The histogenesis of the rat adrenal cortex: a study based on histologic analysis of mosaic pattern in chimeras

A series of chimeric rats was used to investigate the histogenesis of the adrenal cortex. These animals were produced by amalgamating preimplantation embryos of two congenic strains which express different alloantigens of the major histocompatibility system. The manner in which cells assort themselves during embryogenesis and organ renewal is amenable to analysis by using radiolabeled antibodies directed to the class I antigens. Mosaic pattern analysis of the adrenal cortex of these rats revealed that a clonal pattern of division was maintained across all three histogenic zones of the organ, even in highly unbalanced chimeric combinations. No apparent relationship existed between the proportion of cell types and the area of contiguously similar lineage (patch size). In the series examined, the percentage of cells derived from the PVG-RT1a lineage varied from 5 to 85%. The number of parallel cords varied from 6 to 28. The pattern analysis of informative corners of cross sections of the gland supports the thesis that organ maintenance is the result of cell division from the outside of the cortex toward the inside medullary surface.     

A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting.

This paper describes the use of lipoprotein blotting to detect low-density-lipoprotein (LDL) receptors in rat and rabbit liver and adrenal glands and in bovine adrenal glands. Using this technique we show that the rabbit and rat liver LDL receptors have Mr values of 128000 and 145000 respectively. Mr values for the rabbit, rat and bovine adrenal receptors are 131000, 142000 and 132000 respectively. Differences between the bovine adrenal and rat liver receptors are not due to differences in the degree of sialylation. Lipoprotein blotting can be used to detect dietary- and drug-induced changes in the concentrations of LDL receptors. When rabbits are fed on a cholesterol-rich diet, liver LDL receptors cannot be detected, consistent with the suppression of hepatic LDL receptors by cholesterol feeding. Pharmacological doses of 17 alpha-ethinyloestradiol cause a marked increase in hepatic LDL-receptor activity in the rat. This is accompanied by a corresponding increase in the number of LDL receptors detected by lipoprotein blotting. The Mr of the induced receptor is identical with that of the receptor from control rats, which suggests that the induced receptors are produced by the same gene as LDL receptors normally present in the liver.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

Three dimensional multi-scale modelling and analysis of cell damage in cell-encapsulated alginate constructs

One of the major challenges in scaffold guided regenerative therapies is identifying the essential cues such as mechanical forces that induce cellular responses to form functional tissue. Developing multi-scale modelling methods would facilitate in predicting responses of encapsulated cells for controlling and maintaining the cell phenotype in an engineered tissue construct, when mechanical loads are applied. The objective of this study is to develop a 3D multi-scale numerical model for analyzing the stresses and deformations of the cell when the tissue construct is subjected to macro-scale mechanical loads and to predict load-induced cell damage. Specifically, this methodology characterizes the macro-scale structural behavior of the scaffold, and quantifies 3D stresses and deformations of the cells at the micro-scale and at a cellular level, wherein individual cell components are incorporated. Assuming that cells have inherent ability to sustain a critical load without damage, a damage criterion is established and a stochastic simulation is employed to predict the percentage cell viability within the tissue constructs. Bio-printed cell-alginate tissue constructs were tested with 1%, 5% and 10% compression strain applied and the cell viability were characterized experimentally as 23.2±16.8%, 9.0±5.4% and 4.6±2.1%. Using the developed method, the corresponding micro-environments of the cells were analyzed, the mean critical compressive strain was determined as 0.5%, and the cell viability was predicted as 26.6±7.0, 13.3±4.5, and 10.1±2.8. The predicted results capture the trend of the damage observed from the experimental study.     

A study of cell migration in the adrenal cortex of the rat using bromodeoxyuridine

Abstract Bromodeoxyuridine (BrdU) was administered by a single intraperitoneal injection to immature (14 days) male and female and adult male Sprague-Dawley rats. Animals were killed at intervals from 2 hr to 28 days following injection. Labelled cells in the adrenal cortex were identified by an indirect immunoperoxidase technique using a monoclonal antibody to BrdU. At 2 hr, labelling was maximal in the outer zona fasciculata and zona glomerulosa in both prepubertal and adult rats. The numbers of immunopositive cells were greater in the 14 day rats. In both groups, the front of immunopositive cells moved deeper into the cortex with time. These results support the centripetal migration theory of adrenal growth.     

An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions

The NAD- and NADP-dependent malic enzymes from rat liver and adrenal mitochondrial fractions were separated and partially purified by gel filtration on Sepharose 6B. Two activity peaks were observed. The first contained a malic enzyme capable of reducing either NAD or NADP. This enzyme showed sigmoid kinetics in plots of activity versus the malate concentration. Succinate was an allosteric activator and ATP was a competitive inhibitor of malate. The second peak showed hyperbolic kinetics in plots of activity versus the malate concentration and was unaffected by either succinate or ATP. The relative activities of the two malic enzymes were quite constant in the adrenal mitochondrial fractions. In the liver mitochondrial fractions, the activity of the first peak varied and was sometimes absent while the activity of the second peak was quite constant. The kinetic properties of the first malic enzyme implicate it as an important regulator of malate oxidation.     

Post-mortem visualization of peroxisomes in rat and in human liver

Summary This paper describes spontaneous post-mortem changes of peroxisomal staining in normal liver and kidney of rats and in human autopsy liver. At room temperature, regional staining loss is observed at 18h after death in rat kidney, at 24h in human liver and at 48 h in rat liver. Preservation at 4°C delays this phenomenon. In human liver, the peroxisomal volume density is decreased at both temperatures at 48 h. After freezing of fresh tissue in dry ice, peroxisomal staining is decreased homogeneously. Under the electron microscope, peroxisomal alterations suggest a loss of catalase activity. These changes do not necessarily preclude the study of peroxisomal features since, even after 48 h at room temperature, peroxisomes are still well stained in the less affected regions. Catalase and three -oxidation enzymes, namely acyl-CoA oxidase, bifunctional protein (with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) and 3-oxoacyl-CoA thiolase, could be visualized immunocytochemically in human autopsy livers up to 48 h after death. However, the study of certain peroxisomal features such as catalase activity and peroxisomal distribution, may be hampered as the post-mortem period is prolonged.     

Metabolism of 3 H-digitoxigenin by rat liver, adrenal, and ovary homogenates

The metabolism of ³H-digitoxigenin was studied in rat liver, adrenal, and ovary homogenates under identical conditions. The major metabolite formed by liver and ovarian preparations was 3-epidigitoxigenin. Male liver homogenates showed higher epimerizing activity than female liver or ovary homogenates. In the adrenal preparations, the major metabolite formed was 3-digitoxigenone, and no sex difference was observed in its rate of formation. Adrenal and liver homogenates produced small amounts of digitoxigenin polar metabolites. The polar metabolites formed by the adrenal preparations were tentatively identified as 5-hydroxydigitoxigenin and 16β-hydroxydigitoxigenin.     

Nucleolar alterations induced by 4-aminopyrazolo(3,4-d)pyrimidine in adrenal cortex and liver cells of rat

Summary The nucleolar ultrastructural changes produced in adrenal zona fasciculata cells and hepatocytes of rats by 4-aminopyrazolo(3,4-d) pyrimidine (4-APP), an inhibitor of the lipoprotein synthesis, are described. Male rats were injected intraperitoneally for three consecutive days with 4-APP in sodium phosphate buffer (50 mg/Kg/day). On the 4th day, the animals were sacrificed and their adrenals and liver were processed for EM. The nucleoli of the adrenal zona fasciculata cells showed nucleolar fragmentation with loss of their normal reticular appearance, separation of fibrillar and granular components, extensive vacuolization and evidence of fibrillar centers. The hepatocyte nucleoli also exhibited fragmentation. These changes seem to be the morphological counterpart of alterations in rRNA synthesis and processing, since 4-APP inhibits de novo purine synthesis and thus interferes with its incorporation into RNA and DNA.     

Identification of specific vasopressin binding sites in cell membranes of the adenohypophysis, liver, kidney and adrenal glands of the rat

3H vasopressin specifically binds to the binding sites in liver, kidney and adenohypophysis with Bmax = 11.8 +/- 5.6, 1.7 +/- 0.5 and 4.9 +/- 1.2 pmol/g tissue and Kd = 1.5 +/- 0.5, 0.66 +/- 0.21 and 0.84 +/- 0.21 nM, correspondingly. Specific binding increases in the presence of Mg2+ and Ni2+ and decreases at high temperature (37 degrees). The presence of high affinity binding sites for 3H vasopressin was shown in the rat adrenals, binding was saturable and reversible. Concentration of vasopressin binding sites in adrenals is 6-8 fold less than in adenohypophysis. It is supposed that vasopressin receptors in adrenals may participate in the regulation of corticosteroid secretion.     

Comparison of some physicochemical and kinetic properties of S-adenosylhomocysteine hydrolase from bovine liver, bovine adrenal cortex and mouse liver

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) was purified to apparent homogeneity from bovine liver, bovine adrenal cortex and mouse liver. All enzymes were tetramers, composed of two types of subunit present in the proportion 1:1, as judged by SDS-polyacrylamide gel electrophoresis. The partition coefficient was exactly the same for these enzymes on high-performance gel permeation chromatography, and they co-sedimented in density gradients, suggesting the same molecular size and form of S-adenosylhomocysteine hydrolase from these sources. The bovine enzymes differed from the mouse liver enzyme with respect to isoelectric point (pI = 5.35, versus pI = 5.7), affinity for DEAE-cellulose, and migration of subunits on SDS-polyacrylamide gel electrophoresis with SDS from some commercial sources. The enzymes were not substrates for cAMP-dependent protein kinase. The apparent Km values for adenosine (0.2 microM) and S-adenosylhomocysteine (0.75 microM) were the same for all three enzymes. The ratio between Vmax for the synthesis and hydrolysis of S-adenosylhomocysteine was about 4 for the mouse liver enzyme, and about 6 for the bovine enzymes. It is concluded that only subtle kinetic and physicochemical differences exist between S-adenosylhomocysteine hydrolase from these bovine and mouse tissues. This suggests that differences in experimental procedures rather than species- and organ-differences of S-adenosylhomocysteine hydrolase are responsible for the variability in kinetic and physicochemical parameters reported for the mammalian hydrolase.     

Effects of vinblastine and colchicine on the rat adrenal cortex: morphometric and cytochemical studies

The effects of administration of anti-microtubular drugs--vinblastine and colchicine--on the ultrastructure of the zona fasciculata cells of young rat adrenal were studied. Young male rats were injected with vinblastine and sacrificed 2 hr later or with colchicine and sacrificed 3 hr after drug administration. Animals injected with isotonic saline in same experimental conditions served as controls. Ultrastructural alterations provoked by both drugs, vinblastine or colchicine, were identical and were most prominent in the Golgi areas. They appeared enlarged and crowded with round, or slightly elongated light vesicles, acid phosphatase, and osmium negatives. The Golgi dictyosomes, although keeping their normal morphology, were less numerous and presented cisternae which were narrower and shorter than controls. Electron-dense vesicles, round or elongated, and acid phosphatase positive--lysosomes--were observed in great number in the Golgi areas, intermingled with light vesicles. The relative volume of light vesicles and lysosomes of the treated animals was significantly increased when compared with controls, but the relative volume of dictyosomes was significantly decreased. Also the numerical density of light vesicles and lysosomes of the injected rats was significantly increased when compared with controls. These alterations are highly suggestive of the Golgi involvement in the adrenal secretory process.