Ultrastructural aspects of the paraventricular nuclei in the rat

Summary The ultrastructural aspects of the paraventricular nucleus and its neuropil are described in the normal rat.Two types of neurons can be distinguished morphologically. The first type contains numerous dense-core vesicles (mean diameter: 140 nm). The cisternae of the endoplasmic reticulum are arranged parallely at the periphery of the cell body.The second type of neuron contains a few dense-core vesicles (mean diameter: 75 nm) and the endoplasmic reticulum is randomly distributed in the cytoplasm. In the neuropil, two types of dense-core vesicles are observed in separated axons. The histogram of the distribution of their mean diameter clearly indicates a double population of vesicles.The signification of the second type of neuron in the paraventricular nucleus is discussed and its possible relation to TRF synthesis is evoked.This work was supported by a grant from the Belgian National Fund for Scientific Research.The author wish to thank Mrs. Hunninck-Couck for her devoted and skillful technical assistance.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei

Summary Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C. 2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

Demonstration of a DNase-sensitive network associated with the nuclear pore complexes in rat liver nuclei

Rat liver nuclei have been studied by transmission electron microscopy after resuspension in a phosphate-buffered salt solution containing SO²⁻ ₄ as the quantitatively dominant anion. Owing to the high solubility of chromatin in the presence of SO²⁻ ₄ instead of Cl⁻ at isotonicity, nuclei are depleted for chromatin by DNase I digestion in this buffer, eliminating the need for high-salt extraction. This shows that at least 75% of the nuclear pore complexes are associated with fibrogranular structures, which ramify as a network throughout the nucleus, interconnecting the nuclear lamina, interchromatin granule clusters and nucleoli. Perichromatin granules are located in this material proximal to the nuclear pore complexes. Most of the chromatin is removed without major impact on the network, but below a level of 25% residual chromatin there is a considerable reduction of this material, and only about 15% of the connections to the nuclear pore complexes are resistant to digestion with DNase I or streptodornase A and B. The percentage of nuclear pore complexes connected to the network is further reduced by salt extraction and RNase treatment. These results suggest that DNA is an integral part of the network, which presumably plays a role in nucleo-cytoplasmic transport of RNA and protein. Received: 1 September 1998; in revised form: 17 December 1998 / Accepted: 17 December 1998     

Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat

Summary Fine structural localization of an ACTH-like substance was obtained in neurons of the rat arcuate nucleus using immuno-electron microscopy, whereas it could not be confirmed that ACTH-containing cell bodies are present in the supraoptic nucleus. The immunoreactive cells of the arcuate nucleus appeared to be more numerous than the unreactive neurons. Immunostaining was carried out before embedding in resin. Empty vesicles of irregular shape were found in dendrites of immunoreactive arcuate neurons, but their significance and nature remain enigmatic. The reaction product was distributed uniformly throughout the cytoplasm of the ACTH-positive cells, except that the mitochondria, rough endoplasmic reticulum and Golgi vesicles and cisternae were devoid of PAP molecules. This distribution differed from the localization reported in ACTH-secreting cells of the rat anterior pituitary, where the reaction product was found in the rough endoplasmic reticulum and Golgi complex as well as in secretory granules.     

Glycoprotein mannosylation in rat liver nuclei

Nuclei and non-nuclear membranes were tested for their ability to transfer in vitro (14C)mannose from GDP-(14C)mannose to endogenous glycoprotein acceptors in the presence and in the absence of exogenous retinyl-phosphate. Electrophoretic analysis shows that retinylphosphate is responsible for the labeling of a few endogenous acceptors only in the non-nuclear membranes; in the nuclei the mannosylation reaction is not retinylphosphate dependent and the electrophoretic profile of the labeled protein acceptors is different from that of the non-nuclear membranes.     

Histone acetylation in rat liver nuclei

Rat liver nuclei were incubated for various lengths of time in the presence of increasing concentrations of acetyl CoA. The rate of acetylation varied strongly according to the acetyl CoA concentration. A very small part of the histone was acetylated. After incubation in the presence of increasing acetyl CoA concentrations, four apparent Km could be determined. Electrophoretic analysis showed that only histone H3 was acetylated which suggests that each Km corresponds to a sequential acetylation of its lysine residues. This could be correlated with the possible role of histone acetylation in the control of gene activity.     

Ultrastructural study of glial gap junctions in the thalamic nuclei of rat

Despite a growing interest in gap junctions (GJs) of mammalian brain, their distribution and role in cell ensembles of thalamus remains unknown. The aim of this work was ultrastructural and immunoelectron study of glial GJs in ventral posteromedial (VPM) and posteromedial (POM) thalamic nuclei and thalamic reticular nucleus (RTN) of rats. GJs were identified by standard techniques of transmission electron microscopy and by pre-embedding immunohistochemistry protocol using anti-connexin-43 antibodies with Dako EnVision System + Peroxidase (DAB) detecting system. It was found that glial cells surround thalamocortical axons and axo-spiny synapses and form numerous elongated gap junction plaques located near chemical synapses. A single axon-spiny chemical synapse can be surrounded by several (up to 4) gap junctions that seem to form peculiar networks of glial cells united by GJs. Closely adjacent gap junctions disposed at an angle from 30° to 140° to each other were revealed. Immunoelectron labeling demonstrated that gap junction plaques located around chemical synapses have an astroglial origin. Despite the accumulation of osmiophilic material in the contact zone, ultrastructural signs of GJs were clearly identified. Due to the formation of intercellular glia-glial GJs astroglia may acquire a function of spatial buffer to regulate extracellular concentration of potassium and other ions, providing intracellular and extracellular ion homeostasis. We believe that astroglial processes joined into a network by GJs play a key role in the circulation of information and can modulate subcortical neuronal ensembles. We suggest that a close spatial location of astroglial GJs and asymmetrical chemical synapses is reflected in the functional organization of specific and nonspecific thalamic nuclei, which are the main centers of the afferent and efferent inputs of the cerebral cortex.     

Ultrastructural aspects of the reversible nature of sclerotic changes in the liver

Experiments on mice have demonstrated ultrastructural changes in collagen and hepatocytes during reverse development of liver cirrhosis. Progressive lysis of collagenous fibers has been noted. Changes in hepatocytes point to a rise in the synthetic and endocytosis activity in these cells. It is suggested that exocellular lysis of collagen in the process under consideration is initiated by collagenase whereas subsequently it takes place under the action of lysosomal enzymes secreted by hepatocytes to the exocellular space.     

Histone-specific acetyltransferase from the rat liver nuclei

Certain properties of histone-specific acetyltransferases A, B and C, obtained from the rat liver are determined. pH optimum for enzyme A is within the range of 7.5-8.5, for B--7.8 and for C--7.5. The maximal activity for enzymes A, B and C is observed with the 60 micrograms/ml concentration of the substrate. The activity is inhibited by N-maleimide, iodacetamide and chloromercuribenzoate. The results obtained show that a number of similar properties are typical of the above enzymes.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

Synthetic activity of isolated rat liver nuclei : I. Ultrastructural study at various steps of isolation

The incorporation of labelled amino acids into hot TCA-insoluble material was used as a measure of protein elaboration in nuclear fractions and compared with the kinetics and distribution of labelling by means of electronmicroscope autoradiography.The method of isolation and the various controls were designed in order to exclude reasonably that this incorporation could be due to cytoplasmic contamination.The pattern of incorporation was found to be characteristic, as compared with microsomal systems. It was insensitive to RNAse and did not require a cytoplasmic pH 5 fraction or an exogenous energy-yielding system.The localization of the activity in the autoradiographs was not random, but clearly associated with definite regions of the nucleus. The nucleolus was 3 times as radioactive as the rest of the nucleus.These results can be interpreted in favor of the concept that protein synthesis occurs in the nucleus. The theoretical limitations of this conclusion are discussed.