VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [³H]acetate as a precursor of ACh. Little vesicular [³H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [³H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [³H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.     

Penetration and entrapment of large particles in erythrocytes by electrical breakdown techniques

Human erythrocytes suspended in isotonic solutions were subjected to haemolysis by application of an electric field pulse to the cell suspension. The field strengths used were 12 and 16kV/cm, respectively; the pulse duration 40 microseconds. The lysed cells showed resealing properties. The permeability change of the membrane generated by the field pulse and by the subsequent osmotic processes were large enough to facilitate the penetration and entrapment of ferritin and Latex particles (diameter: 0.091 and 0.176 micron, respectively) as revealed by electron microscopy. Correct identification of the Latex particles in the electron-micrographs indicated that LOYTER et al. [J. Cell Biol. 66, 292 (1975)], who recently demonstrated the entrapment of Latex spheres in erythrocytes prepared by osmotic haemolysis mistook electron-dense bodies probably consisting of denaturated protein for Latex particles. Under conditions of osmotic haemolysis, carried out according to BODEMANN and PASSOW, particles could only occasionally be detected within the membrane itself and never within the cell interior, suggesting that the electrical haemolysis method is much more effective in the generation of large holes in the membrane.     

Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium.

A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.     

Amide isosteres of lexitropsins: synthesis, DNA binding characteristics and sequence selectivity of thioformyldistamycin.

The synthesis and properties of an amide isostere of the antibiotic distamycin, thioformyldistamycin 3 is described. Compound 3 exists predominantly in the E conformation of the thioamide group in freshly prepared DMSO solution but is converted into the Z form, predicted by molecular mechanics to be more stable, on standing for 24 h. The coalescence temperature in DMSO is 110 degrees C by 1H-NMR. The thioformyl moiety of 3 is resistant to both peptidase action and acid treatment. Complementary strand MPE footprinting on a EcoRI/Hind III restriction fragment of pBR322 DNA demonstrated that either E or Z forms of 3 give a single set of footprints very similar to that of the parent antibiotic with strongest protection at TAAG and TATTAT with moderately strong protection at ATTT and AAAA. The strength of binding of 3 and distamycin from delta Tm measurements to either poly.d(AT) or calf thymus DNA is comparable. Molecular modeling predicted a preferred conformation for 3 wherein the C = S bond has a torsional angle of 110 degrees with the pyrrole ring. The energy difference between this conformation and the E form is less than 1 kcal/mole. In contrast the E-form has an energy 17.3 kcal/mole greater than the Z and a value of 26.3 kcal/mole was calculated for the energy barrier between the two isomers.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

The mechanism of electrical breakdown in the membranes ofValonia utricularis

Summary The dielectric breakdown in the membranes of cells ofValonia utricularis was investigated using intracellular electrodes and 500-sec current pulses. Electrical breakdown, which occurs when the membrane potential reaches a well-defined critical value, is not associated with global damage to the cell or its membranes (the membrane reseals in <5 sec). It was thus possible to investigate the effect of temperature on dielectric breakdown in single cells. It was found that the critical potential for breakdown was strongly dependent on temperature, decreasing from 1000 mV at 4°C to 640 mV at 30°C. The decrease in the breakdown potential with increasing temperature and the very short rise-time of the breakdown current (1 sec) suggests that the Wien field dissociation does not play a major role in the breakdown process. It is shown that the nonlinearI–V characteristics observed at different temperatures can be accurately accounted for with no adjustable parameters, by considerations of the mechanical compression of the membrane due to stresses induced by the electric field. Electrical breakdown on this scheme results from an electromechanical instability in the membrane. On this basis the present results indicate that the elastic modulus of the region of the membrane where breakdown occurs, decreases by a factor of 2 with increasing temperature from 4 to 30°C. On the assumption of a thickness of 4.0 nm and a dielectric constant of 5, the elastic modulus is estimated to have a value of 5×10⁶ Nm^–2 at 20°C.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

The dopamine circuit as a reward-taxis navigation system

Studying the brain circuits that control behavior is challenging, since in addition to their structural complexity there are continuous feedback interactions between actions and sensed inputs from the environment. It is therefore important to identify mathematical principles that can be used to develop testable hypotheses. In this study, we use ideas and concepts from systems biology to study the dopamine system, which controls learning, motivation, and movement. Using data from neuronal recordings in behavioral experiments, we developed a mathematical model for dopamine responses and the effect of dopamine on movement. We show that the dopamine system shares core functional analogies with bacterial chemotaxis. Just as chemotaxis robustly climbs chemical attractant gradients, the dopamine circuit performs ‘reward-taxis’ where the attractant is the expected value of reward. The reward-taxis mechanism provides a simple explanation for scale-invariant dopaminergic responses and for matching in free operant settings, and makes testable quantitative predictions. We propose that reward-taxis is a simple and robust navigation strategy that complements other, more goal-directed navigation mechanisms.