A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes

The "direct coloring" thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 micron. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the detection of multiple forms of AChE in polyacrylamide or agarose gels.     

The dopaminergic innervation of the goldfish pituitary

Summary The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Clathrin-associated proteins contain bound nucleotide

An alcohol dehydrogenase isolated from Zymomonas mobilis was found to be activated by ferrous ions but not by zinc, after inactivation with metal-complexing agents. Cobaltous ions also re-activated to a lesser extent. It is suggested that in this species the alcohol dehydrogenase naturally contains iron. Kinetic studies on the iron-treated enzyme indicate an 'alcohol activation' phenomenon, which may have physiological relevance in overcoming product inhibition during fermentation.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Irreversible Activation of the Opiate Receptor of Neuroblastoma × Glioma Hybrid Cells by an Alkylating Benzomorphan Derivative

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.