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.     

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β₄subunit is present in association with α₆chain on both normal and transformed epithelial cells. Recently α₆β₄heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β₄subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α₆β₄has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β₁(TGF-β₁) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α₆β₄expression is modulated by TGF-β₁, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β₁cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β₄subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β₁.     

Different forms of U15 snoRNA are encoded in the introns of the ribosomal protein S1 gene of Xenopus laevis.

Recent cloning and sequencing of one of the two Xenopus gene copies (S1b) coding for the ribosomal protein S1 has revealed that its introns III, V and VI carry a region of about 150 nt that shares an identity of 60%. We show here the presence in Xenopus oocytes and cultured cells of a 143-147 nt long RNA species encoded by these three repeated sequences on the same strand as the S1 mRNA and by at least one repeat present in the S1 a copy of the r-protein gene. We identify these RNAs as forms of the small nucleolar RNA U15 (U15 snoRNA) because of their sequence homology with an already described human U15 RNA encoded in the first intron of the human r-protein S3 gene, which is homologous to Xenopus S1. Comparison of the various Xenopus and human U15 RNA forms shows a very high conservation in some regions, but considerable divergence in others. In particular the most conserved sequences include two box C and two box D motifs, typical of most snoRNAs interacting with the nucleolar protein fibrillarin. Adjacent to the two D boxes there are two sequences, 9 and 10 nt in length, which are perfectly complementary to an evolutionary conserved sequence of the 28S rRNA. Modeling the possible secondary structure of Xenopus and human U15 RNAs reveals that, in spite of the noticeable sequence diversity, a high structural conservation in some cases may be maintained by compensatory mutations. We show also that the different Xenopus U15 RNA forms are expressed at comparable levels, localized in the nucleoli and produced by processing of the intronic sequences, as recently described for other snoRNAs.     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

A Chronobiological Approach to Circulating Levels of Renin, Angiotensin-Converting Enzyme, Aldosterone, ACTH, and Cortisol in Addison's Disease

This study deals with a chronobiological approach to the circadian rhythm of the renin-angiotensin-aldosterone system (RAAS) and the ACTH-cor-tisol axis (ACA) in patients with Addison's disease (PAD). The aim is to explore the mechanism(s) for which the circadian rhythmicity of the RAAS and ACA takes place. The study has shown that both the RAAS and ACA are devoid of a circadian rhythm in PAD. The lack of rhythmicity for renin and ACTH provides indirect evidence that their rhythmic secretion is in some way related to the circadian oscillation of aldosterone and cortisol. This implies a new concept: a positive feedback may be included among the mechanisms which chronoregulate the RAAS and ACA.     

Role of fungal patchiness on vegetal detritus in the trophic interactions between two brackish detritivores,Idotea baltica and Gammarus insensibilis

We investigated patterns of resource partitioning between two brackish crustaceans (I. baltica and G. insensibilis) on trophic mosaics of fungal species colonizing vegetal detritus. Laboratory feeding experiments were carried out to assess consumption rates and diet selection of single individuals belonging to five populations. Adults of two co-occurring population of both G. insensibilis and I. baltica and one population of G. insensibilis, occurring alone in another habitat patch, were studied. Each individual were offered both fragments of Cymodocea nodosa conditioned by 8 fungal species and sterilized fragments as sole food source for 7 days. Both species preferred "conditioned" detritus but potential resource use was greater in G. insensibilis than in I. baltica. Individual niche breadth increased and phenotypic variability reduced in the allotopic sample. Trophic similarities were lower between individuals of co-occuring populations than between I. baltica and the allotopic G. insensibilis. The results suggest that co-occurrence is an important factor contributing to the enhancement of phenotypic variability and, consequently, to a trophic generalization at population level in G. insensibilis. It emphasizes the role of fungal patchiness on detritus in regulating resource partitioning between the two species.     

Expression of ribosomal-protein genes in Xenopus laevis development

Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 2-5), become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs.     

Serum angiotensin-converting enzyme activity in pre-eclamptic pregnancy: evidence for a relative hypermesorACEemia

The circadian rhythm of serum angiotensin-converting enzyme (ACE) activity was investigated in pregnant women with normal and pre-eclamptic gestation. The chronobiological approach was able to document the occurrence of a circadian rhythm for serum ACE activity in normal pregnancy. Such a rhythm is characterized by a decreased mesor and amplitude and a shifted crest. The circadian rhythm for serum ACE activity was not detectable in pre-eclamptic pregnancy. Such an abrogation is accompanied by a negligible decrease of mesor suggesting the occurrence of a relative hyperACEemia. This disorder could play a role in pregnancy-induced hypertension.     

Messenger RNA for ribosomal proteins in dormant and developing Artemia salina embryos

Ribosomal proteins from Artemia salina have been separated in a two-dimensional acrylamide gel system and assigned to the small and large ribosomal subunits. Poly(A)-containing RNA was prepared from dormant cysts and from polysomes of 30 min, 1 h, 5 h and 12 h embryos and hatching larvae. The mRNA from different stages was translated in a wheat-germ lysate and its template activity for ribosomal proteins was analyzed. It was observed that mRNA activity for ribosomal proteins is stored in the cytoplasm of dormant cysts and that it is found associated with polysomes of 30 min and of later stages.