Extensive Endoplasmic Reticulum Research Articles

Non-canonical unfolded protein response transcription factor CREB3L2 regulates survival of long-lived plasma cells

Abstract Differentiation of activated B cells into antibody-secreting cells (ASCs) involves extensive endoplasmic reticulum (ER) & Golgi biogenesis and remodeling that enable increased antibody synthesis and secretion. These processes are mediated by the unfolded protein response (UPR). Canonical UPR pathway genes are regulated by the transcription factors (TFs) XBP-1 and ATFs, which are activated upon ER or Golgi stress. Using scRNA-seq to analyze the generation of splenic and bone marrow (BM) plasma cells (PCs), we discovered that the Creb3l2gene, encoding a non-canonical UPR TF, is specifically expressed in PCs, with highest levels in long-lived PCs (LLPCs). CREB3L2 has been shown to regulate transcription of genes encoding components critical to secretory cells of the pancreas and pituitary gland. To analyze the function of CREB3L2 in ASCs, we pursued a two-pronged strategy (i) using CRISPR/Cas9-RNP editing in the murine CH12 B lymphoma cell line to generate out-of-frame deletions in exon 2 of the Creb3l2gene (Creb3l2−/−) and (ii) generating Creb3l2B-cell conditional KO (cKO) mice carrying a floxed allele of the Creb3l2gene by inserting loxpsites flanking exon 7 with a CD19-cre driver (Creb3l2fl/flCD19 cre/+). RNA-seq of Creb3l2−/−CH12 cells shows CREB3L2 positively regulated genes including Cd93and Cd28& its signaling adapter SLP-76 (Lcp2), that are involved in plasma cell survival in the BM niche. Expression of CD28/SLP-76 and CD93 proteins were strongly diminished in Creb3l2−/−CH12 cells. Consistent with these findings, Creb3l2cKO mice had reduced NP-specific LLPCs (90 d.p.i) after NP-KLH immunization. Thus, CREB3L2 appears to selectively regulate the survival of LLPCs by controlling the expression of CD28/SLP-76 and CD93. Supported by grants from NIAID (U01 AI141990, R01 AI145064).

Protective Effect and Mechanism of Xbp1s Regulating HBP/O-GlcNAcylation through GFAT1 on Brain Injury after SAH.

(1) SAH induces cellular stress and endoplasmic reticulum stress, activating the unfolded protein response (UPR) in nerve cells. IRE1 (inositol-requiring enzyme 1) is a protein that plays a critical role in cellular stress response. Its final product, Xbp1s, is essential for adapting to changes in the external environment. This process helps maintain proper cellular function in response to various stressors. O-GlcNAcylation, a means of protein modification, has been found to be involved in SAH pathophysiology. SAH can increase the acute O-GlcNAcylation level of nerve cells, which enhances the stress capacity of nerve cells. The GFAT1 enzyme regulates the level of O-GlcNAc modification in cells, which could be a potential target for neuroprotection in SAH. Investigating the IRE1/XBP1s/GFAT1 axis could offer a promising avenue for future research. (2) Methods: SAH was induced using a suture to perforate an artery in mice. HT22 cells with Xbp1 loss- and gain-of-function in neurons were generated. Thiamet-G was used to increase O-GlcNAcylation; (3) Results: Severe neuroinflammation caused by subarachnoid hemorrhage leads to extensive endoplasmic reticulum stress of nerve cells. Xbp1s, the final product of unfolded proteins induced by endoplasmic reticulum stress, can induce the expression of the hexosamine pathway rate limiting enzyme GFAT1, increase the level of O-GlcNAc modification of cells, and have a protective effect on neural cells; (4) Conclusions: The correlation between Xbp1s displayed by immunohistochemistry and O-GlcNAc modification suggests that the IRE1/XBP1 branch of unfolded protein reaction plays a key role in subarachnoid hemorrhage. IRE1/XBP1 branch is a new idea to regulate protein glycosylation modification, and provides a promising strategy for clinical perioperative prevention and treatment of subarachnoid hemorrhage.

In Vitro Stimulation of IRE1α/XBP1-Deficient B Cells with LPS.

During immune responses, pathogen-specific B cells differentiate into plasma cells. Plasma cells synthesize and secrete large amounts of immunoglobulin (Ig) molecules which play a central role in immunity against pathogens. The synthesis, proper folding, and secretion of these Ig molecules require expansion of the extensive endoplasmic reticulum (ER) network. Accumulation of unfolded or misfolded proteins in the ER is sensed by three sensors: IRE1/XBP1, PERK, and ATF6, which coordinate with each other and initiate the unfolded protein response (UPR) pathway to expand the ER network and its protein folding and secretion capability. The expansion and maintenance of the ER network in plasma cells is triggered by activation of the IRE1/XBP1 branch of the UPR pathway. Here, we discuss the methods to stimulate the differentiation of B cells into plasma cells, measure the activation of the XBP1 pathway, and quantify the ER network.

DDRGK1, a crucial player of ufmylation system, is indispensable for autophagic degradation by regulating lysosomal function

DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive endoplasmic reticulum (ER) stress. Recently, emerging evidence indicates that the ufmylation system is correlated with autophagy, although the exact mechanism remains largely unknown. To explore the regulation mechanism of DDRGK1 on autophagy, in this study, we established an immortalized mouse embryonic fibroblast (MEF) cell lines harvested from the DDRGK1F/F:ROSA26-CreERT2 mice, in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen (4-OHT) treatment. Here, we show that DDRGK1 deficiency in MEFs has a dual effect on autophagy, which leads to a significant accumulation of autophagosomes. On one hand, it promotes autophagy induction by impairing mTOR signaling; on the other hand, it blocks autophagy degradation by inhibiting autophagosome–lysosome fusion. This dual effect of DDRGK1 depletion on autophagy ultimately aggravates apoptosis in MEFs. Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function, including impaired Cathepsin D (CTSD) expression, aberrant lysosomal pH, and v-ATPase accumulation, which might be a potential trigger for impairment in autophagy process. Hence, this study confirms a crucial role of DDRGK1 as an autophagy regulator by controlling lysosomal function. It may provide a theoretical basis for the treatment strategies of various physiological diseases caused by DDRGK1 deficiency.

Palmitoylation of Membrane-Penetrating Magainin Derivatives Reinforces Necroptosis in A549 Cells Dependent on Peptide Conformational Propensities.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike β-protofibrils. At the close concentrations (∼7.5 μM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 μM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

ORP9 knockdown delays the maturation of junction-related endocytic structures in the testis and leads to impaired sperm release†.

The release of late spermatids from the seminiferous epithelium requires the internalization of intercellular junctions by Sertoli cell specific structures called "tubulobulbar complexes" (TBCs). These large, endocytic devices likely evolved from classic clathrin-mediated-endocytosis (CME) machinery, but have several important morphological differences to CME vesicles. Most notable among these differences is that extensive endoplasmic reticulum (ER) membrane contact sites (MCSs) occur with TBCs and not with clathrin-coated pits. One of the well-established functions of ER MCSs is lipid exchange. Previously, we have established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. Here we use an in vivo knockdown approach to probe function. The testes of Sprague-Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution stimulated emission depletion, and electron microscopy. The knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3day intervals. Compared to controls, sections from ORP9 siRNA-injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release.

Chrysophanol Triggers Cell Death via Unfolded Protein Response and Endoplasmic Reticulum Stress in Oral Cancer FaDu Cells

Oral cancer is a type of head and neck cancer that can be life threatening. Unfolded protein response and endoplasmic reticulum stress play a critical role in carcinogenesis. However, prolonged activation of unfolded protein response also activates apoptosis. Thus, a new treatment strategy maybe activation of extensive unfolded protein response and severe endoplasmic reticulum stress in tumor cells. We examined the pharmacological effects of chrysophanol on an oral cancer cell line FaDu, a hypopharyngeal squamous cell carcinoma specifically for its ability to cause cell death via unfolded protein response and endoplasmic reticulum stress. We measured the expression of binding immunoglobulin protein, C/EBP homologous protein, phosphorylated inositol-requiring enzyme-1α, and activating transcription factor 6 after treatment with chrysophanol in absence or presence of APY29 (an inhibitor of phosphorylated inositol-requiring enzyme-1α). Chrysophanol caused cell death via upregulations of binding immunoglobulin protein that is also known as 78-kDa glucose-regulated protein, C/EBP homologous protein, phosphorylated inositol-requiring enzyme-1α, and activating transcription factor 6. This activity was reversed by APY29. Thus, chrysophanol could be a useful antitumor agent for the management of oral cancer.

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress

Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet-induced ER stress using Zip14-/- (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.

Phenotypic, ultra-structural, and functional characterization of bovine peripheral blood dendritic cell subsets.

Dendritic cells (DC) are multi-functional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets directly ex vivo, without further in vitro manipulation. Multi-color flow cytometric analysis revealed that three DC subsets could be identified. Bovine plasmacytoid DC were phenotypically identified by a unique pattern of cell surface protein expression including CD4, exhibited an extensive endoplasmic reticulum and Golgi apparatus, efficiently internalized and degraded exogenous antigen, and were the only peripheral blood cells specialized in the production of type I IFN following activation with Toll-like receptor (TLR) agonists. Conventional DC were identified by expression of a different pattern of cell surface proteins including CD11c, MHC class II, and CD80, among others, the display of extensive dendritic protrusions on their plasma membrane, expression of very high levels of MHC class II and co-stimulatory molecules, efficient internalization and degradation of exogenous antigen, and ready production of detectable levels of TNF-alpha in response to TLR activation. Our investigations also revealed a third novel DC subset that may be a precursor of conventional DC that were MHC class II+ and CD11c−. These cells exhibited a smooth plasma membrane with a rounded nucleus, produced TNF-alpha in response to TLR-activation (albeit lower than CD11c+ DC), and were the least efficient in internalization/degradation of exogenous antigen. These studies define three bovine blood DC subsets with distinct phenotypic and functional characteristics which can be analyzed during immune responses to pathogens and vaccinations of cattle.

Paneth cells

Paneth cells

Bacterial leaf glands in Styrax camporum (Styracaceae): first report for the family

In this study, we recorded, for the first time, the occurrence of leaf glands in a member of Styracaceae and their association with bacteria. Samples of Styrax camporum Pohl shoot apices and leaves at different developmental stages were prepared according to the conventional techniques for light and electron microscopy. Glands are emergences constituted by epidermal and parenchyma cells and are differentiated into a secretory body on a short nonsecretory stalk supplied with phloem. Actively secreting glands occur from leaf primordia to mature leaves and produce mucilage that accumulates inside schizogenous intercellular spaces. The epidermal secretory cells have abundant cytoplasm rich in hyperactive dictyosomes, an extensive endoplasmic reticulum, and modified plastids. Bacteria enter the gland via the intact surface and proliferate in the intercellular spaces of the glands. Once inside the intercellular spaces of the glands, bacteria enter the cells owing to the weakening of the anticlinal and inner periclinal cell walls and by phagocytosis. Strands of actin filaments occur near the endocytical vesicles containing degenerating bacteria. Accumulations of phenolic compounds and callose could explain the absence of bacteria in the stalk cells. The presence of bacteria inside the leaf glands of S. camporum is a regular and cyclic trait. The significance of the bacteria (not yet identified) and the type of interaction between these two organisms remain unknown.

Ultrastructural organization of the genital tracts in amphigonic females ofEuceraphis betulaeKoch (Aphididae: Calaphidinae)

The ultrastructure of the genital tracts in amphigonic females of Aphidoidea is described for the first time, using Euceraphis betulae Koch (Aphididae: Calaphidinae) as a representative. The female reproductive apparatus consists of two ovaries, each one with three/four meroistic telotrophic ovarioles; two sac-like accessory glands lie laterally to a sac-like seminal receptacle, opening into the dorso-medial part of the common oviduct by means of a spermathecal duct. A marked secretory activity takes place in the epithelial cells of all the investigated tracts as shown by ultrastructural observation of many organelles involved in this process. No evident golgian area was observed in the cytoplasm of these cells. Extensive smooth endoplasmic reticulum, whose probable role is here discussed, was observed in epithelial cells of the wall of the accessory gland. Spermathecal duct and seminal receptacle had peculiar features that could be related to different secretory activities carried out by these two parts of the spermatheca.

NK Cells Kill Myeloma Cells By Increasing ER Stress and Decreasing Autophagy Levels. NKG2D and NKP30 Are Involved In These Processes

Multiple myeloma (MM) plasma cells are equipped with an extensive endoplasmic reticulum (ER) for antibody production. Misfolding of newly synthesized immunoglobulins leads to ER stress and cell death. Plasma cells evade this by enhancing protein degradation through the proteasome pathway and the autophagy machinery. Because of this, MM cells are sensitive to proteasome inhibitors (PIs). However, autophagy induced by PIs and by reactive oxygen species (ROS) can increase drug resistance and lead to eventual relapse.We have previously demonstrated in vitro and in vivo sensitivity of MM cells to cord blood-derived natural killer (NK) cells, and have observed that this effect may involve NKG2D and NKP30.In this study we used confocal microscopy, chromium cytotoxicity assays and flow cytometry and we aimed to determine if there is a unique mechanism of NK cell-mediated cytotoxicity on MM cells involving ER stress, ROS and its dependence on NK cell receptors NKG2D and NKp30.We first demonstrated a difference in NK-mediated killing of MM cells vs K562 cells. As expected inhibition of GranzymeB (GrB) and Caspase3 pathway yielded a 13% reduction in NK-cytotoxicity against K562 cells; however, a 12% increase in killing was seen against ARP1 cells (p<0.001), indicating a GrB-Caspase3 independent mechanism for NK cell cytotoxicity against ARP1 cells. We observed a decrease in ROS levels after NK treatment for both cell lines (p<0.001), but inhibition of GrB and Caspase 3 abrogated the decrease in ROS only for K562 (p<0.001). By confocal microscopy we observed that NKG2D and NKP30 were transferred to tumor cells and entered the endocytic vesicle pathway. These receptors also appeared to play a more relevant role in the killing of MM cells (33%, 49% and 73% in ARP1 vs 4%, 3% and 19% in K562 cells of killing reduction after blocking NKG2D, NKP30 and both together, p<0.001). Regarding ER stress in ARP1 cells, we saw that after NK treatment there was a significant decrease in endocytic vesicle markers (EEA1 and Rab11, p<0.001) indicating a decrease in the transport of properly folded proteins. There was also a decrease in Rab7 and in Lysosomes (indicated by Lyso-Tracker probes) (p<0.001) and a dramatic increase for DNA-damage-inducible transcript 3 (CHOP) expression (p<0.001) indicating a decreased level of misfolded protein degradation and higher ER stress after exposure to NK cells. Blocking NKG2D abrogated the NK effect on ER stress (indicated by CHOP levels) and ROS levels in ARP1 cells. Blocking of NKP30 abrogated the NK effect on protein degradation activity (Rab7 levels), and ROS levels. To confirm that these receptors mediate killing of ARP1 cells by increasing ER stress, we performed cytotoxicity assays blocking these receptors and adding the PI bortezomib. Bortezomib did not improve NK cell killing at baseline in MM cells. However, bortezomib was able to partially reverse the deleterious effect of blocking NKG2D or NKp30 (33% enhanced killing for NKG2D-blocked and 57% for NKP30-blocked). Finally, we observed that NKG2D and NKP30 were also transferred secondarily to neighboring ARP1 from ARP1 cells which had been primarily synapsed to NK cells (p<0.001). In addition, these secondary ARP1 cells demonstrated decreased ROS levels after this contact. In a cytotoxicity assay, those ARP1 cells which had previously synapsed with NK cells demonstrated 7% of killing against new ARP1 cells.In summary, we describe a new mechanism of killing of MM cells by NK cells mediated by increased ER stress, decreased autophagy and dependent on NKG2D and NKP30. Furthermore, this cytotoxicity can be transmitted between MM cells. These findings could lead to new cellular therapeutic strategies for MM treatment. Disclosures:Shah:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Topographic and ultrastructural variations in isthmus segment of oviduct during oestrous cycle in Caprines

In the present study, the morphological variations in the isthmus segment of goat (Capra hircus) oviduct were studied using scanning and ultrastructural techniques during luteal and follicular phases of oestrous cycle. Topographic analysis of the isthmus epithelium in the follicular phase demonstrated irregular distribution of ciliated cells on the epithelium. A few secretory cells possessed bulbous apical processes which were concealed by the cilia. Cyclic changes were noticed in the secretory cells of isthmus. Under electron microscopy the isthmus region of the oviduct during the follicular phase revealed presence of almost blunt processes at their apical surfaces. The secretory cells were attached to the basal lamina and were characterized by the presence of secretory granules, numerous ribosomes, extensive smooth endoplasmic reticulum, well-developed golgi zones in the cytoplasm, and a microvillus luminal surface. The parallel use of these two methods in isthmus segment of the oviduct has enabled us to analyze the marked cyclic variations in the topography and fine structure during follicular and luteal phases of the oestrous cycle. The results of the present investigation on topography and ultrastructure will be of great help in explaining different bottlenecks of gamete interaction, maturation, fertilization, and early embryo development.

Guttation: path, principles and functions

Guttation is a process of natural secretion of fluid from leaves via specialised structures called ‘hydathodes’, which are located at the tips, margins, and adaxial and abaxial surfaces of leaves. Hydathodes form natural openings but, unlike stomata, are open permanently and offer little resistance to the flow of fluid out of leaves. Each hydathode is formed of colourless cells, and appears as stomata-like pores in the epidermis or epithem, also known as ‘transfer tissue’. The cells of epithem are soft and made of loosely arranged thin-walled parenchyma cells and without chloroplast, and are involved in absorption and secretion. Internally, they are connected by tracheary endings to a large chamber with masses of thin-walled parenchymatous tissue surrounded by a sheath layer. Ultrastructurally, the epithem cells have a dense cytoplasm, numerous mitochondria, an extensive endoplasmic reticulum system, many small Golgi-derived vesicles, numerous peroxisomes, and are interconnected by abundant plasmodesmata. Functionally, there are two types of hydathodes, namely, epidermal ones that actively exude fluid, and epithemal hydathodes that passively exude fluid. Natural guttation is often observed during early morning or late hours of the day. However, it can also be induced as desired in intact or excised plants under pneumatic pressure. Earlier notions regarding harmful effects on plants of guttation have now been addressed by botanical and physiological research discoveries regarding the basic and practical utility of guttation. This knowledge could lead to new health care applications on the one hand and ease global food-security concerns on the other.

Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal

Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

The Thioxotriazole Copper(II) Complex A0 Induces Endoplasmic Reticulum Stress and Paraptotic Death in Human Cancer Cells

The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible--by direct measurements--to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

Oxidative injury induces selective rather than global inhibition of proteasomal activity

Oxidative injury has been found to be associated with proteasomal inactivity. In this study, the extent of oxidative damage and its effects on proteasomal function has been critically assessed. Left anterior descending coronary artery was occluded (ischemia) and reperfused with or without preconditioning in male Sprague–Dawley rats. For further validation, H9c2 cardiac myoblasts cultures were used. We demonstrate that ischemia–reperfusion causes extensive endoplasmic reticulum stress as evident from the degradation of GRP78 transcript followed by its rapid induction. Western blot analysis and immunohistochemistry showed that increasing duration of ischemia and reperfusion causes accumulation of phosphorylated IκB (p-IκB), thereby suggesting proteasomal inactivity. However, similar analysis for Nrf2, a key mediator of antioxidant defense, showed sustained activation, suggesting intact proteasomal function. Preconditioning of the myocardium preserves the degradation of p-IκB, suggesting effective functioning of proteasome after preconditioning. Further analysis with specific proteosomal inhibitors like epoxomicin (100 nM, inhibits chymotrypsin-like activities of proteasomes) and lactacystin (2 μM, inhibits chymotrypsin as well as some trypsin-like activities of proteasomes) suggests that degradation of p-IκB and Keap-1 in the proteasome occurs by independent mechanisms. This study gives further insight into interrelationship between oxidative injury and catalytic function of the proteasome in heart, where oxidative injury causes selective rather than global inhibition of proteasome.

The balancers of Triturus italicus: an ultrastructural approach

Balancer morphology and ultrastructure, with emphasis on changes occurring during development, are described for Triturus italicus, using stereomicroscopy and light microscopy, as well as scanning and transmission electron microscopy. Morphological differentiation of balancers involves elongation, swelling of the distal region of the organ and appearance of secretory cones. The extensive granular endoplasmic reticulum and the well‐developed Golgi apparatus characterize the inner cell layer of the mature balancer. Other characteristics include the presence of numerous rounded lipid‐like granules and extensive innervation. In the cytoplasm, numerous bundles of tonofilaments can be found, and their amount increases as development progresses. As already stated in previous studies, the general morphology of balancers shows great constancy.