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Phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) (identical with sterol carrier protein 2) belong to the large and diverse family of intracellular lipid-binding proteins. Although these two proteins may express a comparable phospholipid transfer activity in vitro, recent studies in yeast and mammalian cells have indicated that they serve completely different functions. PI-TP (identical with yeast SEC14p) plays an important role in vesicle flow both in the budding reaction from the trans-Golgi network and in the fusion reaction with the plasma membrane. In yeast, vesicle budding is linked to PI-TP regulating Golgi phosphatidylcholine (PC) biosynthesis with the apparent purpose of maintaining an optimal PI/PC ratio of the Golgi complex. In mammalian cells, vesicle flow appears to be dependent on PI-TP stimulating phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. This latter process may also be linked to the ability of PI-TP to reconstitute the receptor-controlled PIP2-specific phospholipase C activity. The nsL-TP is a peroxisomal protein which, by its ability to bind fatty acyl-CoAs, is most likely involved in the β-oxidation of fatty acids in this organelle. This protein constitutes the N-terminus of the 58 kDa protein which is one of the peroxisomal 3-oxo-acyl-CoA thiolases. Further studies on these and other known phospholipid transfer proteins are bound to reveal new insights in their important role as mediators between lipid metabolism and cell functions.

A novel disulfide reductase activity was observed in the amoeba, Dictyostelium discoideum. Both 5,5'-dithiobis(2-nitro-benzoate) (DTNB) and insulin were substrates for reduction. NADH was utilized in preference to NADPH. The activity comigrated with NADPH-dependent cytochrome c reductase on sucrose gradients, suggesting localization in the endoplasmic reticulum (ER). The buoyant density of the disulfide reductase was not shifted when ribosomes were released from the ER nor was the activity solubilized when membranes were shocked with low osmotic buffer. It therefore appears that the reductase was membrane-bound in the lumen of the smooth ER.

The Sprague Dawley myelin mutant, the taiep rat, demonstrates a defect in CNS myelination which worsens with age and which is associated with abnormal accumulations of microtubules in oligodendrocytes. Quantitative and qualitative electron microscopic studies of myelin development and oligodendrocyte morphology were used to describe the temporal development of the defect in this mutant, in three regions of the CNS. The results indicate that the time of onset of myelination is similar in mutant and control rats, however the amount of myelin formed is reduced in the mutant, compared to controls, and there is a loss of myelin from the taiep CNS as the animals age. Thus the myelination defect in taiep has features of both hypomyelination and demyelination. Oligodendrocyte microtubule abnormalities were noted in each region of the taiep CNS at the time of onset of myelination. The earliest changes seen were close associations of oligodendrocyte microtubules with endoplasmic reticulum, with marked accumulations of microtubules filling the cytoplasm of oligodendrocytes from older taiep rats. These findings suggest that the microtubule abnormality in the taiep mutant inhibits both the initial formation and the long-term maintenance of myelin by the oligodendrocyte. In addition, there is also evidence to suggest that although the microtubule abnormality is present in oligodendrocytes throughout the taiep CNS, it results in a more marked defect in the myelination of axons of small diameter.

A single dose of cyclophosphamide (CY) was administered to Sprague-Dawley rats to investigate the effects of CY on thymic cortical epithelial cells (TCE) at the ultrastructural level. The most striking finding among the alterations in the TCE after CY treatment was a cytoplasmic vacuolization with an increased amount of granular and membranous content. The granular content appeared not only as dense bodies but also as loosely aggregated forms or finely dispersed granules. The membranous structures appeared in various forms including vesicular, tubular, vacuolar and irregular membranous structures and myelin figures. Some of the membranous structures contained granular material. Several vacuoles were closely associated with the endoplasmic reticulum (ER). The morphological alterations of the ER were also remarkable. The Golgi apparatus, mitochondria and vesicles increased in number. The cytoplasm became densely granulated due to an increased number of ribosomes and an increased amount of granular material. The tonofilaments lost their original array and increased in amount. The cell surface exhibited many cytoplasmic processes like microvilli. It seems that the above features result not only from some damage by CY, but also signs of a hyperfunctional state of the TCE, probably due to their important functions in repopulation and maturation of the cortical thymocytes during recovery after CY-induced acute thymic involution, including the secretion of some humoral factors.

The presenilin 1 (PS1) gene has been identified by positional cloning. More than 30 mutations were detected in this gene which cosegregate with Alzheimer's disease (AD). Understanding their role in disease pathogenesis requires a characterization of the PS1 protein. We have generated a set of antibodies against the three major hydrophilic domains of the deduced amino acid sequence. Analyzing cultured cells and brain samples, we identified the endogenous PS1 polypeptide as well as amino- and carboxy-terminal fragments. These metabolites were much more abundant than the full-length molecule, indicating substantial processing. Overexpression of human PS1 markedly increased the full-length polypeptide but hardly altered the amount of the metabolites. Instead, additional proteolytic fragments appeared suggesting a different metabolism of the excess PS1, which may impede studies in transfected cells. Our results indicate a tight regulation of the endogenous PS1 metabolites. PS1 and its fragments are shown to be integral membrane proteins of the endoplasmic reticulum. The mechanisms regulating the generation of the metabolites, their potential function, and role in AD remain to be studied.

Our aims were i) to determine which lipids colocalize with newly synthesized apolipoprotein (apo) B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilization and/or translocation of this protein; and ii) to determine the intracellular sites of assembly of lipids into very low density lipoprotein (VLDL). In order to do this, we have developed a new method for the separation of ER-derived microsomes on self-generated gradients of iodixanol. Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles and the heavier peak contained rough vesicles. Each peak was collected in a number of subfractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (smooth endoplasmic reticulum (SER) fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apoB and VLDL lipids determined by ELISA or high performance thin-layer chromatography, respectively. The biosynthetic relationships of apoB and lipids were investigated, in timed or chase experiments, by incubation of isolated rabbit hepatocytes with radiolabeled precursors of apoB or lipids, followed by isolation and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol, and cholesteryl ester co-localize with apoB into the lumen of the RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesized triacyl-glycerol, phospholipid, cholesterol, and cholesteryl ester were also transferred to the lumen of the RER and were chased into the SER lumen. Double-labeling experiments showed that cholesteryl ester produced from newly synthesized cholesterol (labeled with [3H]mevalonate and [14C]oleate) was almost exclusively present in the RER, while cholesteryl ester in the SER was labeled only with [14C]oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.

Equilibrium [3H]ryanodine binding assay was applied to total membrane fractions of six rodent species, including the Mexican volcano mouse Neotomodon alstoni alstoni, Wistar rat Rattus norvegicus albinus, golden hamster Mesocritus auratus, gerbil Meriones unguiculatus, guinea-pig Cavia porcellus, and ground squirrel Spermophillus mexicanus. The organs selected for this study were: skeletal muscle, heart, brain and liver. The constants derived from Scatchard analysis show slight variations in their Kd, ranging from 3 to 15 nM, except in the gerbil's skeletal muscle (38 nM) and the hamster's brain (27 nM). Remarkably, the Bmax calculated in guinea-pig muscle was as high as that reported for the rabbit fast twitch muscle (4.6 pmol/mg of protein) using the same membrane fraction preparation. For all the other skeletal muscles, Bmax was similar to the corresponding heart Bmax values (from 0.5 to 1 pmol/mg of protein). Gerbil cardiac Bmax was the highest (1.1 pmol/mg of protein). The ground squirrel was the rodent with more cerebral ryanodine binding sites (0.26 pmol/mg of protein), whereas the rat and the volcano mouse showed the lowest values (0.12 pmol/mg of protein). The richest sources of hepatic ryanodine receptor were the guinea-pig and rat livers (approximately equal to 0.35 pmol/mg of protein), whereas the lowest Bmax corresponded to the hamster liver (0.018 pmol/mg of protein). These results allow us to detect the similarities and differences of the ryanodine receptor binding constants from four different tissues of some of the rodents most widely used as biomedical laboratory animals.

Isolated organ perfusion is used in clinical practice for chemotherapy in adults with malignant tumors. However, it has not been performed in children because of the size mismatch with the adult circuits. The authors have previously studied isolated liver perfusion in small animals using the self-regulating extracorporeal membrane oxygenation circuit. The present study was designed to investigate the biochemical and morphological changes in the liver during isolated liver perfusion with double bypass using automatic blood pumps. Isolated liver perfusion was performed with bypass between the hepatic and portal veins in seven weanling Yorkshire swine weighing 8.2 to 12.2 kg, at a flow rate of 20 mL/min/kg for up to 4 hours. Venous blood from the intestine and lower body was bypassed to the superior vena cava. As a result, perfusate glutamic pyruvic transaminase and lactate concentrations did not change during liver perfusion. On gross inspection, the surface of the liver was mottled. Microscopically, normal histology of the hepatic parenchyma and portal tract structures was preserved. Transmission electron microscopy showed no gross structural abnormalities in most of the hepatocytes for up to 4 hours. However, swelling of the mitochondria and smooth endoplasmic reticulum was seen occasionally in a very small number of the hepatocytes after more than 3 hours of perfusion. Glycogen granules decreased with time in some animals. Isolated liver perfusion at 20 mL/min/kg of perfusion flow can be performed safely for up to 4 hours with nearly intact hepatocellular function and morphology.

Recent studies have shown high levels of calcium in activated dendritic spines, where the smooth endoplasmic reticulum (SER) is likely to be important for regulating calcium. Here, the dimensions and organization of the SER in hippocampal spines and dendrites were measured through serial electron microscopy and three-dimensional analysis. SER of some form was found in 58% of the immature spines and in 48% of the adult spines. Less than 50% of the small spines at either age contained SER, suggesting that other mechanisms, such as cytoplasmic buffers, regulate ion fluxes within their small volumes. In contrast, >80% of the large mushroom spines of the adult had a spine apparatus, an organelle containing stacks of SER and dense-staining plates. Reconstructed SER occupied 0.001–0.022 μm3, which was only 2–3.5% of the total spine volume; however, the convoluted SER membranes had surface areas of 0.12–2.19 μm2, which were 12 to 40% of the spine surface area. Coated vesicles and multivesicular bodies occurred in some spines, suggesting local endocytotic activity. Smooth vesicles and tubules of SER were found in continuity with the spine plasma membrane and margins of the postsynaptic density (PSD), respectively, suggesting a role for the SER in the addition and recycling of spine membranes and synapses. The amount of SER in the parent dendrites was proportional to the number of spines and synapses originating along their lengths. These measurements support the hypothesis that the SER regulates the ionic and structural milieu of some, but not all, hippocampal dendritic spines.

In this study, 11,284 mg/m3 (2600 ppm) of xylene was administered for 8 hours a day to pregnant rats by means of inhalation, starting from the sixth day of their pregnancies. Furthermore, while a group of non-pregnant rats inhaled the same amount of xylene during the same period, the control group inhaled clean air. Consequently, in addition to the embryotoxic effects of xylene, the effects on the various tissues of the mothers and their litters were observed light and electron microscopes. No external anomalies were observed in any of the rats born at the end of the 21st day, and there were no macroscopic defects in their organs either. While following xylene inhalation no structural defects in the kidney and pancreas was found, expansions in the smooth endoplasmic reticulum of the liver tissues, increases in the lysosomes, and defective mitochondrion structures were found in the pregnant and non-pregnant rats. It was noticed that xylene in particular caused structural defects in the liver of the fetus. Compared to the control groups, increases were observed in the activities of the AST, ALT, ALP, and Arginase enzymes in the liver.

In previous studies, 2,4-dithiobiuret (DTB) caused a delayed onset neuromuscular weakness in rats which was associated with decreased quantal content, alterations in postsynaptic ion channel properties, and abnormalities in nerve terminal ultrastructure. The latter include features typical of degenerating or diseased motor endplates as well as a marked proliferation of smooth endoplasmic reticulum (SER), swelling of mitochondria and evidence for a decreased in intraterminal calcium concentrations at early stages of intoxication (Jones, 1989, Acta Neuropathol. 78:72). These in vivo studies do not allow us to distinguish between the initial effects of DTB on the nerve terminal and those evolving as a result of disuse or secondary to its action on the muscle fiber or Schwann cells. To begin to distinguish between primary and secondary effects of DTB, we examined DTB-treated rat PC12 cells for comparable changes. The direct effects of DTB on PC12 cells included signs of general toxicity. Cell death in sparsely- plated cultures increased from 8-9% in controls to 13.7% at 10 microM for 24 hr exposure, and continued to increase in a concentration-dependent fashion to 25% mortality at 25 microM. However, between 25 and 100 microM there was little additional increase in mortality. 10 to 40 microM DTB slightly decreased the ability of both differentiated and undifferentiated cells to adhere to a substrate. This effect was independent of cell mortality. In moderately-differ-entiated cells having processes up to 10 cell diameters and several varicosities, concentrations of DTB as high as those invoking increased cell mortality and comparable to those affecting the rat neuromuscular junction did not cause abnormalities in the structure of the SER. No masses of tubulovesicular profiles were seen with transmission electron microscopy, and large changes in the quantity or distribution were not detected at the light microscope with the fluorescent stains DiOC6 or rhodamine B. Other signs of neuronal degeneration (blebbing of the plasmalemma, large intracellular droplets, mitochondrial abnormalities) preceded or accompanied any evidence for abnormalities in the SER. Thus the effect of DTB on the SER at the rat motor nerve terminal may occur secondary to a more general toxic action on other cell types, or may be dependent on a level of neuronal activity not achieved in sparsely- plated cultures, or may require a greater degree of differentiation of the neuronal cells than provided by the PC12 cell model used in this study.

The morphogenesis of a Dutch PRRS field strain virus (Lelystad virus) was studied and compared to that of a U.S. field strain VR2332 and its attenuated vaccine strain JJ1882. Porcine lung alveolar macrophages (PLAM) and CL2621 cells were infected with high doses of virus (MOI = 10). At 4, 6, 9, 12, 18, 24, and 48 h post infection (hpi) cells were fixed for electronmicroscopy or for detection of viral antigens by immunoperoxidase staining. From 6 hpi on, viral antigens were detected in the cytoplasm and from 9 hpi on completely assembled virus particles could be detected in infected cells. The three strains were similar in assembly of new virus particles, envelopment at the smooth endoplasmic reticulum, and egress from infected cells. However, distinct differences were seen in replication time of the three strains in various cell types. The Lelystad virus replicated very fast and efficiently in PLAM while VR2332 and JJ1882 replicated preferably in CL2621 cells. JJ1882 replicated faster in CL2621 cells than VR2332 did, probably because of increased adaptation to the cell-line. Although the U.S. and European strains differ at the level of the genome, morphogenesis is not visibly altered. There is however a distinct difference in preferred cell type between the European strain and the two U.S. strains.

Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens in egg production were examined by light and electron microscopy. Based on oocyte and germinal vesicle (GV) diameters and organelle morphology and distribution, the sequential development of the primary oocyte was divided into five stages. No Balbiani body was observed in Stage I oocytes (80 microns in diameter). Pleomorphic mitochondria were localized around the GV and multivesicular bodies were scattered in the ooplasm. By Stage II (81 to 150 microns), the Balbiani body was observed adjacent to the GV. Pleomorphic mitochondria, macrobodies, and smooth endoplasmic reticulum (SER) were associated with the Balbiani body. Lipid droplets were predominantly localized to the periphery of the oocyte. The Balbiani body was partially dispersed by Stage III (151 to 350 microns) and associated organelles appeared in clusters in the ooplasm. Golgi and SER were observed immediately subjacent to the oolemma. Stage IV oocytes (351 to 500 microns) were characterized by the absence of the Balbiani body, a more centrally located GV, and the redistribution of the mitochondria to the periphery of the oocyte. Throughout the ooplasm was vesicular SER. By Stage V (501 to 800 microns), zonation of the organelles was completed with the mitochondrial ring immediately subjacent to the oolemma and a concentric layer of lipid droplets subjacent to the mitochondrial ring. The GV was in the periphery of the oocyte. Organelle and inclusion redistribution and organelle pleomorphism were presumed to be reflective of increasing metabolic and transport requirements of the growing oocyte in the mature turkey hen.

Electron microscopy of the maturing neurons and developing and maturing synapses in the substantia nigra of 14 human embryos/foetuses of 8-24 weeks of gestation are reported. At 8 weeks, cells were immature with very little cytoplasm and cellular organelles. Contact sites of processes appeared more electron dense than the other areas. At 12 weeks, many of the cells had acquired more cytoplasm and cellular organelles and could be identified as neurons. Asymmetric synapses with clear, round synaptic vesicles also were identifiable at this age. Such synapses, first to appear in the developing substantia nigra, are reported to be formed by recurrent collateral nigro-striatal fibres. Substance P fibres from the striatum also are contributing to this type of synapse. At 15-16 weeks, not only was the number of such synapses increased, but many appeared morphologically mature. Symmetric synapses having clear round vesicles along with a few dense core vesicles also appeared at this stage, suggesting striatal input. By 24 weeks of gestation, most of the neurons had cytological features comparable to that of the mature neurons. There was an increase in the total number of synapses and the individual variety from 15 to 24 weeks of gestation. The present study indicates that synaptogenesis starts at 8 weeks and continues beyond 24 weeks of gestation.

A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils. The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types. Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes. The cells showed little evidence of intercellular contact even when confluent. The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase. The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament. These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics. Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis. The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis.

We studied the possible impairment of fast axonal transport in patients with amyotrophic lateral sclerosis (ALS) to gain some insight into the pathogenesis of the disease.We carried out an ultrastructural investigation of the proximal axons (axon hillock and initial segment) of the anterior horn neurons on samples from 11 ALS patients; specimens from 12 age-matched individuals who died of nonneurological diseases served as controls. Eighty-seven proximal axons that emanated directly from normal-appearing neurons were examined in each group of subjects. Increased smooth endoplasmic reticulum (SER) and the formation of bundles of fibrillary SER with a single unit membrane were not uncommonly observed in the initial segment of the patients with ALS. In some instances, there was loss of the parallel SER arrangement along the longitudinal axis. When viewed in transverse sections, the bundles had a tubular appearance. These morphologic changes of SER were exclusively demonstrated in patients with ALS. A marked increase or accumulation of mitochondria and lysosomes was more common in the proximal axons, particularly in the axon hillock, of ALS patients than of control subjects. The accumulation of these membrane-bounded cytoplasmic organelles suggests that fast axonal transport is impaired in the proximal axons of individuals with ALS. In addition, there were Lewy body-like hyaline inclusions, lipofuscin granules, and multiple membranous structures in the proximal axons. The presence of these unusual structures may also be a reflection of axonal transport dysfunction. By contrast, in the central chromatolytic neurons, there was not only a decrease in the number of neurofilaments in the axon hillock and initial segment, but also of mitochondria, lysosomes, and SER. In some instances, none of these cytoplasmic organelles was seen. These findings support the notion that the outflow of cytoplasmic constituents from the anterior horn cell body into the proximal axon may be impaired in central chromatolytic neurons.NEUROLOGY 1996;47: 535-540

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

Mature male rainbow trout (Oncorhynchus mykiss) were exposed for 28 days to 0, 1, 5, and 20 micrograms/liter disulfoton, i.e., to concentrations well below any macroscopically visible effect due to the primary acute toxic mechanism of acetylcholine esterase inhibition. In an attempt to reveal sublethal injury of disulfoton in rainbow trout, ultrastructural and stereological parameters were recorded in the liver as the central organ of xenobiotic metabolism in fish. Quantitative methods were definitely not able to replace qualitative techniques because, except for mitochondria, peroxisomes, and hepatocellular lipid inclusions, stereological analysis revealed only insignificant variations of hepatocellular components, whereas hepatocytes displayed a complex pattern of numerous delicate qualitative alterations. Effects were most evident within cisternae of the rough endoplasmic reticulum (RER), thus suggesting modifications of protein metabolism. Structural alterations included degenerative effects such as dilation and vesiculation of RER cisternae, formation of concentric RER arrays and augmentation of smooth endoplasmic reticulum, dilation of Golgi cisternae, and the development of cytoplasmic myelinated bodies as well as stacks of membranous material within mitochondria. Structural integrity and augmentation of peroxisomes and mitochondria as well as increased activity of the Golgi system were indicative of adaptive/compensative reactions following disulfoton treatment. In fact, adaptive effects seemed more pronounced than degenerative phenomena resulting in only minor disturbances in hepatocyte structure following disulfoton exposure. Because most effects had to be classified as unspecific responses to environmental or xenobiotic stressors, no distinct mode of sublethal action can be suggested for disulfoton.

The stereology work on the hepatocyte smooth endoplasmic reticulum (SER) and mitochondria was performed in order to bring evidence of changes in the hepatocyte ultrastructure during acute ethylene glycol poisoning. Hepatocytes of Wistar rats were examined 1, 5, and 14 days after acute intoxication. On the ground of measurements made with a computer image analysis system, the following stereological parameters, which characterize the examined organelles, were calculated: volume density of the SER cisterns, surface density of the SER membranes, numerical density of the mitochondria and volume density of mitochondria. It has been stated that on the first day after intoxication proliferation of SER takes place, and this shows active participation of smooth reticulum enzymes in the metabolism of ethylene glycol. The decrease of SER quantity on 5th and 14th day after intoxication may probably be the result of synthesis handicap of structural proteins. The decrease of mitochondria indexes during the 1st day can be the result of considerable sensibility of those organelles to toxic action of ethylene glycol metabolites and metabolite acidosis being the result of intoxication. The progressive increase of mitochondria quantity on the 5th and 14th day shows that gradually the compensatory mechanisms are initiated.

As part of a comparative morphological study, the fine structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in the galah (Eolophus roseicapillus), an Australian cockatoo. The RPE consists of a single layer of low cuboidal cells joined basally by a series of zonulae occludentes. Basally (sclerally) the retinal epithelial cells display numerous deep infoldings while apically (vitreally) microvillar processes interdigitate with photoreceptor outer segments. Internally the RPE cells show a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and numerous polysomes but very little rough endoplasmic reticulum (RER). Numerous mitochondria are located basally. In the light-adapted specimens studied, the melanosomes of the RPE are almost exclusively located within the apical processes indicating that retinomotor movement of this pigment probably occurs. Phagosomes and lysosome-like bodies are present as are myeloid bodies which may show ribosomes on their outer surface. The choriocapillaris endothelium is thin but only minimally fenestrated facing Bruch's membrane. Most fenestrations of the choriocapillaris display a single-layered diaphragm while the remainder have a double-layered diaphragm. Bruch's membrane (complexus basalis) is typical of avian species in that it is pentalaminate with the central lamina densa displaced towards the choriocapillaris.

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.

Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteine proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.

The retinal pigment epithelium (RPE) as well as the choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the barred owl (Strix varia). The RPE consists of a single layer of cuboidal cells joined laterally by a series of tight junctions that forms part of the blood-ocular barrier. Basally (sclerally) the retinal epithelial cells display numerous deep infoldings while apically (vitreally) microvillar processes interdigitate with the photoreceptor outer segments. Internally the RPE cells show a large vesicular nucleus, plentiful smooth endoplasmic reticulum (SER) and polysomes but very little rough endoplasmic reticulum (RER). Numerous pleomorphic (including ring-shaped) mitochondria are basally located. In the light-adapted state the small melanosomes are almost exclusively located within the apical process indicating that retinomotor movements probably occur. Phagosomes and lysosome-like bodies are present as are myeloid bodies which may show ribosomes on their outer surface. Bruch's membrane is typical of avian species in that it is pentalaminate and the central lamina densa is displaced towards the choroid. The choriocapillaris endothelium is thin but only minimally fenestrated facing Bruch's membrane. Most fenestrations present show a single-layered diaphragm while others display a double-layered diaphragm as noted in other avian species.

Structural heterogeneity and contact interactions of vascular dendritic cells (VDCs) in early atherosclerotic lesions of the human aortic intima were studied using electron microscopy. VDCs were identified through their ultrastructural features which were typical of dendritic cells. From their morphological characteristics, two phenotypes of VDCs (types I and II) were distinguished. VDCs were found to be distributed throughout atherosclerotic lesions, and through their processes were seen to be in contact with each other and with other intimal cells. VDCs developed multiple contacts with intimal macrophages, smooth muscle cells and foam cells. Smooth muscle cells contacting VDCs were mainly of synthetic phenotype suggesting the involvement of VDCs in the regulation of cell phenotypes. Occasionally, contacts between VDCs and lymphocyte-like cells were also observed. We speculate that through cell-to-cell contacts, VDCs are involved in immune reactions in atherosclerotic lesions.

Chicken chondrocytes in culture synthesize aggrecan proteoglycan as a 370 kDa precursor that is glycosylated and secreted into the medium with a half-life of 30 min. In metabolic studies the 370 kDa precursor was shown to render a degradation intermediate of 190 kDa, which appeared with no measurable lag phase; it was dependent on temperature (> 20 °C) and inhibited by certain serine and serine/cysteine protease inhibitors such as leupeptin and PMSF. By contrast, degradation was unaffected by treatment of the cells with brefeldin A or with lysosomotropic agents. Aggrecan precursors were detected by immunofluorescence analysis within a subcompartment of the endoplasmic reticulum (ER), previously characterized as a smooth-membrane-bound subregion [Vertel, Velasco, LaFrance, Walters and Kaczman-Daniel (1989) J. Cell Biol. 109, 1827–1836]. Analysis of the subcellular fractions derived from chondrocytes indicated that the degradation intermediate was concentrated in the ER subcompartment. Degradation was dependent on the Ca2+ concentration and the redox state in the ER. Treatment of the cells with agents or conditions that alter the degradation rate of aggrecan precursors, such as protease inhibitors, decreased temperature or dithiothreitol, also modified the retention of these molecules in the ER subcompartment, as seen by immunofluorescence. These results indicate that a fraction of the 370 kDa aggrecan precursor is targeted to a smooth ER subcompartment where it undergoes degradation.

PCB 156 (2,3,3',4,4',5-hexachlorobiphenyl) congener was given to weanling Sprague-Dawley rats in diets prepared by mixing it in 4% corn oil. The animals were placed in eight groups, each comprising 10 males or females and received diets that contained 0.01, 0.1, 1, 10 ppm PCB; in addition, two control groups of rats of each gender were given diets mixed with corn oil. Thirteen weeks after commencement of dosing, animals were euthanized and liver specimens were harvested and prepared for transmission electron microscopy. Hepatocyte architectural alterations comprised augmentation of smooth reticulum profiles and mitochondria with unorthodox cristae in animals regardless of gender from 1 and 10 ppm groups. Hepatic microsomal aminopyrine-N-demethylase was elevated significantly in both genders at highest (10 ppm) congener concentration. Based on our previous work, PCB 156 is estimated to be more toxic than PCB 153 or 28 in terms of liver morphologic expressions.

Structure of smooth endoplasmic reticulum and cytoskeleton of goldfish Mauthner neurons were studied together with the behavior of fish after partial deafferentation of one of the dendrites with following long-term natural stimulation of another one and soma. Strong disturbance of cytoskeleton and proliferation of smooth endoplasmic reticulum especially of its transversely localised component accompanies exhaustion of Mauthner neurons caused by stimulation in normal absolute afferentation. On behavioral level it is manifests in significant suppression of fish motor activity. Similar stimulation of partly deafferentated Mauthner neurons turns to be more sparing both for fish behaviour and Mauthner neurons structure. Proliferation of endoplasmic reticulum was not in fact observed and in the cytoskeleton bundles of filaments were detected which implies improvement of its physico-chemical characteristics and increase of resistance to external factors. Probably, this relative resistance of partly deafferentated Mauthner neurons of fish to long-term natural stimulation is conditioned predominantly by postsynaptic mechanisms and is likely linked with reorganization in cytoskeleton, caused by preliminary change of Mauthner neurons innervation.

Lead is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, we have examined the effects of lead on the biosynthesis of steroid hormones by Leydig cells in the rat. To determine whether lead has a direct action on Leydig cells, we have compared the concentrations of testosterone secreted by Leydig cells in ex vivo experiments after animals had been injected with high doses of lead and in vitro experiments with Leydig cells from normal rats maintained in culture in presence or absence of lead. In ex vivo experiments male Sprague-Dawley rats were injected i.p. with lead acetate (8 mg lead/kg/day, 5 days a week for S weeks) or with sodium acetate. Testosterone production by Leydig cells isolated and maintained in culture for 48 h was then assessed under basal conditions or after stimulation by human chorionic gonadotrophin (hCG). Both basal and hCG-stimulated testosterone production dropped by 59% and 37%, respectively, with Leydig cells from lead-exposed rats. For in vitro experiments, cultures of Leydig cells from control rats were exposed to various concentrations of lead acetate for different periods. Dose and time-dependent reductions of testosterone level were observed in the culture medium. The effective doses of hCG for maximal and half-maximal testosterone production did not change, indicating that the sensitivity of Leydig cells to hCG was not impaired by exposure to lead in vitro. Progesterone production was also decreased after this exposure. The negative effect of lead on testosterone and progesterone production was correlated with the lower expression of the enzymes cytochromes P450scc (CYP11 A1) and P450c17 (CYP17) and 3β-hydroxysteroid dehydrogenase (3β-HSD) involved in steroid hormone biosynthesis, as shown by immunohistochemistry. Ultrastructural alterations of the smooth endoplasmic reticulum observed after lead administration might be correlated with the lower expression of the microsomal enzymes P450cl7 and 3β-HSD. Our results indicate that lead can adversely affect the Leydig cell function by impairing directly steroidogenesis.

The slow wave frequency of the canine colon has previously been hypothesized to be paced by an intracellular biochemical clock. We investigated the relationship between the endoplasmic reticulum (ER) Ca++ and the periodicity of the biochemical clock. Cyclopiazonic acid, a specific inhibitor of the ER Ca++ pump, dose-dependently decreased the pacemaker frequency. Similarly, chelating cytosolic Ca++ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) also decreased the pacemaker frequency. These observations suggest that delaying the Ca++ uptake into the ER decreases the pacemaker frequency. The pacemaker frequency was similarly decreased by neomycin [inhibiting inositol 1,4,5-triphosphate (IP3) synthesis] and by caffeine at concentrations higher than 5 mM (inhibiting the IP3-sensitive Ca++ channels in the ER membrane). Hence the IP3-sensitive Ca++ stores are involved in the biochemical clock. Ryanodine (up to 60 microM) did not affect the pacemaker frequency, which indicates that a ryanodine-sensitive store, if it exists, is not coupled to the biochemical clock. Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae that is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in interstitial cells of Cajal, slightly less in branching smooth muscle cells and far less in circular muscle cells. In summary, on the basis of these electrophysiological and morphological observations, we hypothesize that the Ca++ refilling cycle of the IP3-sensitive calcium stores associated with the plasma membrane determines the frequency of the pacemaker activity generated by the submuscular interstitial cells of Cajal-smooth-muscle network of the canine colon.

The effects of nicardipine, a Ca2+ channel antagonist, on the abnormal excitability of hippocampal CA3 neurons in spontaneously epileptic rats (SER), a double mutant (zi/zi, tm/tm), were examined to elucidate whether or not the abnormality was due to that of Ca2+ channels. An intracellular recording study was performed using brain slice preparations of SER 12–15 weeks of age, when SER showed both tonic convulsions and absence-like seizures. Bath application of nicardipine (10 nM) completely inhibited the depolarizing shifts lasting for 60–120 ms and accompanying repetitive firings on mossy fiber stimulation in SER. However, this drug did not affect the single action potential induced by the mossy fiber stimulation in CA3 neurons of SER and normal Wistar rats. In the CA3 pyramidal neurons of SER, the Ca2+ spikes induced by the depolarizing pulse applied in the cell in the presence of tetrodotoxin and tetraethylammonium had a different configuration from that in normal Wistar rats. Nicardipine also inhibited the Ca2+ spikes in SER CA3 neurons at a concentration (1 nM) that had no effect on those in normal Wistar rats, while the Ca2+ spikes in Wistar rat CA3 neurons were inhibited by 10 nM nicardipine. These findings suggest that the abnormal excitability of CA3 pyramidal neurons in SER might be attributed to abnormalities of the Ca2+ channels, and that the Ca2+ channel antagonist may be effective as an antiepileptic drug.

In order to shed more light on the controversial tissue histogenesis of the stromal cells (SC), light microscopic, immunohistochemical and electron microscopic studies were performed on surgical specimens of hemangioblastomas (36, 26 and 7 cases, respectively). SC were immunoreactive for vimentin, S-100 protein, and neuron specific enolase (NSE) in all cases. Occasional SC were also positive for desmin, smooth muscle actin, Factor VIII, Ulex europaeus lectin receptors, GFAP, and Factor XIIIa. However, majority of these cells were negative with all the endothelial and smooth muscle cell markers used. Electron microscopy demonstrated several different types of SC that were reminiscent of pericytes, smooth muscle cells and abnormal endothelium as well as the intermediate forms between all the above cell types. Few SC were found lining the vascular lumina. Some SC formed small cavities reminiscent of early capillaries. However, typical Weibel-Palade bodies were not found in these SC. It is concluded that SC represents a heterogeneous population of lipidized cells, derived predominantly from the vasogenic mesenchyme. Although immunohistochemistry failed to reveal any consistent antigenic property of SC, ultrastructural findings strongly support the hypothesis that these cells are modified or abnormally differentiated endothelial cells and pericytes.

Objective To compare demographic, embryologic, pregnancy, and newborn outcomes after intracytoplasmic sperm injection (ICSI) cycles with or without mature oocytes (metaphase II [MII]) showing visible aggregates of tubular smooth endoplasmic reticulum (aSERT) and to describe the ultrastructure of this dysmorphism. Design Retrospective study. Setting Private fertility center and university cell biology and genetics departments. Patient(s) There were 721 ICSI cycles, 520 carrying morphologically normal MII (control group) and 60 containing aSERT-MII (study group). Intervention(s) None. Main Outcome Measure(s) Embryologic and clinical and live birth outcomes, including malformations and ultrastructural characterization of aSERT-MII. Result(s) Compared with the control group there was a significant decrease in the fertilization, embryo cleavage, and blastocyst rates in the study group. The only child born after transfer of embryos derived from aSERT-MII presented a major cardiovascular malformation. Ultrastructurally, large aSERT were surrounded by abnormal-shaped mitochondria and clusters of small dense bodies formed by very small vesicles, and they had curvilinear dense tubules in the interior. The same pathology was observed in small peripheral aSERT. Conclusion(s) The presence of large aSERT, showing attainment of the periphery, demonstrated that the cytoplasm is pathologic. The compromised embryo development and implantation was associated with decreased clinical outcomes and newborn malformations. Therefore, oocytes with large aSERT should not be used for embryo transfer.

Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.

Summary Sebastian platetet syndrome is an hereditary thrombocytopenia with giant platelets and inclusion bodies in the granulocytes consisting of dispersed filaments, clusters of ribosomes and a few segments of rough and smooth endoplasmic reticulum at the ultrastructural level, similar to those observed in Fechtner syndrome (a variant of the AIport syndrome) — Sebastian ptatelet syndrome lacks the additional clinical features such as high frequency deafness, congenital cataract, and chronic interstitial nephritis. Here we report the fourth case worldwide and the first of an Arabian ancestry.

The development of Antechinus stuartiifrom the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2–8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell–zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2–4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.

In gland cells of the exocrine pancreas of the guinea pig and the anterior pituitary of the hamster both of which were fed in normal condition, electron microscopy explored the occurrence of curious concentric lamellae which are probably composed of multiple apposition of many cup-shaped cisternae of the smooth-surfaced endoplasmic reticulum. Cavities of the cisternae are empty and almost collapsed in the case of the guinea pig pancreas, but in the hamster pituitary they contain a relatively large amount of homogeneous substance. It must be emphasized that these bodies, in both of the pancreas and the pituitary, are not whorls or spirals but they are actually concentric circles as seen in some plane of sectioning. In another plane of sections such as those vertical to the former, the structure may be seen like an up-to-down cut-surface of onions. Most of the membranes of these concentric lamellae are smooth-surfaced, but peripheral parts may be studded with ribosomes. The structures reported here may be one of the variations in arrangement of the endoplasmic reticulum, but functional or pathological (if this structure is an abnormal being) significance of these lamellar bodies may not be decided at the present level of accumulation of knowledges concerning this structure.

Translocational pausing is a mechanism used by certain specialized secretory proteins whereby discrete domains of a nascent chain destined for the endoplasmic reticulum lumen are transiently exposed to the cytosol. Proteoliposomes reconstituted from total endoplasmic reticulum proteins properly assemble translocationally paused intermediates. The capacity of the translocon to correctly pause the nascent chain is dependent on a glycoprotein fraction whose active component is TRAM. In the absence of TRAM, the normally sealed ribosome-membrane junction still opens in response to a pause transfer sequence. However, nascent chain domains that are not exposed to the cytosol in the presence of TRAM are so exposed in its absence. Thus, TRAM regulates which domains of the nascent chain are visible to the cytosol during a translocational pause.

The endoplasmic reticulum (ER) has distinct morphological domains composed of sheets and tubules, which differ in their characteristic membrane curvature. Key proteins may drive the formation of these structural morphologies, which in turn could generate the rough and smooth functional domains of the ER.

Graphical abstract, Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR− rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane.