Effect of praseodymium nitrate on hepatocytes and Kupffer cells in the rat.

Intravenous administration of the rare earth metal salt, praseodymium nitrate, induced hepatic damage in the rat, as assessed by morphologic examination (light and electron microscopy) and biochemical parameters (serum glutamic-pyruvic transaminase (EC 2.6.1.2) and glutamic-oxalacetic transaminase (EC 2.6.1.1) activity as well as hepatic triglyceride content). Praseodymium hepatotoxicity was only attained with lower doses (10, 20, or 40 mg/kg), whereas a larger dose (80 mg/kg) was inactive in this respect. As detected by electron microscopy, lower doses of the metal salt caused hepatocytic alterations consisting of degranulation and dilatation of rough endoplasmic reticulum, accumulation of smooth endoplasmic reticulum as well as numerous lipid droplets. No abnormalities were detected in the cell organelles following administration of a large dose of the metal salt; however, vacuoles containing markedly electron-dense material were seen in the cytoplasm of the hepatocytes and the sinusoidal Kupffer cells.     

ACETYLCHOLINESTERASE IN FROG SYMPATHETIC AND DORSAL ROOT GANGLIA : A Study by Electron Microscope Cytochemistry and Microgasometric Analysis with the Magnetic Diver

The localization and chemical determination of acetylcholin esterase in the frog sympathetic and dorsal root ganglia were studied by a combination of the methods of electron microscopy, histochemistry, and microgasometric analysis with the magnetic diver. The Koelle-Friedenwald copper thiocholine histochemical method was modified by eliminating the sulfide conversion and by treatment of the tissue with potassium permanganate. In fixed tissue, enzymatic activity was demonstrated on the inner surface of the endoplasmic reticulum, nuclear envelope, subsurface cisternae, and agranular reticulum of the perikaryon and axon. In briefly fixed tissue, end product appeared also at the axon-sheath and the sheath-sheath interface. Activity at the synaptic junction was most readily obtained in unfixed tissue. Isolated neurons recovered from the diver following chemical analysis were studied with the electron microscope. Cells having a high enzyme activity showed a badly ruptured or absent neural plasmalemma and sheath. In this case the measured activity was apparently due to the enzyme present in the endoplasmic reticulum. Neurons having low activity exhibited an intact plasmalemma and sheath. This may reflect the effectiveness of the neural plasmalemma and sheath as a penetration barrier. The effects of fixation on enzyme activity are discussed. Electron microscopic examination of cells following microgasometric analysis is shown to be essential for the interpretation of the biochemical data.     

AChE synthesis in cholinergic neurons: electron histochemistry of enzyme translocation

Summary Endoplasmic reticulum of cholinergic nerve cells exhibits acetylcholinesterase activity. In central neurons that exert an ephemeric acetylcholinesterase activity only during some ontogenetical states, enzyme reaction product is present also in the Golgi system. Neurotubular and neurofilamentar structures exert an acetylcholinesterase in terminal axons of young animals. From these electron histochemical studies it is concluded that enzyme protein molecules, synthesized in the endoplasmic reticulum, are translocated to the active sites (surface membranes) via axonal filaments (or tubules), or will be extruded from the neuron via the Golgi system.Our thanks are due to Dr. P. Röhlich, head of the Budapest Electron Microscope Laboratory, for his generous help in various aspects of the electron microscopic work.     

Effect of alpha-adrenergic blockers on calmodulin association with the nuclear matrix of rat liver cells during proliferative activation

A transient peak of cytosolic calmodulin (CaM) was produced during the prereplicative phase of rat liver cell proliferation following partial hepatectomy. After accumulating in the cytosol, CaM apparently translocated into the nuclei, associating with the nuclear matrix. The administration of alpha 1-adrenergic blockers to hepatectomized rats prevented the association of CaM with the nuclear matrix without affecting the increase in the total nuclear CaM. The inhibitory effect of the alpha 1-antagonists was reversed by the simultaneous injection of the alpha-agonist noradrenaline. Since the activation of alpha 1-adrenergic receptors results in the release of Ca2+ from endoplasmic reticulum stores, the results suggest that the association of CaM with the nuclear matrix during proliferative activation is mediated by Ca2+ released from endoplasmic reticulum and show that the association with the matrix is independent of its intranuclear accumulation.     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver.

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.     

Biochemical changes after hepatic injury by allyl alcohol and N-hydroxy-2-acetylaminofluorene.

Administration of hepatotoxic doses of allyl alcohol and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) TO adult male rats produced periportal necrosis and functional derangement of the hepatic endoplasmic reticulum within 24 h. The rates of N-demethylation of ethylmorphine and p-hydroxylation of aniline were decreased 6 h following allyl alcohol administration, but cytochromes P-450 and b5 were unchanged. In contrast, administration of NOH-AAF decreased cytochromes P-450 and b5 and the rate of aniline p-hydroxylation, but did not change the rate of N-demethylation of ethylmorphine or the activities of cytochrome c reductase and glucose-6-phosphatase. No decrease was observed in the activity of the cytosol enzyme, DT diaphorase, following allyl alcohol treatment. The changes by these periportal hepatotoxins were compared with those produced both by central and midzonal hepatotoxins and with changes occurring in the liver after surgical partial hepatectomy.     

幼龄皱纹盘鲍唾液腺和消化腺的超微结构与组织化学

以透射电镜观察和组织化学方法研究了45日龄皱纹盘鲍的唾液腺和消化腺。唾液腺由粘液细胞和纤毛细胞组成,粘液细胞含发达的粗面内质网和大量的粘原颗粒,分泌中性和酸性混合粘多糖。消化腺由消化细胞和嗜碱性细胞组成,消化细胞呈现活跃的内吞和细胞内消化,并具蛋白酶和非特异性酯酶活性。嗜碱性细胞含发达的粗面内质网和大量含铁的折光小体,折光小体的电子密度较低。     

Changes in prolactin cells caused by partial hepatectomy in the rat.

In order to study ultrastructure alterations in prolactin (PRL)-secreting cells, PRL cells of the anterior pituitary glands of partially hepatectomized female rats were observed by the protein A-gold procedure with the electron microscope. Simultaneously, their serum PRL and estradiol (E2) levels were measured by radioimmunoassay. After about 70% hepatectomy PRL cells were remarkably changed, that is active granule extrusion, prominent Golgi vesicles and a hypertrophic rough endoplasmic reticulum were observed. These changes were most remarkable on day 3 after the operation. Significant increases in serum PRL and E2 were also seen following partial hepatectomy. It may be assumed that the morphological changes in PRL cells and the elevation of serum PRL were probably due to surgical stress and to the diminution of E2 receptors in the liver after partial hepatectomy in the rat.     

The development of hepatic drug-metabolizing enzymes in perinatal guinea pigs: a biochemical and morphological study.

The development of four functionally diverse, hepatic enzymes (p-nitroanisole O-demethylase, aniline hydroxylase, carboxyleterase, and glucuronyltransferase (with alpha-naphthol as the aglycone acceptor)) was studied in perinatal Hartley guinea pigs from 8 days prepartum to 28 days postpartum. A good correlation was observed between the activities measured in resuspended Ca2+-aggregated microsomes and the quantities of hepatic smooth endoplasmic reticulum visible by electron microscopic examination at the different stages of development. The study demonstrated that, postnatally, the guinea pig developed competent enzymatic systems as rapidly as did other laboratory species but that, prenatally, these same enzyme(s) systems were much further advanced than those in other species.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.     

Immunocytochemical localization of monoamine oxidase type B in rat liver

We used an immunohistochemical method to examine the localization of monoamine oxidase type B (MAOB) in rat liver. At the light microscopic level, MAOB was highly expressed in rat liver. It was intense around portal area, and weak around central area. All the hepatocytes examined had MAOB immunoreactivity. For the first time, using a double-labeling immunofluorescence histochemical method for laser microscopy, we report that no MAOB is found in endothelial cells, hepatic stellate cells, or Kupffer's cells. When examined under transmission electron microscopy, MAOB was localized to the mitochondrial outer membrane of hepatocytes. No apparent localization of MAOB was found in the rough endoplasmic reticulum, the crystal membrane of mitochondria, the nuclear envelope, or the plasma membrane.     

Morphology of the reproductive system of the armadillo. The spermatogonia

Spermatogonia of the nine-banded armadillo, Dasypus novemcinctus mexicanus, were studied morphologically using light and electron microscopy and examined histochemically using light microscopy. Immature flat type spermatogonia have ovoid or irregular nuclei with loosely condensed chromatin. Free ribosomes are abundant while profiles of rough endoplasmic reticulum are scarce. Smooth endoplasmic reticulum is a prominent feature occasionally taking an unusual cylindrical form. Mature spermatogonia exhibit rounder nuclei with greater degrees of chromatin clumping. Smooth endoplasmic reticulum is no longer prominent whereas profiles of rough endoplasmic reticulum are quite common. Occasional lysosomal configurations are found in mature spermatogonia. The majority of spermatogonial cells exhibit weak to moderate reactivity when stained with the periodic acid-Schiff (PAS) reaction. Certain cells in each tubular cross section stain vividly with this reaction and the PAS positivity is removable with salivary amylase. Because of nuclear characteristics, position of the cell immediately upon the basal lamina, intensity of the PAS reaction and the relative paucity of the vividly staining cells, it is suggested that they are members of the immature spermatogonial cell line, perhaps acting as stem cells. None of the several other histochemical procedures employed was capable of selectively demonstrating these cells.     

An ultrastructural and histochemical study of autoimmune aspermatogenesis in the rat testis

Summary Immune aspermatogenesis was induced in young rats by the method of Freundet al. (1954) and testes were studied by electron microscopic and histochemical methods. At time of sacrifice the testes of several animals were markedly atrophic as demonstrated by reduction in weight. Sections of seminiferous tubules exhibited primarily profiles of Sertoli cells but germinal elements were sparse or absent. The ultrastructure of Sertoli cells appeared to be normal except for the presence of areas of dilated smooth endoplasmic reticulum and fragments of phagocytized germ cells in the cytoplasm.Light microscopic sections showed an apparent hyperplasia of intertubular tissue. Electron micrographs revealed a moderate to extreme vesiculation of the smooth endoplasmic reticulum in many interstitial cells. Macrophages and lymphocytes were often observed in contact with these cells.There was increased localization of non-specific esterase and acid phosphatase associated with lipid bodies of the tubules and in intertubular areas.This work was supported in part by USPHS Grant FR 5391.     

Elevated synthesis of human alpha 1-antitrypsin hinders the secretion of murine alpha 1-antitrypsin from hepatocytes of transgenic mice

alpha 1-Antitrypsin (AAT) is a major hepatic secretory protein. The elevated synthesis of human AAT within hepatocytes of transgenic mice results in its accumulation within a subset of distended cisternae of the rough endoplasmic reticulum. The protein does not accumulate in large insoluble aggregates as is the case for the human PiZ AAT variant. Furthermore, the accumulated protein is not associated with immunoglobulin heavy chain binding protein. Transgenic animals exhibiting an elevated synthesis and subsequent intrahepatic accumulation of human AAT exhibit reduced serum levels of murine AAT as a result of its hindered secretion and accumulation within the rough endoplasmic reticulum. Interestingly, the secretion of murine transferrin and albumin which represent glycosylated and non-glycosylated hepatic secretory proteins, respectively, is unaffected. Overall, these results demonstrate that the elevated synthesis of human AAT can hinder the export of murine AAT from the hepatic rough endoplasmic reticulum in an apparently specific manner.     

The problem of diabetic angiolopathy in sand rats (Psammomys obesus)

No diabetic angiolopathy was found in the retinas, kidneys and skeletal muscles of protodiabetic and overtly diabetic sand rats. The terminal blood vessels were investigated using histological, enzyme histochemical, immunofluorescence microscopic, autoradiographic and electron microscopic methods. There seems to be little or no connection in sand rats between the diabetic metabolism syndrome and the metabolic process leading to angiolopathy.     

The effect of toluene inhalation exposure on catecholamine contents in rat sympathetic neurons

Adult male rats were exposed to toluene in short-term exposure by inhalation for 48 h (2000 ppm, continuously), and in long-term inhalation for 3 months (1000 ppm, 8 h daily). The formaldehyde-induced fluorescence (FIF) technique for histochemical demonstration of catecholamines (CA) was used to detect changes in the catecholamine stores. The concentration of CA in the sympathetic neurons of superior cervical ganglia and adrenal medulla was measured by the FIF technique combined with microfluorimetry. The short-term toluene exposure induced a statistically significant reduction of CA contents in sympathetic neurons. After long-term exposure, no change in the CA level could be demonstrated either in sympathetic ganglion or adrenal medulla. In electron microscopic studies no clear pathological changes were detected after toluene exposure.     

Histological, histochemical, and fine structural observations on the spleen of seals

Spleens of three species of Antarctic seals with different diving habits (Weddell seal, crabeater seal, and fur seal) have been studied with histological, histochemical, and electron microscopic methods. The spleens can be classified as nonsinusoidal, with capsule and trabeculae rich in innervated smooth muscle cells. The trabecular system is particularly well developed in the deep- and long-diving Weddell seal. As in other mammals the pulp can be divided into white and red pulp. In the white pulp, periarteriolar lymphatic sheaths and secondary lymphatic nodules occur; both are surrounded by a marginal zone rich in macrophages and eosinophils. The nodules can be observed frequently, which is in accordance with abundance of plasma cells in the red pulp. Well-developed white pulp and numerous plasma cells and eosinophils obviously reflect a high load of nematodes, which have mainly been found in lung and stomach. Additionally, in the red pulp morphological evidence for the following functions has been found: destruction of erythrocytes, erythropoiesis, and thrombopoiesis. In respect to blood flow through the red pulp, we interpret our observations in the following way: terminal branches of arterioles open into the space between the fibroblastic reticulum cells; blood draining from here is collected into pulp veins, which are mainly found near the trabeculae. Thus, the seals have an open vascular compartment in their spleens, as also occurs in the cat. The red pulp is innervated by numerous nerve fibers that seem to include both cholinergic and adrenergic ones. The target cells of these fibers seem to be the fibroblastic reticulum cells, whose state of contraction may influence the direction of blood flow through the red pulp.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.