A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii

The immunohistochemical distribution of serotonin-containing nerve fibres and cells has been described in the brain of the Antarctic fish, Trematomus bernacchii. The largest serotonergic system was associated with the diencephalic and rhombencephalic ventricles. In particular, serotonin-positive cells have been found in the lateral recess and neuropile zone of the diencephalic ventricle, where we have identified the serotonergic portion of the paraventricular organ. Numerous serotonin cells were localized in the dorsal nucleus of the raphe, the dorsal tegmental nucleus and the central gray. Two large cell groups, arranged in a pair of well-defined columns and connecting the central gray with the dorsal reticular formation, were immunostained in the region of the trigeminal nuclei. In addition, few positive cells have been found in the preoptic area and the cerebellar valvula, and few serotonergic nerve fibres, probably belonging to the lateral lemniscus, have been identified. The distribution of serotonin elements in the brain of T. bernacchii has been compared with that described in other fish, where it showed some modifications in the immunoreactive pattern. Finally, the lack of a serotonergic system at the level of the reticular superior formation has been reported; however, it was not possible to rule out a phylogenetic or environmental explanation.     

A 45,X male with Y-specific DNA translocated onto chromosome 15.

A 20-year-old male patient with chromosomal constitution 45,X, testes and normal external genitalia was examined. Neither mosaicism nor a structurally aberrant Y chromosome was observed when routine cytogenetic analysis was performed on both lymphocytes and skin fibroblasts. Y chromosome-specific single-copy and repeated DNA sequences were detected in the patient's genome by means of 11 different recombinant-DNA probes of known regional assignment on the human Y chromosome. Data indicated that the short arm, the centromere, and part of the long-arm euchromatin of the Y chromosome have been retained and that the patient lacks deletion intervals 6 and 7 of Yq. High-resolution analysis of prometaphase chromosomes revealed additional euchromatic material on the short arm of one of the patient's chromosomes 15. After in situ hybridization with the Y chromosome-specific probe pDP105, a significant grain accumulation was observed distal to 15p11.2, suggesting a Y/15 chromosomal translocation. We conclude that some 45,X males originate from Y-chromosome/autosome translocations following a break in the proximal long arm of the Y chromosome.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.     

Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

TxA2 production by human arteries and veins

Human arterial and venous segments from patients under-going operations when incubated in Tris buffer both alone and with arachidonic acid were able to produce thromboxane B2 (assessed by radioimmunoassay). Thromboxane B2 (TxB2) production was progressive in time (till 40 min.) and was enhanced by the addition of 1mM norepinephrine. Contamination of tissues by platelet was checked and platelets did not contribute to thromboxane formation. The investigation of the conversion of 1-14C arachidonic acid by vascular tissue indicated that human vascular tissues produce the metabolites of the cyclooxygenase dependent pathway and that prostacyclin is the main metabolite with a PGI2/TxA2 ratio of 4:1. The arterial wall was found to possess an active lipoxygenase dependent pathway. Thromboxane production by intimal cells was negligible and the main source of thromboxane was the media. The production of thromboxane did not change in relation to age, but arterial segments from men produced significantly larger amounts of thromboxane than those from women.     

Improved bromodeoxyuridine/DNA analysis by anti-BudR monoclonal antibody versus right angle light scatter

Summary Dynamic cell cycle analysis is based on the incorporation of labelled precursors into DNA. Although antibodies to BrdU are very useful for analysing in flow cells which synthesize DNA, this approach has two main limitations. First, the detection of low incorporating cells is often difficult; second, four parameter flow cytometry is not able to correlate cell cycle to any other cellular marker. We have developed a methodology that, employing an IgG_H+L as a second antibody and side scatter instead of propidium iodide fluorescence, allows a better discrimination of BudR⁺ cells. This approach allows the collection of an extra-fluorescent signal, and the analysis of specific cellular markers within the cell cycle.