Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation.

Misdirected immunity gives rise to the autoimmune tissue inflammation of rheumatoid arthritis, in which excess production of the cytokine tumor necrosis factor (TNF) is a central pathogenic event. Mechanisms underlying the breakdown of self-tolerance are unclear, but T cells in the arthritic joint have a distinctive metabolic signature of ATPlo acetyl-CoAhi proinflammatory effector cells. Here we show that a deficiency in the production of mitochondrial aspartate is an important abnormality in these autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide, causing ADP deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of transmembrane TNF. ERrich T cells were the predominant TNF producers in the arthritic joint. Transfer of intact mitochondria into T cells, as well as supplementation of exogenous aspartate, rescued the mitochondria-instructed expansion of ER membranes and suppressed TNF release and rheumatoid tissue inflammation.

Are unusual ultrastructural features occurring in the pollen endomembrane system of Cyperaceae and other angiosperms?

Cyperaceae representatives present peculiar microsporogenesis and microgametogenesis, which raises the question of how regular the sedge pollen is. In order to answer this question, preanthesis pollen grains of Cyperaceae individuals were analyzed under different tools such as light and transmission electron microscopy, which included cytochemistry and immunogold procedures. The results showed that maturing pollen in Cyperaceae presents some unusual endomembrane behaviors. Endoplasmic reticulum and dictyosomes are concerned in classic secretion pathways in vegetative cells, and possibly the late breakdown of degenerative microspores. However, cortical and concentric endoplasmic reticulum are also present and are possibly related to other functions aside the biosynthetic pathway. Unconventional secretion of large membrane-bound bodies containing cell wall precursors was also observed and confirmed by immunogold. However, since these bodies most likely receive material from dictyosomes, as observed in silver nitrate reaction, the "unconventional" status of this secretion is debatable. Reports of the literature show that these unusual endomembrane formations are not exclusive of the sedge pollen, but little attention have been given to them so far. This could represent an opportunity to re-examine our understanding on the endomembrane system in pollen cells in general.

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

Tiao Geng decoction for treating menopausal syndrome exhibits anti-aging effects likely via suppressing ASK1/MKK7/JNK mediated apoptosis in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: TG-decoction (Tiao Geng decoction) is the extract of a Chinese herb mixture that has been used for treating menopausal symptoms for over 30 years. We have previously reported anti-aging and anti-oxidative effects of the TG-decoction on hypothalamic neurons in ovariectomized (OVX) rats. AIM OF THE STUDY: The present study further investigates the effects of TG-decoction on the prevention of aging-related ultrastructural changes in menopausal hypothalamic neurons and the likely molecular mechanism. MATERIALS AND METHODS: A total of 120 four-month-old female SPF Sprague Dawley rats were divided into six groups. Five groups were ovariectomized (OVX) and one group served as a sham control. Three OVX groups received TG-decoction at three different doses. The remaining two OVX groups served as positive and negative controls by receiving estradiol valerate and saline solution. The sham group received saline. After one month, aging-related ultrastructural alterations in hypothalamic neurons were evaluated using transmission electron microscopy. Nissl staining was used to assess the pathomorphological changes of the hypothalamic neurons. Cell apoptosis was evaluated by TUNEL. Expression of Bcl-2 family genes was studied using qRT-PCR. Expression of the apoptosis-related proteins ASK1, MKK7, JNK, c-Jun, Bax, Casp3 and Bcl-2 was studied using western blotting. RESULTS: Ovariectomy of female rats led to visible damage and aging-like alterations in the mitochondria and endoplasmic reticulum as well as large deposits of lipofuscin in hypothalamic tissue. TG-decoction treatment prevented this visible damage and lipofuscin deposition, increased the number of nerve cells and normally-shaped Nissl bodies, and reduced the number of TUNEL-positive cells. Expression of Bcl-2 gene was increased, while Bax gene reduced. Expression of the proteins ASK1, MKK7, JNK, c-Jun, Bax and Casp3 was reduced, while that of Bcl-2 was increased. CONCLUSION: TG-decoction reduces aging-related ultrastructural changes in hypothalamic neurons, likely by suppressing ASK1/MKK7/JNK-mediated apoptosis in neuronal mitochondria or nuclei.

The Effects of Endoplasmic-Reticulum-Resident Selenoproteins in a Nonalcoholic Fatty Liver Disease Pig Model Induced by a High-Fat Diet.

The present study aimed to investigate the intervention of selenium in the oxidative stress and apoptosis of pig livers, which were induced by a high-fat diet, and the effects of four endoplasmic reticulum (ER)-resident selenoproteins in the process. A 2×4 design trial was conducted that included two dietary fat levels (BD = basal diet and HFD = high-fat diet) and four dietary Se supplementation levels (0, 0.3, 1.0, and 3.0 mg/kg of the diet, in the form of sodium selenite (Na2SeO3)). Our results indicated that the HFD significantly increased the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, as well as the degree of steatosis, the content of malondialdehyde (MDA), the apoptotic rate, and the level of mRNA caspase-3 in the liver compared to their BD counterparts (p < 0.05). Moreover, these parameters in the HFD groups were more significantly reduced (p < 0.05) for a Se concentration of 1.0 mg/kg than for the other concentrations. Further, for both the BD and HFD, the groups supplemented with 1.0 mg/kg Se showed the highest mRNA level of selenoprotein S. In conclusion, the consumption of an HFD can induce oxidative damage and apoptosis in the liver. This shows that the supplementation of Se at 1.0 mg/kg may be the optimum concentration against damage induced by HFD, and Sels may play a key role in this process.

Network Pharmacology-Based Prediction and Verification of Qingluo Tongbi Formula to Reduce Liver Toxicity of Tripterygium wilfordii via UGT2B7 in Endoplasmic Reticulum.

BACKGROUND The hepatotoxicity of Tripterygium wilfordii Hook. f. (TWHF) limits its clinic utilization. Qingluo Tongbi formula (QTF) was formulated based on a basic Chinese medicine theory. Previous studies have confirmed the safety and efficacy of QTF in treating rheumatoid arthritis. Therefore, we considered that TWHF could be detoxified based on its reasonable compatibility with QTF. We investigated the detoxicity mechanism of QTF in reducing the liver toxicity of TWHF. MATERIAL AND METHODS We used network pharmacology to determine the relevant metabolism targets of TWHF, focusing on the phase II metabolic enzymes uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1), UGT1A6, and UGT2B7. Based on the molecular mechanisms of these predictions and the results of the network analysis, we designed experiments to verify our hypothesis in vivo. We used western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), double immunofluorescence, and laser confocal microscopy to detect the expression of UGTs. Finally, we used transmission electron microscopy to observe the endoplasmic reticulum structure. RESULTS The results confirmed that QTF reversed the TWHF-induced reduction of UGT content in liver microsomes, upregulated UGT1A1 and UGT1A6 but not UGT2B7 in the liver tissue. UGT2B7 expression in the liver and liver microsomes was inconsistent. QTF upregulated the expression of UGT2B7 in the endoplasmic reticulum, and QTF upregulated UGT2B7 expression levels in the endoplasmic reticulum compared with TWHF, which reduced liver toxicity. Structural changes were observed in the endoplasmic reticulum. CONCLUSIONS The Chinese traditional medicine compound QTF can achieve the effect of detoxification by upregulating the expression of UGT2B7 in the endoplasmic reticulum.

[Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease].

OBJECTIVE: To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. METHODS: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. RESULTS: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P < 0.05), reduced accumulation of the lipid droplets (P < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P < 0.001) and P-PERK (P < 0.01) in FFAs-induced cells with PKC-δ knockdown. CONCLUSIONS: HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.

Ginsenoside Rg1 Prevents Doxorubicin-Induced Cardiotoxicity through the Inhibition of Autophagy and Endoplasmic Reticulum Stress in Mice.

Ginsenoside Rg1, a saponin that is a primary component of ginseng, has been demonstrated to protect hearts from diverse cardiovascular diseases with regulating multiple cellular signal pathways. In the present study, we investigated the protective role of ginsenoside Rg1 on doxorubicin-induced cardiotoxicity and its effects on endoplasmic reticulum stress and autophagy. After pre-treatment with ginsenoside Rg1 (50 mg/kg i.g.) for 7 days, male C57BL/6J mice were intraperitoneally injected with a single dose of doxorubicin (6 mg/kg) every 3 days for four injections. Echocardiographic and pathological findings showed that ginsenoside Rg1 could significantly reduce the cardiotoxicity induced by doxorubicin. Ginsenoside Rg1 significantly inhibited doxorubicin-induced formation of autophagosome. At the same time, ginsenoside Rg1 decreased the doxorubicin-induced cardiac microtubule-associated protein-light chain 3 and autophagy related 5 expression. Ginsenoside Rg1 can reduce endoplasmic reticulum dilation caused by doxorubicin. Compared with the doxorubicin group, the expression of cleaved activating transcription factor 6 and inositol-requiring enzyme 1 decreased in group ginsenoside Rg1. Treatment with ginsenoside Rg1 reduces the expression of TIF1 and increases the expression of glucose-regulated protein 78. In the ginsenoside Rg1 group, the expression of p-P70S6K, c-Jun N-terminal kinases 1 and Beclin1 declined. These results indicate that ginsenoside Rg1 may improve doxorubicin-induced cardiac dysfunction by inhibiting endoplasmic reticulum stress and autophagy.

Effect of dehydroepiandrosterone on the liver of perimenopausal rat: multiple doses study.

Dehydroepiandrosterone (DHEA) is a widespread nutritional "anti-aging" supplement. Exogenous supplementation of DHEA is now being commonly used to augment ovarian stimulation in perimenopausal women with diminished ovarian reserve. Whether DHEA causes side effects in such age is, however, unknown. Thus, this study investigates the effects of pharmacological doses of DHEA supplementation on the liver of perimenopausal rats. DHEA supplementation to perimenopausal rats resulted in slight hepatomegaly and steatosis, hepatocytic hypertrophy, mitochondrial swelling, elevation in serum alanine aminotransaminase levels, in addition to the accumulation of lipid droplets and lipolysosomes in a dose-dependent manner. In conclusion, long-term administration of high doses of DHEA causes ultrastructural alterations and changes in the levels of cholesterol and triglyceride in hepatocytes of perimenopausal rats. DHEA at a dose of 50 mg/kg improves health and decreases the body weight, with the least side effects on the liver of perimenopausal rats.

Survival-Related Autophagic Activity Versus Procalcific Death in Cultured Aortic Valve Interstitial Cells Treated With Critical Normophosphatemic-Like Phosphate Concentrations.

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.

Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia.

MAIN CONCLUSION: Calreticulin is involved in stabilization of the tip-focused Ca 2+ gradient and the actin cytoskeleton arrangement and function that is required for several key processes driving Petunia pollen tube tip growth. Although the precise mechanism is unclear, stabilization of a tip-focused calcium (Ca2+) gradient seems to be critical for pollen germination and pollen tube growth. We hypothesize that calreticulin (CRT), a Ca2+-binding/buffering chaperone typically residing in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, is an excellent candidate to fulfill this role. We previously showed that in Petunia pollen tubes growing in vitro, CRT is translated on ER membrane-bound ribosomes that are abundant in the subapical zone of the tube, where CRT's Ca2+-buffering and chaperone activities might be particularly required. Here, we sought to determine the function of CRT using small interfering RNA (siRNA) to, for the first time in pollen tubes growing in vitro, knockdown expression of a gene. We demonstrate that siRNA-mediated post-transcriptional silencing of Petunia hybrida CRT gene (PhCRT) expression strongly impairs pollen tube growth, cytoplasmic zonation, actin cytoskeleton organization, and the tip-focused Ca2+ gradient. Moreover, reduction of CRT alters the localization and disturbs the structure of the ER in abnormally elongating pollen tubes. Finally, cytoplasmic streaming is inhibited, and most of the pollen tubes rupture. Our data clearly show an interplay between CRT, Ca2+ gradient, actin-dependent cytoplasmic streaming, organelle positioning, and vesicle trafficking during pollen tube elongation. Thus, we suggest that CRT functions in Petunia pollen tube growth by stabilizing Ca2+ homeostasis and acting as a chaperone to assure quality control of glycoproteins passing through the ER.

Endoplasmic reticulum pathology and stress response in neurons precede programmed necrosis after neonatal hypoxia-ischemia.

The endoplasmic reticulum (ER) is tasked, among many other functions, with preventing excitotoxicity from killing neurons following neonatal hypoxia-ischemia (HI). With the search for delayed therapies to treat neonatal HI, the study of delayed ER responses becomes relevant. We hypothesized that ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal HI. Since necrostatin-1 (Nec-1), an inhibitor of programmed necrosis, provides delayed neuroprotection against neonatal HI in male mice, Nec-1 is an ideal tool to study delayed ER responses. C57B6 male mice were exposed to right carotid ligation followed by exposure to FiO2=0.08 for 45 min at p7. Mice were treated with vehicle or Nec-1 (0.1 µl of 8 µmol) intracerebroventricularly with age-matched littermates as controls. Biochemistry assays at 3 and 24h and electron microscopy (EM) and immunohistochemistry at 96 h after HI were performed. EM showed ER dilation and mitochondrial swelling as apparent early changes in neurons. With advanced neurodegeneration, large cytoplasmic fragments containing dilated ER "shed" into the surrounding neuropil and calreticulin immunoreactivity was lost concurrent with nuclear features suggestive of programmed necrosis. Nec-1 attenuated biochemical markers of ER stress after neonatal HI, including PERK and eIF2α phosphorylation, and unconventional XBP-1 splicing, consistent with the mitigation of later ER pathology. ER pathology may be an indicator of severity of neuronal injury and potential for recovery characterized by cytoplasmic shedding, distinct from apoptotic blebbing, that we term neuronal macrozeiosis. Therapies to attenuate ER stress applied at delayed stages may rescue stressed neurons after neonatal HI.

Protective effect of carbenoxolone on ER stress-induced cell death in hypothalamic neurons.

Hypothalamic endoplasmic reticulum (ER) stress is known to be increased in obesity. Induction of ER stress on hypothalamic neurons has been reported to cause hypothalamic neuronal apoptosis and malfunction of energy balance, leading to obesity. Carbenoxolone is an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor that converts inactive glucocorticoid into an active form. In addition to its metabolic effect via enzyme inhibitory action, carbenoxolone has shown anti-apoptotic activity in several studies. In this study, the direct effects of carbenoxolone on ER stress and cell death in hypothalamic neurons were investigated. Carbenoxolone attenuated tunicamycin induced ER stress-mediated molecules such as spliced XBP1, ATF4, ATF6, CHOP, and ROS generation. In vivo study also revealed that carbenoxolone decreased tunicamycin-induced ER stress in the hypothalamus. In conclusion, the results of this study show that carbenoxolone has protective effects against tunicamycin induced-ER stress and apoptosis in hypothalamic neurons, suggesting its direct protective effects against obesity. Further study is warranted to clarify the effects of carbenoxolone on hypothalamic regulation of energy balance in obesity.

Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells.

In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 µg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1α (IRE1α) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca(2+) leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress.

Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

From zero to six double bonds: phospholipid unsaturation and organelle function.

Cellular phospholipids (PLs) differ by the nature of their polar heads as well as by the length and unsaturation level of their fatty acyl chains. We discuss how the ratio between saturated, monounsaturated, and polyunsaturated PLs impacts on the functions of such organelles as the endoplasmic reticulum, synaptic vesicles, and photoreceptor discs. Recent experiments and simulations suggest that polyunsaturated PLs respond differently to mechanical stress, including membrane bending, than monounsaturated PLs owing to their unique conformational plasticity. These findings suggest a rationale for PL acyl chain remodeling by acyltransferases and a molecular explanation for the importance of a balanced fatty acid diet.

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure.

Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15-30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG-Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.

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.

Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.

Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca(2+) reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca(2+) waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~4.5h post-fertilization: hpf), and ending at the shield stage (~6.0hpf) these distinct classes of waves propagated at mean velocities of ~50 and ~4µm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca(2+) wave propagation occurred only when this was <~8µm; an IND >~8µm was coincidental with wave termination at shield stage. Treatment with the IP3R antagonist, 2-APB, the Ca(2+) buffer, 5,5'-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca(2+) waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca(2+) waves are generated mainly via Ca(2+) release from IP3Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.