Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections

Estrogen has been shown to affect ventromedial hypothalamic (VMH) nerve cell nucleoli in ovariectomized rats, by causing an increase in the number of electron-dense aggregates associated with nucleoli. In order to characterize these nucleolus-associated structures and other nuclear components, we examined the ultrastructure of ventromedial hypothalamic nucleoli and nuclei revealed by enzyme digestions (pepsin, RNase and DNase) in resinless thin sections. Digestion by pepsin did not cause obvious alterations in the morphology of the nucleolus or its related structures. Pepsin treatment followed by RNase, however, reduced the density of the nucleolus, while that of the nucleolus-associated structure and other related structures remained unchanged. Conversely pepsin treatment followed by DNase, reduced the density of nucleolus-associated and other chromatin structures, but had no effect on the density of the nucleolus. Pepsin treatment followed by RNase and then DNase treatment, reduced the density of the nucleolus and nucleolus-associated structures. A residual nucleolus and nucleolus-associated structure remained after this treatment. Stereo viewing of resinless sections shows that the nucleolus, its associated structures, and other related structures, are associated with fine filaments that may comprise the nuclear matrix. The nucleolus-associated structure containing DNA may direct RNA synthesis at an increased rate in estrogen-treated hypothalamic cells.     

Crosslinking of uteroglobin by transglutaminase

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.     

Cell attachment and spreading on extracellular matrix-coated beads

Parietal yolk sac cells M1536-B3 grown on cytodex 2 beads deposited an extracellular matrix on the surface of the beads. Cell-free matrix-coated beads were isolated by treatment of the cell monolayer with cytochalasin B (CB) at a concentration of 10 μg/ml of phosphate-buffered saline (PBS). The matrix when analysed by electrophoresis on polyacrylamide gels (PAGE) revealed that the major components were laminin and entactin. The matrix-coated beads were used to study the attachment, spreading, and growth of African Green monkey BSC-40, human mammary MCF-7, mouse fibroblast L929, rat liver clone 9, and rat hepatoma H-4-II-E cells in defined serum-free growth medium. The different cell lines exhibited varying responses to matrix-coated vs uncoated beads with respect to rate of attachment, spreading, and growth. One of the most consistent responses observed was the enhancement of cell spreading on matrix-coated beads. The results suggested that the matrix-coated beads will provide a readily available and valuable tool for studies on cell surface-extracellular matrix interactions and the physiological consequences of those interactions.     

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.     

Alteration by estrogen of the nucleoli in nerve cells of the rat hypothalamus

Summary Estrogen is accumulated from the blood by nerve cells in the ventromedial nucleus of the hypothalamus and can facilitate female reproductive behavior by acting on this region of the brain. This cell group was examined in ovariectomized female rats, given estrogen or control treatment, by use of light and electron microscopy. A significantly greater portion of the nerve cells in the estrogen-treated animals had protuberances on their nucleolar surfaces, apparent under the light microscope. The fine structure of such protuberances included dense, aggregated material, which is shown to contain DNA by the sodium tungstate staining technique. Because increased numbers of such protuberances were found in nuclei of cells of the experimental group where previous studies demonstrated a significant increase in ultrastructural signs of biosynthetic activity, they may be associated with increased RNA synthesis. Thus, they could indicate, ultrastructurally, increased synthetic rates for RNA in nerve cells through which estrogen promotes reproductive behavior.     

Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.     

Synthesis of plasma lipoproteins by the isolated perfused liver from the fasted and fed pig.

Livers from fasted or fed pigs were perfused for 5 h with Krebs-Ringer bicarbonate buffer containing human erythrocytes, bovine serum albumin, glucose, and amino acids. Liver viability was estimated by color, consistency, portal pressure, bile flow, electrolyte changes, and glucose levels in the perfusate, urea synthesis, [1-14C]leucine incorporation into protein, oxygen uptake, and histological examination. It was shown that the liver was maintained in good condition throughout the perfusions. The apolipoprotein B (apoB) and apolipoprotein A-I (apoA-I) in the perfusate were measured by solid phase radioimmunoassay. In the fasted state, the amount of apoB released was greatest in the low density lipoprotein (LDL) fraction and the amount was especially high during the 1st h. There was no increase of apoB in this fraction by feeding. The apoB in the very low density lipoprotein (VLDL) fraction was less than that in the LDL fraction in the fasted state, and it increased more than 2-fold in the fed animals. The amount of apoA-I was greatest in the 1.21 bottom fraction and was relatively small in the high density lipoprotein (HDL) fraction. The HDL fraction contained approximately one-twentieth as much apoA-I as the 1.21 bottom fraction in the fasted condition. In the fed state, apoA-I in the HDL fraction increased markedly, although the amount was still less than in the 1.21 bottom fraction.     

A preliminary crystallographic study of isocitrate dehydrogenase from Azotobacter vinelandii

Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4₂2₁2 with cell dimensions $\text{a = 122.1}\text{A}\text{?}$, $\text{c = 163.9}\text{a}\text{?}$. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Capillaries of the adrenal cortex possess aminopeptidase A and angiotensin-converting-enzyme activities.

The enzymes required to convert the prohormone angiotensin I into angiotensins II and III, secretagogues of aldosterone, are enriched in association with capillary endothelium isolated from rat adrenal cortex. Thus the secretion of aldosterone may be controlled, in part, by processing of peptides occurring within the adrenal gland itself.