Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.     

EPR study of the redox interactions in cytochrome c3 from Desulfovibrio vulgaris Miyazaki

We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovibrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovibrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: -250 mV less than or equal to e41 less than or equal to -220 mV, -325 mV less than or equal to e2 less than or equal to -320 mV, -335 mV less than or equal to e3 less than or equal to -330 mV, -360 mV less than or equal to e4 less than or equal to -355 mV, -5 mV less than or equal to I12 less than or equal to 20 mV, -10 mV less than or equal to I13 less than or equal to 5 mV, -15 mV less than or equal to I23 less than or equal to -5 mV, -15 mV less than or equal to I24 less than or equal to -10 mV, -25 mV, less than or equal to I34 less than or equal to -15 mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Growth,aluminium uptake and mucous cell morphometrics of early life stages of brown trout,Salmo trutta,in low pH water

Synopsis Young (7–10 days after hatching) brown trout (Salmo trutta) exposed for 5 days to pH 5 in high calcium water and at 2 temperatures (12°, 4°C) in the laboratory displayed no alterations in growth or in mucous cell concentration and volume, compared to the control group kept at pH 7.2. Contamination of acid-stressed young with 230 μg All^-1 resulted in significant growth depression and Al accumulation, but in no changes of mucous cell morphometrics. Field tests in low calcium water produced high mortality at low pH (5.1), but showed consistent effects on mucous cells as in laboratory experiments. Three-month-old juveniles of brown trout, subjected to decreased pH values at 12° and in high calcium water for 8 days exhibited mucous cell hyperplasia (without hypertrophy) within 3 h of the acid addition. After 120 h sloughing of the integument occurred with full recovery not possible within a 4-day-recovery period. Although the results presently apply only to hard water conditions, the differences between juveniles and recently hatched young in tolerance to pH- and Al-mediated stress may also be of importance for soft waters affected by acid rain.     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.