Dilated Endoplasmic Reticulum Research Articles

The effect of ultraviolet radiation of pancreatic exocrine cells in mole rats: An ultrastructural study

The purpose of this study was to investigate the ultrastructural effects of ultraviolet C (UVC) radiation on the pancreatic exocrine cells of mole rats. The mole rats were divided into two groups as control and test groups. Control group did not receive any radiation. The other group was irradiated with UV radiation for 14 and 28 days. The pancreatic tissue samples were prepared and then analyzed through transmission electron microscope. Depending on the radiation exposure, it is likely to say that the zymogen granules decreased to more than 70 per cent for the control group and the dilation of rough endoplasmic reticulum and vacuolization of mitochondria increased in the pancreatic exocrine cells at the 14 days of radiation as compared with the control group; however, they were easily observed in the 28 days of radiation exposure. Particularly in the 28 days of radiation, the zymogen granules decreased and vacuolated, and the rough endoplasmic reticulum was frequently shortened and dilated. These findings clearly demonstrated the effects of UVC radiation on pancreatic exocrine cells in an exposure-period dependent manner.

PCB77 Inducing Renal Tubular Cell Apoptosis

PCBs are a family of persistent environmental toxicants with a wide spectrum of toxic features, such as neurotoxic, hepatoxicity, immunotoxicity, endocrine disruption effects, and oncogenic effects. The kidney is the most important organ involved in the elimination of toxins and drugs. To date, little has been done to investigate the potential influence of nephrotoxicity of 3,3’,4,4’- tetrachlorobiphenyl (PCB77). By assessing cell viability and apoptotic cell death in renal tubular epithelial (NRK-52E) cells cultures, we found that PCB77 could decrease cellular viability at least at 30 μM concentration after 3 h exposure. PCB77 was demonstrated to promote DNA breakage resulting in apoptosis. Moreover, apoptotic subcellular morphological changes administration of PCB77 was observed using transmission electron microscopy. Appearance swelling of mitochondria, endoplasmic reticulum dilation and chromatin agglutinate, and other apoptosis cells morphological characteristics could be visible. Due to increased PCB77 concentration, cells viability was decreased. Collectively, our findings identified the morphological mechanism that PCB77-induced nephrotoxicity via promoting renal tubular epithelial cells apoptosis. It is suggested that using and production of PCB77 should be carefully managed to reduce public health risks.

The hsp70 inhibitor VER155008 induces paraptosis requiring de novo protein synthesis in anaplastic thyroid carcinoma cells

In this study, we evaluated the effect of the hsp70 inhibitor VER155008 on survival of anaplastic thyroid carcinoma (ATC) cells. In ATC cells, VER155008 increased the percentages of dead cells and vacuolated cells. VER155008 did not lead to the cleavage of caspase-3 protein regardless of pretreatment with z-VAD-fmk. VER155008 increased LC3-II protein levels but the protein levels were not changed by autophagy inhibitors. VER155008 caused the dilatation of endoplasmic reticulum (ER), and the increased mRNA levels of Bip and CHOP, suggesting paraptosis. VER155008-induced paraptosis was attenuated by pretreatment with cycloheximide. In conclusion, VER155008 induces paraptosis characterized by cytoplasmic vacuolation, independence of caspase, dilatation of ER and induction of ER stress markers in ATC cells. Moreover, VER155008-induced paraptosis requires de novo protein synthesis in ATC cells.

S3-4 - NASH (Nonalcoholic steatohepatitis): A case of multiorganelle failure

The clinical term 'multiorgan failure' lends itself, modified to 'multiorganelle failure', to the cascading events in cellular systems leading to hepatocyte injury, cell death, inflammation and fibrosis and ultimately to cirrhosis in NASH (non-alcoholic steatohepatitis). NASH is one of the most common forms of liver disease and constitutes the severe form of NAFLD (non-alcoholic fatty liver disease). The key features that distinguish potentially progressive NASH from relatively stable non-NASH fatty liver (NNFL, often referred to as simple steatosis) are cellular ballooning, inflammation and fibrosis. These findings, together with steatosis or accumulation of greater than normal hepatic lipid, usually constitute histological NASH seen on liver biopsy or in laboratory samples. Cellular ballooning is not specific to NASH but it is perhaps the most emblematic finding on histological samples. The ballooned hepatocyte has evidence of cytoskeletal injury (depletion and condensation as Mallory-Denk bodies), accumulation of partially oxidized small fat droplets, mitochondrial morphological changes presumably related to organelle dysfunction, dilated endoplasmic reticulum and autophagosomes - an attempt at cellular repair. Ballooning itself likely results from a combination of cytoskeletal injury resulting in loss of normal cell shape and from accumulation of injured and somewhat derelict small fat droplets and dilated endoplasmic reticulum. Cellular injury in NASH and especially cellular ballooning can be viewed as a process of 'multi-organelle failure' beginning with generation of super oxide and failure to contain the subsequent oxidative injury and by-products in an environment rich in lipid fuel. These events lead to activation of imunologic pathways. Dysfunction of the small fat droplet appears to be a central mechanism and the oxidative injury can be viewed as the process of rancidification - the chemical decomposition of oils, lipids and fats.

Unfolded Protein Response (UPR) Predicts and Exacerbates the Development of Alcohol-Induced Pancreatitis

Introduction: Pancreatitis affects ˜0.04-5% of the U.S. population. The exact pathogenesis of pancreatitis, either acute or chronic, is not fully understood; to date, there are no pathogenesis-based treatment options for pancreatitis. The unfolded protein response (UPR) is a coordinated adaptive response to limit the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which perturbs calcium homeostasis, energy balance, and redox state; all these components of cellular stress have been reported in pancreatitis, yet the role of UPR in pancreatitis is yet to be fully understood. We hypothesized that redox stressors, such as alcohol, will increase UPR; we further predicted that UPR exacerbation will lead to advanced pancreatic tissue damage and thus development of pancreatitis. Methods: We analyzed pancreas tissue from humans with alcoholic pancreatitis and C57Bl6 mice with alcohol+/-LPS-induced pancreatitis; tissue was analyzed by histology, enzyme biochemically, proteins by Western and RNA by PCR assays. Results: We report significant UPR activation in humans with pancreatitis, manifested as increased expression of PERK, eIF2α, Ire1α-Xbp-1, and XBP1 target protein disulfide isomerase (PDI) RNA levels. We identified similarly increased expression of PERK, eIF2α, Ire1α-Xbp-1, and PDI RNA in pancreas tissue of mice fed alcohol, yet the pancreas histology in these mice looked normal and there was no elevation of serum lipase. Challenges of alcohol-fed mice with LPS caused disorganized and dilated ER, loss of zymogen granules, accumulation of vacuoles, increased acinar cell death detected by microscopy, and lead to elevated serum lipase/activated zymogen suggestive of pancreatitis. More importantly, alcohol+LPS had additive effects on increased expression of PERK, eIF2α, Ire1α-Xbp-1, and PDI RNA levels, lead to increased production of several pro-inflammatory mediators (TNFa, IL-6), increased P-c-JUN, CHOP, and cleavage of caspase-3. These findings suggest that UPR occurs in stressed pancreatic tissue, in our case by exposure to alcohol, prior to biochemical signature (elevated pancreatic enzymes and altered histology) of pancreatitis. Further, multi-hit oxidative stressors, such as combination of alcohol and LPS, exacerbate alcohol-induced UPR and facilitate translation to florid pancreatitis with elevated serum levels of pancreatic enzymes, signs of inflammation, and tissue remodeling on histology. Conclusion: Our findings point to an early role of UPR in pancreatitis even prior to evident biochemical signature in the serum and tissue, and identify UPR-based therapeutic targets of potential translational importance.

Dietary high-fat lard intake induces thyroid dysfunction and abnormal morphology in rats.

Excess dietary fat intake can induce lipotoxicity in non-adipose tissues. The aim of this study was to observe the effects of dietary high-fat lard intake on thyroid in rats. Male Sprague-Dawley rats were fed a high-fat lard diet for 24 weeks, and then the rats were fed a normal control diet (acute dietary modification) or the high-fat lard diet for another 6 weeks. The serum lipid profile, total thyroxine (TT4), free thyroxine (FT4) and thyrotropin (TSH) levels were determined at the 12, 18, 24 and 30 weeks. High-frequency ultrasound scanning of the thyroid glands was performed at the 24 or 30 weeks. After the rats were sacrificed, the thyroid glands were collected for histological and immunohistochemical analyses. The high-fat lard diet significantly increased triglyceride levels in both the serum and thyroid, and decreased serum TT4 and FT4 levels in parallel with elevated serum TSH levels. Ultrasonic imaging revealed enlarged thyroid glands with lowered echotexture and relatively heterogeneous features in the high-fat lard fed rats. The thyroid glands from the high-fat lard fed rats exhibited enlarged follicle cavities and flattened follicular epithelial cells under light microscopy, and dilated endoplasmic reticulum cisternae, twisted nuclei, fewer microvilli and secretory vesicles under transmission electron microscopy. Furthermore, the thyroid glands from the high-fat lard fed rats showed markedly low levels of thyroid hormone synthesis-related proteins TTF-1 and NIS. Acute dietary modification by withdrawal of the high-fat lard diet for 6 weeks failed to ameliorate the high-fat lard diet-induced thyroid changes. Dietary high-fat lard intake induces significant thyroid dysfunction and abnormal morphology in rats, which can not be corrected by short-term dietary modification.

Effects of chronic tramadol administration on testicular tissue in rats: an experimental study.

In a prospective experimental study, the effects of chronic tramadol administration on gonadotrophic and sex hormones, histopathological and morphometrical alterations in rat testicular tissue were investigated in a laboratory setting. Tramadol was given alone to adult male albino rats. Gonadotrophic and serum sex hormone levels were measured and testicular pathological and morphometric changes were observed in treated vs. After 30 days of treatment, tramadol induced a decrease in LH, FSH and testosterone serum levels. Histologically, degenerative changes in the seminiferous tubules were observed. They showed shrinkage, separation of tubular basement membrane, disorganisation and vacuolisation of spermatogenic layers. Morphometric analysis revealed significant decrease in the mean values of the tubular diameter and epithelial height. Ultrastructural abnormalities were detected in all cells of spermatogenic lineage in addition to the appearance of apoptotic cells. Sertoli cell vacuolation, huge lipid droplets and disrupted Sertoli cell junctions were observed. Leydig cells showed euchromatic nuclei and dilated smooth endoplasmic reticulum. In view of these findings, it is concluded that tramadol induces alterations in sex hormonal levels in conjunction with disruption of the normal histological structure of rat testis. This might lead to the risk of male infertility. Therefore, tramadol should be used with caution with appropriate dose monitoring.

L-Carnitine attenuates H2O2-induced neuron apoptosis via inhibition of endoplasmic reticulum stress

Both oxidative stress and endoplasmic reticulum stress (ER stress) have been linked to pathogenesis of neurodegenerative diseases. Our previous study has shown that l-carnitine may function as an antioxidant to inhibit H2O2-induced oxidative stress in neuroblastoma SH-SY5Y cells. To further explore the neuroprotection of l-carnitine, here we study the effects of l-carnitine on the ER stress response in H2O2-induced SH-SY5Y cell injury. Our results showed that l-carnitine pretreatment could increase cell viability; inhibit apoptosis and ROS accumulation caused by H2O2 or tunicamycin (TM). l-carnitine suppress the endoplasmic reticulum dilation and activation of ER stress-associated proteins including glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), JNK, Bax and Bim induced by H2O2 or TM. In addition, H2O2-induced cell apoptosis and activation of ER stress can also be attenuated by antioxidant N-acetylcysteine (NAC), CHOP siRNA and the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, our results demonstrated that H2O2 could trigger both oxidative stress and ER stress in SH-SY5Y cells, and ER stress participated in SH-SY5Y apoptosis mediated by H2O2-induced oxidative stress. CHOP/Bim or JNK/Bim-dependent ER stress signaling pathways maybe related to the neuroprotective effects of l-carnitine against H2O2-induced apoptosis and oxidative injury.

Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase.

BackgroundHepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment.MethodsIn this regard, after establishing and consequently exploring Hep88 mAb’s tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb’s specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1–3 days was investigated using a transmission electron microscope.ResultsThe result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction.ConclusionThese fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.

Toxicity of new emerging pollutant tris-(2,3-dibromopropyl) isocyanurate on BALB/c mice.

The emerging heterocyclic brominated flame retardant tris-(2,3-dibromopropyl) isocyanurate (TBC), widely used in reinforced plastics, has demonstrated toxicity to fish. However, little is known about its toxicity in rodents. This study aims to determine the effect of TBC on growth, biochemical parameters in serum, organs and related gene expression of both male and female BALB/c mice after gastro-gavage administration of 0, 2, 10 and 50 mg kg⁻¹ TBC for 28 days. Results indicated that exposure to TBC had no effects on basic growth and food intake of mice, but significantly increased serum alanine aminotransferase levels in male mice. Histopathological analyses showed that focal necrosis (2, 10 and 50 mg kg⁻¹ TBC-exposed groups) and ballooning degeneration (10 and 50 mg kg⁻¹ TBC-exposed groups) were found in mouse liver, whereas transmission electron microscopy revealed dose-dependent hepatocyte apoptosis, mitochondrial degeneration and endoplasmic reticulum dilation. Histopathological and ultrastructural assessments in the lung showed dose-dependent hyperplasia of pulmonary alveolar epithelium, bronchial congestion, infiltration of inflammatory cells and mitochondrial swelling following TBC exposure. Our results also indicated that mitochondria are one of the major target cytoplasmic organelles for TBC, suggesting that damage in mitochondria is one of the pathways that led to toxic effects in the liver and lung of TBC-treated groups. Moreover, TBC effectively activated the gene expression of p53 in mice liver. Our findings provide strong evidence that TBC induces significant toxicity in mice organs, especially in liver and lung, which play vital roles in detoxification and gas exchange, respectively. This research will contribute to characterize the toxic effects of TBC, which was introduced as one of the candidates for brominated flame retardant replacement.

Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L−1) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8–1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6–1.8-fold-changes at the 250 mg L−1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3–1.6-fold increases at the 250 mg L−1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.

THE PROTECTIVE EFFECT OF CAMEL’S MILK, IN COMPARISON TO THYMOQUINONE, ON STREPTOZOTOCIN-INDUCED DAMAGE IN THE PANCREAS OF ALBINO RATS

Streptozotocin has been used extensively to produce an experimental animal model for diabetes mellitus. The present study was undertaken to investigate the protective effect of camel milk, in comparison to thymoquinone, against streptozotocin-induced damage in the pancreas. The rats were divided into four groups as follows. (1) Control group, (2) Diabetic group with a single intraperitoneal injection of streptozotocin (50 mg/kg body weight), (3) Diabetic group treated with camel’s milk (CM) orally and daily at a dose of 2 ml/rat, (4) Diabetic group treated with thymoquinone (TQ) orally and daily at a dose of 50 mg/kg body weight. After 2 months, the animals were sacrificed and blood samples were collected for the detection of serum glucose levels. The pancreas was prepared for histopathological, immunohistochemical and ultrastructural studies. The histological examination of diabetic pancreas showed vacuolation and shrinkage of the islets of Langerhans. The exocrine part of the gland showed vacuolization and flattening of the lining epithelium of the acini. Dense collagen fibres around the acini, blood vessels and pancreatic ducts were also detected. The immunoreactivity or anti-insulin antibodies was markedly decreased. Electron microscopic examination of the diabetic group showed marked changes in pancreatic acini represented by dilatation of the rough endoplasmic reticulum, decrease of secretory granules, vacuolization of the cytoplasm, damage of the mitochondria, and dilatation of the perinuclear space. The β-cells of the diabetic rats showed decrease in the number of secretory granules, heterochromatic nuclei and cytoplasmic vacuolation. CM and TQ improved the histological and ultrastructural changes of the β-cells and exocrine acini epithelium, which almost reverted to their normal structure. These factors increased insulin immunoreactivity and decreased the elevated glucose concentrations. However, insulin immunoreactivity was found in the majority of the pancreatic islets in diabetic rats treated with CM or TQ.

A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.

Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

Connective tissue alterations in Fkbp10-/- mice.

Osteogenesis imperfecta (OI) is an inherited brittle bone disorder characterized by bone fragility and low bone mass. Loss of function mutations in FK506-binding protein 10 (FKBP10), encoding the FKBP65 protein, result in recessive OI and Bruck syndrome, of which the latter is additionally characterized by joint contractures. FKBP65 is thought to act as a collagen chaperone, but it is unknown how loss of FKBP65 affects collagen synthesis and extracellular matrix formation. We evaluated the developmental and postnatal expression of Fkbp10 and analyzed the consequences of its generalized loss of function. Fkbp10 is expressed at low levels in E13.5 mouse embryos, particularly in skeletal tissues, and steadily increases through E17.5 with expression in not only skeletal tissues, but also in visceral tissues. Postnatally, expression is limited to developing bone and ligaments. In contrast to humans, with complete loss of function mutations, Fkbp10(-/-) mice do not survive birth, and embryos present with growth delay and tissue fragility. Type I calvarial collagen isolated from these mice showed reduced stable crosslink formation at telopeptide lysines. Furthermore, Fkbp10(-/-) mouse embryonic fibroblasts show retention of procollagen in the cell layer and associated dilated endoplasmic reticulum. These data suggest a requirement for FKBP65 function during embryonic connective tissue development in mice, but the restricted expression postnatally in bone, ligaments and tendons correlates with the bone fragility and contracture phenotype in humans.

Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts.

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Identification and Characterization of Maizefloury4as a Novel Semidominant Opaque Mutant That Disrupts Protein Body Assembly

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19-kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ER-associated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 α-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

The protective effect of mild hypothermia pretreatment against injury to primary cultured cortical neurons induced of rat by glutamate

To investigate the effect of mild hypothermia preconditioning against ischemia/reperfusion (I/R) injury of cultured primary cortical rats neurons, and to compare the protective effect of mild hypothermia only and with its combination with drugs. Cortical neurons of neonatal Sprague-Dawley (SD) rat within 24 hours after birth were harvested and cultured in vitro. On the 3rd day, the cells were cultured in a medium containing 2.5 mg/L cytosine arabinoside to inhibit the growth of glial cells and fibroblast. Having cultured for 6 days they were randomly divided into blank control group, glutamate damaged group (cultured with 200 μmol/L glutamate for 0.5 hour after washing), mild hypothermia preconditioning group (cultured under 33.5 centigrade for 24 hours before injury induced by glutamate), mild hypothermia combining with edaravone preconditioning group, and the hypothermia combining with propofol preconditioning group (medium containing 100 μmol/L edaravone and 3 mg/L propofol). They were cultured under 33.5 centigrade for 24 hours before injury induced by glutamate. After 24 hours of culturing in various medium, apoptosis ratio was observed by flow cytometry. Cell surviving rate was determined with methyl thiazolyltriazolium (MTT), c-fos protein expression was assayed, and morphologic change of cells with hematoxylin-eosin (HE) staining under the microscope, and ultrastructure changes were observed after uranyl acetate and lead citrate staining under transmission electron microscope. The apoptosis ratio and c-fos protein in glutamate damaged group were significantly higher than those in blank control group [apoptosis ratio: (9.85±0.76)% vs. (0.94±0.20)%, c-fos: 6.96±0.75 ng/L vs. 1.65±0.59 ng/L, both P<0.01], the cell surviving rate was significantly lower than that in blank control group [(0.20±0.02)% vs. (0.97±0.03)%, P<0.01]. Mild hypothermia preconditioning reversed surviving rate, apoptosis ratio and c-fos protein, and the effect of hypothermia combining with propofol preconditioning was obviously better [cell surviving rate: (0.80±0.04)% vs. (0.20±0.02)%, apoptosis ratio: (2.26±0.54)% vs. (9.85±0.76)%, c-fos: 2.98±0.46 ng/L vs. 6.96±0.75 ng/L, all P<0.01]. The morphology of cortical neurons in blank control group was normal. Most of the cells in glutamate damaged group showed bluish black cytoplasm with pyknic nuclei, with crumpled axons and of them were fractured, and cell number was obviously decreased. In each pre-conditional group, decrease in cell number was unconspicuous, and only a few cells showed apoptosis. Under transmission electron microscope, it was found that cell membrane, mitochondria and rough endoplasmic reticulum were intact in blank control group, but with reduction in organelles, severely swollen mitochondria, even with formation of vacuole or pyknosis, serious dilation of rough endoplasmic reticulum, with loss of cristae with loss of vacuoles or pyknosis, and marked dilatation of internal reticular endoplasm, and loss of cristae with vacuolation and chromatin were observed under electron microscope in glutamate damaged group. Compared with the glutamate damaged group, these pathologic changes were markedly alleviated in protected groups. Mild hypothermia preconditioning can inhibit glutamate-induced injury to cortical neurons. The protective effect of mild hypothermia combined with propofol is better.

The ER/SR Ca2+ sensing protein STIM1 is essential for normal cardiomyocyte function (1150.8)

Store‐operated Ca2+ entry (SOCE) and its molecular mediator, the endoplasmic reticulum (ER) Ca2+ sensor, Stromal interaction molecule 1 (STIM1) have been implicated in hypertrophic signaling. However, since the physiological role of STIM1 in the adult heart remains unknown, we developed a cardiomyocyte‐restricted STIM1 knockout (crSTIM1‐KO) mouse. Using serial echocardiography and histological analysis, we observed a progressive decline in contractile function from 20 weeks of age, which was associated with marked left ventricular dilatation by 36 weeks. Immunoblot analyses revealed that crSTIM1‐KO hearts exhibit increased ER stress prior to contractile dysfunction (i.e., 12 weeks old) as indicated by increased levels of CHOP. Electron microscopy revealed ER dilatation, mitochondrial disorganization and smaller mitochondria in crSTIM1‐KO hearts, which was associated with a decrease in mitofusin 2 and an increase in Drp1 consistent with increased mitochondrial fission. Interestingly, perfused hearts from 20 week crSTIM1‐KO mice exhibited significant reductions in both glucose oxidation and net lactate release. These data demonstrate for the first time that STIM1 plays an essential role in normal cardiomyocyte function in the adult heart and may be mediated through regulation of ER/ mitochondrial function and/or metabolism.Grant Funding Source: Supported by NIH grants HL101192 and HL110366 and William W Featheringill Postdoctoral Fellowship

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.