Vacuolar changes in neuromuscular disorders: a morphometric study

BACKGROUND: Vacuoles in muscle represent a nonspecific alternation and are found in a variety of neuromuscular disorders. To understand their significance, a morphometric study of the vacuolar formation in muscle biopsies from 340 patients was reviewed. METHODS: Vacuolar changes in muscles were found in 24 out of 340 patients with muscle biopsies. The specimens were prepared for histopathological, histochemical and electron microscopic examinations. The location, size, shape, number and content of the vacuoles were recorded. The number of fibers containing vacuoles was also assessed. RESULTS: Observed after modified Gomori trichrome stain, there were rimmed and non-rimmed vacuoles. Rimmed vacuoles were found in limb-girdle myopathy with rimmed vacuoles (5), oculopharyngeal muscular dystrophy (1). inclusion body myositis (2) and neurogenic disorder (1). These vacuoles were usually cleft-like in shape in 5-16% of muscle fibers with a diameter of 3-20 microns. An elevation of acid phosphatase activities and the presence of cytoplasmic debris suggested an autophagic process. In 15 patients with non-rimmed vacuoles, round and oval shapes with variable sizes and numbers were noted. Four with acid maltase deficiency (AMD) had numerous vacuoles containing glycogen, while three with lipid storage disease contained lipid. Interestingly, in AMD the size of the vacuoles was usually more than 10 microns in diameter, but less than 5 microns in lipid storage disease. In other diseases including polymyositis (5), Duchenne muscular dystrophy (2) and hypokalemic periodic paralysis (1), the numbers of vacuoles were usually fewer than three in each fiber. CONCLUSIONS: Vacuolar changes were not specific, but morphometric analysis of the vacuoles may provide some clues in differential diagnosis. The vacuoles were usually numerous in glycogen and lipid storage diseases, while rimmed vacuoles may be found in limb-girdle myopathy with rimmed vacuoles, oculopharyngeal muscular dystrophy, and inclusion body myositis.     

Conformational changes of the smooth endoplasmic reticulum are facilitated by L-glutamate and its receptors in rat Purkinje cells

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week.     

Association of spectrin with a subcompartment of the endoplasmic reticulum in honeybee photoreceptor cells

In order to investigate the mechanisms involved in originating a diverse TCR repertoire in human peripheral blood we analyzed TCRV beta surface expression in different T cell subsets of unrelated individuals. The relative frequencies of 11 distinct V beta chains were determined for immature double positive (DP) as well as for mature CD4 single positive (4SP) and CD8 single positive (8SP) thymocytes, respectively. By comparing these data with expression in peripheral blood T lymphocytes of the same donors we were able to show that usage of TCRV beta in peripheral T cells is significantly (p < 0.001) depending on the pattern in mature SP thymocytes whereas the frequency of TCRV beta families in immature DP thymocytes has no impact (p > 0.2). No association with distinct HLA-haplotypes was observed. Preferential usage of V beta-families in either CD4- or CD8-positive peripheral T cells also correlates with the status in mature thymic precursors (p < 0.001). Altogether, this first combined study of TCR frequencies within different stages of human T cell ontogeny indicates that TCRV beta repertoire is determined mainly through selectional processes within the thymus. Since neither genomically imposed expression nor modulating events in the periphery seem to have strong influence on the relative expression of TCRV beta chains these findings have to be considered in future studies of human diseases.     

Protein folding: a missing redox link in the endoplasmic reticulum

Native disulphide-bond formation during protein folding in the endoplasmic reticulum requires oxidative machinery, the components and mechanism of which are not yet fully understood. Two recent papers have identified a novel protein component that appears to play a key role in this important redox pathway.     

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.     

Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells

Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.     

Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis

1-Cyano-2-hydroxy-3-butene (CHB) has been reported to cause cell death in rat pancreatic acini. In this report, we describe the time-dependent effects of CHB on mouse acinar cell apoptosis and the effects of CHB-induced acinar cell apoptosis on the severity of secretagogue-induced acute pancreatitis in mice. CHB administration to mice resulted in a time-dependent increase in pancreatic apoptosis, which was maximal 12 hours after CHB administration. The severity of pancreatitis was significantly reduced by prior CHB administration and maximal protection was observed when the caerulein injections were started 12 hours after CHB administration. These observations indicate that induction of apoptosis can reduce the severity of pancreatitis and they suggest that induction of pancreatic acinar cell apoptosis may be beneficial in the clinical management of acute pancreatitis.     

Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells. A comparative study

We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+]er) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplamsic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer study of the dynamics of [Ca2+]er. The steady-state [Ca2+]er (500-600 microM) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 degrees C. The behavior of both [Ca2+]er and cytoplasmic [Ca2+] ([Ca2+]c) after the addition of increasing concentrations of agonists and/or Ca2+-ATPase inhibitors, or following incubation in Ca2+-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca2+]er, which is enough to produce a sharp increase in [Ca2+]c. Termination of Ca2+ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca2+]c, a mechanism that appears to be designed to release the minimum amount of Ca2+ necessary to produced the required [Ca2+]c signal. We also show that Ca2+ release is inhibited progressively when [Ca2+]er decreases below a threshold of about 150 microM, even in the absence of Ca2+ pumping or -Ca2+-c increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca2+]er.     

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.     

The role of actin filaments in the organization of the endoplasmic reticulum in honeybee photoreceptor cells

Close to the bases of the photoreceptive microvilli, arthropod photoreceptors contain a dense network of endoplasmic reticulum that is involved in the regulation of the intracellular calcium concentration, and in the biogenesis of the photoreceptive membrane. Here, we examine the role of the cytoskeleton in organizing this submicrovillar endoplasmic reticulum in honeybee photoreceptors. Immunofluorescence microscopy of taxol-stabilized specimens, and electron-microscopic examination of high-pressure frozen, freeze-substituted retinae demonstrate that the submicrovillar cytoplasm lacks microtubules. The submicrovillar region contains a conspicuous F-actin system that codistributes with the submicrovillar endoplasmic reticulum. Incubation of retinal tissue with cytochalasin B leads to depolymerization of the submicrovillar F-actin system, and to disorganization and disintegration of the submicrovillar endoplasmic reticulum, indicating that an intact F-actin cytoskeleton is required to maintain the architecture of this domain of the endoplasmic reticulum. We have also developed a permeabilized cell model in order to study the physiological requirements for the interaction of the endoplasmic reticulum with actin filaments. The association of submicrovillar endoplasmic reticulum with actin filaments appears to be independent of ATP, Ca2+ and Mg2+, suggesting a tight static anchorage.     

Insertion of a bacterial secondary transport protein in the endoplasmic reticulum membrane

The sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) contains 12 hydrophobic potential transmembrane domains. Surprisingly, an alkaline phosphatase fusion study in Escherichia coli has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). To provide independent data on the topology and mode of membrane insertion of CitS, we have investigated its insertion into the endoplasmic reticulum (ER) membrane. By using in vitro translation of model proteins in the presence of dog pancreas microsomes, each of the putative transmembrane segments of CitS was assayed for its potency to insert into the ER membrane, both as an isolated segment as well as in the context of COOH-terminal truncation mutants. All 12 segments were able to insert into the membrane as Ncyt-Clum signal anchor sequences. In a series of COOH-terminal truncation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segments VIII and IX, which, according to the alkaline phosphatase fusion study, are in the periplasm of E. coli, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segments and also demonstrate that the sequence requirements for signal anchor and stop transfer function are different.     

Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells

Proteins entering the secretory pathway may be glycosylated upon transfer of an oligosaccharide (Glc3Man9GlcNAc2) from a dolichol-P-P derivative to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). Oligosaccharides are then deglucosylated by glucosidases I and II (GII). Also in the ER, glycoproteins acquire their final tertiary structures, and species that fail to fold properly are retained and eventually degraded in the proteasome. It has been proposed that in mammalian cells the monoglucosylated oligosaccharides generated either by partial deglucosylation of the transferred compound or by reglucosylation of glucose-free oligosaccharides by the UDP-Glc:glycoprotein glucosyltransferase (GT) are recognized by ER resident lectins (calnexin and/or calreticulin). GT is a sensor of glycoprotein conformation as it only glucosylates misfolded species. The lectin-monoglucosylated oligosaccharide interaction would retain glycoproteins in the ER until correctly folded, and also facilitate their acquisition of proper tertiary structures by preventing aggregation. GII would liberate glycoproteins from the calnexin/calreticulin anchor, but species not properly folded would be reglucosylated by GT, and so continue to be retained by the lectins. Only when the protein becomes properly folded would it cease to be retained by the lectins. This review presents evidence suggesting that a similar quality control mechanism of glycoprotein folding is operative in Schizosaccharomyces pombe and that the mechanism in Saccharomyces cerevisiae probably differs substantially from that occurring in mammalian and Sch. pombe cells.     

Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae

Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles. In a cell-free system, this process is ATP- and cytosol-dependent. While yeast cytosol promotes the export of glycopeptides from mammalian ER in vitro, glycopeptide release cannot be detected in the presence of mammalian cytosol. We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes. Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes. The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.     

Action of cholecystokinin and cholinergic agents on membrane-bound calcium in dispersed pancreatic acinar cells

In dispersed acinar cells prepared from guinea pig pancreas, cellular uptake of 45Ca was moderately rapid and reached a steady state by 60 min. At the steady state, 69% of total cellular 45Ca was membrane-bound. In acinar cells preloaded with 45Ca and then incubated with COOH-terminal octapeptide of cholecystokinin (CCK-OP) or carbamylcholine, total cellular 45Ca decreased by approximately 40% within 5-10 min and then steadily increased to control values by 60 min. Under identical conditions, membrane-bound 45Ca decreased by 40% within 5-10 min and remained constant for the duration of the incubation. Free cellular 45Ca did not change during the initial 30 min but then increased steadily to values three times those in control cells by 60 min. In cells preloaded with 45Ca and then incubated with EDTA, the loss of total cellular radioactivity stimulated by CCK-OP could be accounted for by loss of membrane-bound 45Ca. CCK-OP failed to alter total cellular uptake of 45Ca when both tracer and peptide were added at the beginning of the incubation. Under identical conditions, membrane-bound 45Ca was not altered by CCK-OP during the first 30 min of incubation but was significantly below control values after this time. The effect of CCK-OP on free cellular 45Ca was the same as in cells preloaded with the tracer. These results suggest that CCK-OP causes release of 45Ca from a membrane-bound compartment that equilibrates slowly with extracellular fluid and that the change in free cellular 45Ca is a secondary effect.     

Maleate modifies apical endocytosis and permeability of endoplasmic reticulum membranes in kidney tubular cells

Chromosomal aberrations (CA) were studied in the peripheral blood lymphocytes of 85 healthy male volunteers from Barcelona (Catalonia, Spain). The effect that factors such as age, life style, work exposure and medical treatment had on the cytological endpoints was studied by means of a Poisson regression model. The results obtained indicate a significant positive relationship between the age of the subjects analyzed and the total of chromosome-type aberrations. With respect to the other variables analyzed, a positive association was found between the frequent consumption of analgesics and the incidence of chromatid-type aberrations.     

Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum

Heat shock protein (HSP) 47 is a collagen-binding stress protein localized in the endoplasmic reticulum (ER). In addition to stress-inducibility through heat shock element-heat shock factor interaction, the expression of HSP47 under normal conditions always correlates with that of collagens in various cell types and tissues. Both HSP47 and types I and III collagens are also dramatically induced under pathophysiological conditions such as liver fibrosis. HSP47 transiently associates with procollagen in the ER and dissociates from it in the cis-Golgi compartment. Possible functions of HSP47 as a molecular chaperone specific for procollagen are discussed: prevention of nascent procollagen chains from forming aggregates, effect on the modification of procollagen, inhibition of intracellular degradation of procollagen, quality control mechanisms under stress conditions, and effect on the secretion from the ER to the Golgi compartment.     

The ontogeny of key endoplasmic reticulum proteins in human embryonic and fetal red blood cells

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6-phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate-glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.     

A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Astroglial changes in the cerebral cortex of AIDS brains: a morphometric and immunohistochemical investigation

Sutureless phacoemulsification with implantation of a 7-mm PMMA intraocular lens was performed through a modified scleral tunnel in 100 consecutive patients. This was done to minimize postoperative astigmatism while retaining the advantages of implanting intraocular lenses with large optics. Visual and keratometric results and complications are reported after completion of a follow-up period of 6 months for the first 30 patients. Average uncorrected visual acuity improved from 0.13 preoperatively to 0.30 as early as 1 week postoperatively. Average best-corrected visual acuity improved from 0.23 before surgery to 0.51 as early as 1 week after surgery. No significant changes in visual acuity were recorded thereafter. The absolute value of keratometric astigmatism was not increased significantly at any postoperative examination time. The induced cylinder (Jaffe and Clayman) shifted from -1.27 D x 166 degrees at 1 week to 1.18 D x 91 degrees at 1 month postoperatively without further relevant changes thereafter. Endothelial cell loss did not differ from that reported by other authors after conventional cataract surgery. Corneal thickness was not increased significantly at any postoperative examination time. Implantation of intraocular lenses with large optics through a scleral tunnel allows quick visual rehabilitation as well as early stability of refraction.