Membranes Of Granular Endoplasmic Reticulum Research Articles

ULTRASTRUCTURAL ORGANIZATION OF THE LYMPHATIC CAPILLARIES IN CONJUNCTIVA AND THE LYMPHATIC CHANNELS IN HUMAN CHOROID

The aim of this work was a comparative study of the ultrastructural organization of lymphatic structures in the choroid and conjunctiva of the human eye. Material and methods. Samples of the choroid and conjunctiva of the human eye, obtained perioperatively, were studied by immunohistochemical analysis using specific antibodies to endothelial cells markers of blood and lymphatic vessels, and electron microscopic studies, morphometric processing and statistical analysis of the results were performed. Results and discussion. Within simultaneous immunohistochemical staining of conjunctiva and choroid samples using antibodies to markers of lymphatic vessels (LYVE-1, Prox1, podoplanin) the positively stained typical lymphatic vessels have been revealed in the conjunctiva structure and positively stained channels with narrow elongated cells, fibroblasts and pigment cells in choriocapillar layer of the choroid and the transition zone between choroid and sclera. Electron microscopic study revealed that cells expressing lymphatic markers and forming lymphatic channels in the choroid have an ultrastructural organization that is differ from typical lymphatic vascular endothelial cells: they do not have stropic filaments, contain a greater volume density of the granular endoplasmic reticulum membranes, and a lower volume density of mitochondria and micropinocytotic vesicles. The data obtained indicate that the lymphatic pathways of the intraocular fluid outflow in the human eye choroid have organ-specific structural features.

Endothelial ultrastructural alterations of intramuscular capillaries in infantile mitochondrial cytopathies: “Mitochondrial angiopathy”

Electron microscopy (EM) is a reliable method for diagnosing mitochondrial diseases in striated muscle biopsy in infancy. Ultrastructural alterations in mitochondria of myofibers are well documented, but there are few studies of endothelial involvement in intramuscular capillaries. Quadriceps femoris biopsies of five representative infants and toddlers, ages neonate to 3.5 years, were performed because of clinical and laboratory data consistent with mitochondrial disease without mitochondrial DNA (mtDNA) mutations and likely with nuclear DNA mutations. Pathological studies included histochemistry, EM, respiratory chain enzymatic assay and mtDNA sequencing and deletion/duplication analysis. EM demonstrated frequent and severe alterations of mitochondria in capillary endothelium. The most constant changes included: either too few or fragmented cristae; stacked and whorled cristae; paracrystallin structures that often were large and spheroid with stress fractures; closely apposed membranes of granular endoplasmic reticulum surrounding mitochondria with loss of the normal intervening layer of cytoplasm; long narrow, thin looped microvilli extending into the lumen; and thick microvilli containing large, abnormal mitochondria. We conclude that mitochondrial cytopathies in early life exhibit more severe ultrastructural alterations in the endothelium than in myofibers and that paracrystallin body structure differs, perhaps due to less rigid surrounding structures. This distribution may explain the frequent lack of prominent histochemical and biochemical abnormalities in muscle biopsies of young patients. Endothelial changes do not distinguish the genetic defects. Vascular involvement in brain contributes to cerebral lesions and neuronal death by impairment of molecular and nutrient transport and ischemia; endothelium in muscle may reflect similar changes.

Elevated activity of the oxidative and non‐oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver

(Pre)neoplastic lesions in livers of rats induced by diethylnitrosamine are characterized by elevated activity of the first irreversible enzyme of the oxidative branch of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PD), for production of NADPH. In the present study, the activity of G6PD, and the other NADPH-producing enzymes, phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (ICD) and malate dehydrogenase (MD) was investigated in (pre)neoplastic lesions by metabolic mapping. Transketolase (TKT), the reversible rate-limiting enzyme of the non-oxidative branch of the PPP, mainly responsible for ribose production, was studied as well. Activity of G6PD in (pre)neoplastic lesions was highest, whereas activity of PGD and ICD was only 10% and of MD 5% of G6PD activity, respectively. Glucose-6-phosphate dehydrogenase activity in (pre)neoplastic lesions was increased 25 times compared with extralesional parenchyma, which was also the highest activity increase of the four NADPH-producing dehydrogenases. Transketolase activity was 0.1% of G6PD activity in lesions and was increased 2.5-fold as compared with normal parenchyma. Transketolase activity was localized by electron microscopy exclusively at membranes of granular endoplasmic reticulum in rat hepatoma cells where G6PD activity is localized as well. It is concluded that NADPH in (pre)neoplastic lesions is mainly produced by G6PD, whereas elevated TKT activity in (pre)neoplastic lesions is responsible for ribose formation with concomitant energy supply by glycolysis. The similar localization of G6PD and TKT activity suggests the channelling of substrates at this site to optimize the efficiency of NADPH and ribose synthesis.

In Situ Localization of Transketolase Activity in Epithelial Cells of Different Rat Tissues and Subcellularly in Liver Parenchymal Cells

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.

Ultrastructural localization of arginine vasopressin in coronary vessels of newborn rat.

We report on the ultrastructural distribution of arginine-vasopressin (AVP) in the heart of newborn rats using pre-embedding peroxidase-antiperoxidase immunocytochemistry with a polyclonal AVP antibody for electron microscopy. Positive labelling for AVP was localized in endothelial cells of main coronary arteries and cardiac vessels of smaller diameter (microvessels). Examination of the right coronary artery showed that approximately 58% of the endothelial cells were positive for AVP. Immunoreactivity to AVP in the cytoplasm of arterial endothelium predominated in association with the membranes of granular endoplasmic reticulum and in subplasmalemmal areas. The endothelium of small vessels exhibits less endoplasmic reticulum, but still shows AVP immunoprecipitate in the cytoplasm. It is suggested that endothelial AVP may contribute to vasomotor control of the coronary circulation in the early stages of postnatal development. AVP antibody also labelled some fibroblast/fibroblast-like cells associated with the coronary arteries and microvessels; thus, these cells as well as the endothelium appear to be a source of AVP in the newborn rat heart. The functional significance of these findings is discussed.

Immunocytochemical localization of cytochrome P4501A1 in liver of rainbow trout ( Oncorhynchus mykiss)

Multiple cytochrome P450 isozymes have been characterized in livers of trout ( Oncorhynchus mykiss), scup ( Stenotomus chrysops) and other fishes. Of these, trout P450 LM 41, and scup P450c (or P4501A1) are probably homologous, representing the dominant and possibly only forms of P450 stimulated by inducers of the 3-MC (PAH) type such as β-naphthoflavone (BNF). Immunohistochemistry using scup P4501A1 monoclonal antibodies localized this isozyme within hepatocytes, and presumably sinusoidal endothelial and biliary epithelial cells of scup and trout. In this study ultrastructural localization determined intracellular loci of this isozyme. Juvenile trout ( O. mykiss) were injected once i.p. with 50 μg/g BNF in cod liver oil. After 5 days, livers were processed for electron microscopy. Ultrathin sections were incubated with anti-P4501A1 and 10 nm colloidal gold. Membranes of granular endoplasmic reticulum (GER) were labeled in perinuclear regions of hepatocytes within specific domains and not dispersed along all membranes. Hepatocyte plasma membrane, particularly microvilli, at bile canaliculus was labeled, as was luminal plasma membrane of biliary epithelial cells surrounding passageways. Endothelial plasma membrane at sinusoid and immediately subjacent cytoplasm was labeled, perhaps providing for bioactivation of blood-borne xenobiotics. Localization within biliary epithelial cells could signify the potential to bioactivate PAH, explaining the common finding of biliary as well as hepatocytic tumors of trout liver.

Biologic characterization of human bone tumors. II. Distribution of different collagen types in osteosarcoma--a combined histologic, immunofluorescence and electron microscopic study.

Sixteen cases of typical highly malignant osteosarcoma were investigated by light, electron, and immunofluorescence microscopy to demonstrate the presence of collagen types I-III. It was shown that, in light-microscopically anaplastic areas of the tumor, collagen type III predominates, while only very few membranes of collagen type I are observed. Ultrastructurally, the cells are characterized by numerous free ribosomes in their cytoplasm and only a few membranes of granular endoplasmic reticulum (ER). In osteoblastic areas, collagen type I is increased, while type-III collagen is decreased. The cytoplasm of cells contains markedly more granular ER. An increasing mineralization of matrix is observed. In fibroblastic areas of the tumors, collagen types I and III are codistributed. Tumor cells have a fibroblast appearance with elongated nuclei and well developed granular ER. The chondroblastic areas, characterized by immature neoplastic cartilage, contain varying amounts of collagen type II. Chondroblast-like tumor cells have typical ring-shaped membranes of granular ER in their cytoplasm. The evidence of different collagen types in osteosarcomas lends additional support to the concept that a pluripotent mesenchymal cell is the stem cell of osteosarcomas.

The morphology, occurrence, and distribution of dilated cisternae of the endoplasmic reticulum in tissues of plants of theCruciferae

Morphology, occurrence, and distribution of dilated cisternae of the endoplasmic reticulum (ER) were studied by electron microscopy. The cisternae which contained an electron-dense matrix were intimately associated with the granular ER membranes appearing as tubular necks at the edges of the ER profiles. After budding off from the ER the cisternae still had ribosomes attached to the outside of the bounding membranes. The accumulations were variable in shape, being 0.4 to 1.5μ in width and 4 to 5μ in length.

Observations on the association of helical polyribosomes and filaments with vincristine-induced crystals in Earle's L-cell fibroblasts.

Earle's L-929 fibroblasts from cultures treated with 5-10 microg/ml of vincristine sulfate have a large number of eosinophilic, proteinaceous crystals in their cytoplasm. In electron micrographs, large arrays of helical polyribosomes, stacks of Golgi lamellae, and membranes of granular endoplasmic reticulum are seen in the cytoplasm of these cells. "Stalks" of fine granular material, approximately 300 A wide, are often seen in association with the arrays of the helical polyribosomes. In many sections rows of helical polyribosomes and filaments emerging from individual polyribosomes are seen in intimate contact with the crystals. A gradual reduction in the number of crystals and crystal-bearing cells is seen in cultures removed from the drug-containing medium and reincubated in fresh medium. In electron micrographs, these reincubated cells show large aggregates of filamentous material in the cytoplasm, and in many sections filaments are seen in continuity with the crystals.