Detection of fungal metabolites showing toxic activity through Artemia salina bioassay

The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.     

Kinetics of the Opening and Closing of Individual Excitability-Inducing Material Channels in a Lipid Bilayer

The kinetics of the opening and closing of individual ion-conducting channels in lipid bilayers doped with small amounts of excitability-inducing material (EIM) are determined from discrete fluctuations in ionic current. The kinetics for the approach to steady-state conductance during voltage clamp are determined for lipid bilayers containing many EIM channels. The two sets of measurements are found to be consistent, verifying that the voltage-dependent conductance of the many-channel EIM system arises from the opening and closing of individual EIM channels. The opening and closing of the channels are Poisson processes. Transition rates for these processes vary exponentially with applied potential, implying that the energy difference between the open and closed states of an EIM channel is linearly proportional to the transmembrane electric field. A model incorporating the above properties of the EIM channels predicts the observed voltage dependence of ionic conductance and conductance relaxation time, which are also characteristic of natural electrically excitable membranes.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

Kinetic characteristics of the excitability-inducing material channel in oxidized cholesterol and brain lipid bilayer membranes

The kinetic characteristics of the opening and closing of the excitability-inducing material (EIM) channel in oxidized cholesterol and in brain lipid bilayers are compared. The kinetics of the opening and closing of individual ion-conducting channels in bilayers doped with small amounts of EIM are determined from discrete fluctuations in ionic current. The kinetics for approach to steady-state conductance are determined for lipid bilayers containing many channels. Steady-state and kinetic characteristics for the EIM channel incorporated in brain lipid bilayers can be accounted for by the model developed for the EIM channel incorporated in oxidized cholesterol membranes. Relaxation time, calculated from rate constants of single-channel membranes or directly measured in many-channel membranes is strongly temperature dependent, and is always shorter in brain lipid membranes. Changes in temperature do not affect the interaction of the electric field and the open channel, but the open configuration of the EIM channel in brain lipid bilayers is stablized with increasing temperature. The configurational energy difference between the open and closed channel, calculated from temperature studies, is larger in brain lipid bilayers. The energy barrier which separates the two configurations of the channel is larger in oxidized cholesterol bilayers.     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle.

Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+.     

Phylogeny of the Drosophila obscura species group deduced from mitochondrial DNA sequences

Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., afftnis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).     

Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.