultivated plants. A carefully planned cultivation of Hypericum seems the best option to achieve high and steady biomass yields, but there is a need for phytochemical studies, aimed to identify for multiplication the genotypes with the highest content of the active metabolites.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

Conditioning of beta(1)-adrenoceptor effect via beta(2)-subtype on L-type Ca(2+) current in canine ventricular myocytes

We investigated the roles of beta(1)- and beta(2)-receptors (beta-AR) in adrenergic enhancement of L-type Ca(2+) current (I(CaL)) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I(CaL) relationship (CR), respectively. alpha(1)-AR inhibition with prazosin and beta(2)-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on I(CaL). beta(2)-AR inhibition with ICI-118551 reduced the E(max) of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I(CaL) response to beta(1)-AR stimulation but abolished the enhancement via beta(2)-AR. Zinterol augmented the enhancement of I(CaL) by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta(2)-AR stimulation does not increase I(CaL) but enhances adenylyl cyclase activity when stimulated via beta(1)-AR and with forskolin. beta(2)-AR activity preconditions adenylyl cyclase for beta(1)-AR stimulation.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).