Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Inverted neurons in agyria

Summary An anatomoclinical observation of agyria is reported. The karyotype revealed a partial deletion of the short arm of chromosome 17. The etiology of agyria is reviewed in the light of this chromosomal abnormality. In addition we describe the peculiar pattern of neurons in the cortex: Golgi stain demonstrated many inverted pyramidal cells in the superficial part of the cortical layer. The mechanism of this abnormality is discussed.     

Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli.

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

[³H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.     

Origin of slow negative potential of direct cortical response in cats

To investigate mechanisms of formation of the electrocorticographic slow negative potential (SNP) evoked by direct electrical stimulation of the cortical surface, poststimulus single unit activity in the stimulated area was studied in anesthetized cats and changes in SNP in the depth of the cortex were analyzed. The results showed that membrane hyperpolarization, accompanied by cessation of action potential generation, develops parallel with SNP in neuron bodies in the stimulated area. Investigation of the nature of this hyperpolarization showed that during its development excitability of the neuron and resistance of the postsynaptic membrane fall. It is concluded from the results that this membrane hyperpolarization is an indicator of IPSP development in the neuron bodies. The results of laminar recording showed that SNP may diminish or even disappear in the depth of the cortex without subsequent reversal. Determination of dipoles formed along an axis perpendicular to the cortical surface showed that SNP has one source and two sinks, which are located symmetrically relative to it. The presence of two symmetrical sinks must indicate an active source, formed as a result of hyperpolarization of the neuron membrane. On the basis of the results SNP can be regarded as a field potential formed on the cortical surface as a result of IPSP development in the neuron bodies.Institute of Clinical and Experimental Neurology, Ministry of Health of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 15, No. 3, pp. 314–320, May–June, 1983.