Rough Endoplasmic Reticulum Membranes Research Articles

Zearalenone exposure differentially affects the ovarian proteome in prepubertal gilts during thermal neutral and heat stress conditions.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes endocrine disruption and porcine reproductive dysfunction. Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation. Independently, HS and ZEN both compromise swine reproduction; thus, the hypothesis investigated was two-pronged: that ZEN exposure would alter the ovarian proteome and that these effects would differ in thermal neutral and HS pigs. Pre-pubertal gilts (n = 38) were fed ad libitum and assigned to either thermal neutral (TN: 21.0 ± 0.1°C) or HS (12h cyclic temperatures of 35.0 ± 0.2°C and 32.2 ± 0.1°C). Within the TN group, a subset of pigs were pair-fed (PF) to the amount of feed that the HS gilts consumed to eliminate the confounding effects of dissimilar nutrient intake. All gilts orally received a vehicle control (CT) or ZEN (40 μg/kg/BW) resulting in six treatment groups: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (TZ; n = 6); pair-fed (PF) vehicle control (PC; n = 6); PF ZEN (PZ; n = 6); HS vehicle control (HC; n = 7); or HS ZEN (HZ; n = 7) for 7 d. When compared to the TC pigs, TZ pigs had 45 increased and 39 decreased proteins (P ≤ 0.05). In the HZ pigs, 47 proteins were increased and 61 were decreased (P ≤ 0.05). Exposure to ZEN during TN conditions altered sec61 translocon complex (40%), rough endoplasmic reticulum membrane (8.2%), and proteasome complex (5.4%), asparagine metabolic process (0.60%), aspartate family amino acid metabolic process (0.14%), and cellular amide metabolic process (0.02%) pathways. During HS, ZEN affected cellular pathways associated with proteasome core complex alpha subunit complex (0.23%), fibrillar collagen trimer (0.14%), proteasome complex (0.05%), and spliceosomal complex (0.03%). Thus, these data identify ovarian pathways altered by ZEN exposure and suggest that the molecular targets of ZEN differ in TN and HS pigs.

Intracellular organelles remodeling in myocardial endotheliocytes in COVID-19: an autopsy-based study

ABSTRACT It is known that the unfavorable outcome in patients infected with SARS-CoV-2 may be associated with the development of complications caused by heart damage due to the direct virus action. The mechanism of these cardiovascular injuries caused by SARS-CoV-2 infection has not been fully understood; however, the study of COVID-19-associated myocardial microcirculatory dysfunction can represent the useful strategy to solving this challenge. Thus, here we aimed to study the ultrastructural organization of endothelial cells of myocardial capillaries in patients with COVID-19. The morphology of endotheliocytes of the myocardial blood capillaries in patients with COVID-19 was studied on cardiac autopsy material using transmission electron microscopy. The endotheliocytes of myocardial capillaries in patients with COVID-19 were characterized by the abundant rough endoplasmic reticulum (ER) membranes, the Golgi complex, and free polysomal complexes of ribosomes and lipids. The presence of double membrane vesicles with virions and zippered ER was detected in the cytoplasm of endotheliocytes. The revealed endothelial ultrastructural changes indicate the remodeling of intracellular membranes during SARS-CoV-2 infection. Our findings confirm the formation of virus-induced structures in myocardial endothelial cells considered critical for viral replication and assembly. The data may elucidate the mechanisms of endothelial dysfunction development in patients with COVID-19 to provide potential targets for drug therapy.

Micromorphology of hesperidin crystals and ultrastructure of crystal-bearing cells in Metrodorea nigra, a neotropical Rutaceae species

Researches on the developmental and morphological features of organic crystals in plants are relatively scarce. Hesperidin is a flavanone glycoside found abundantly in Rutaceae, mainly in Citrus fruits. Due to the variety of biological activities, hesperidin is one of the most promising bioflavonoids. We investigated the distribution, micromorphology and intracellular localization of hesperidin crystals in the vegetative organs of Metrodorea nigra, a neotropical Rutaceae tree. Additionally, we analyzed the ultrastructural organization of crystal-bearing cells. Bright field, epifluorescence and polarized-light microscopy, scanning (SEM) and transmission electron microscopy (TEM) analysis were performed. Hesperidin crystals appeared as needle-shaped crystals forming plumose clusters under SEM, and as acicular solitary crystal or aggregate yellow/brown twisted filaments inside cells under light microscope. Hesperidin and calcium oxalate crystals often occurred together in the epidermis and mesophyll tissues. Hesperidin crystals were abundant inside the phloem, while calcium oxalate crystals were absent in the vascular tissues. Phenolic substances occurred in all tissues, but they were abundant within vascular tissues. Flavonoid autofluorescence in blue color was associated with crystals in the cell walls and cytoplasm. The crystal-bearing cells exhibited extensive rough endoplasmic reticulum (RER) and associated vesicles filled with electron-dense materials, proliferation of the tonoplast and abundance of multilamellar bodies containing electron-dense or crystalline materials close to irregular plasmalemma. Our ultrastructural observations allow us to speculate that the RER and tonoplast membranes and associated vesicles appeared to be involved in the packaging and channeling of the materials toward the cell walls. Crystals of hesperidin and calcium oxalate occurred on the surface of common epidermal cells and glandular trichomes. The mechanisms of condensation, crystallization and extrusion of hesperidin crystal through the cell walls remain to be investigate, and could be an interesting subject for future studies.

Distinct transcriptional profiles in plasma exosomes associated with recurrence of nasopharyngeal carcinoma patients with standard treatment.

6534 Background: Nasopharyngeal carcinoma (NPC) is endemic with a high prevalence in Southern China, Asia,cetuximab and North Africa. Exosomes are small vesicles containing a wide range of functional proteins, mRNA and miRNA. In the progression of NPC, the tumor cells constantly release exosomes into the surrounding environment and also into the circulating blood. The aim of this study was to explore the association between RNA expression in plasma exosomes and prognosis of NPC patients after standard treatment. Methods: In this retrospective study, a total of 25 eligible NPC patients were included: 12 patients in the recurrence (R) subgroup and 13 patients in the no recurrence (NR) subgroup. RNA was extracted from the exosomes of plasma specimens which were collected at West China Hospital, Sichuan University. Gene expression profiles were conducted by using the RNA-sequencing platform. The DESeq2 package was used to analyze the differentially expressed genes (DEGs) between R and NR subgroups. The gene set variance analysis (GSVA) was performed to explore C5 gene sets enrichment related to the recurrence after standard treatment. Results: We observed 332 DEGs between R and NR subgroups, which include 125 up-regulated and 207 down-regulated genes (R vs. NR,∣log2fold change∣>1, p<0.05). Moreover, hierarchical clustering analysis of the 332 DEGs revealed that all samples clustered into two subgroups, with cluster 1 containing 82% (9/11) recurrence patients and cluster 2 containing 79% (11/14) no recurrence patients. Further, univariate Cox regression analysis showed that 293 out of 332 DEGs were significantly correlated with DFS ( p<0.05), such as TRAM1, CAPN1, SAT1 and ACTB. GSVA and Log Rank test of survival data demonstrated that a total of 824 pathways/biological processes were significantly different between R andNR subgroups ( p<0.05). Specifically, the top 9 pathways/biological processes, such lipoxygenase pathway, rough endoplasmic reticulum membrane and low density lipoprotein particle clearance, was mainly enriched in the NR subgroup ( p <0.001). Conclusions: Profiling of plasma exosomes RNA in NPC patients reveals distinctive gene expression pattern between patients with or without recurrence. Further functional analysis revealed that top enriched 9 pathways/biological processes may correlate with a favorable prognosis and are worth investigating. Moreover, for the prognosis of patients with NPC, RNA expression of plasma exosomes may be a potentially valuable research object.

Fractionation of Subcellular Compartments from Human Brain Tissue.

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.

Ultrastructure of Highly Ordered Granules in Alveolar Type II Cells in Several Species.

Alveolar Type II cells from seven mammalian species were examined for a protein in the rough endoplasmic reticulum (RER), which showed a multilayered, repeating motif. Each motif, 100 nm in width, comprised two parallel outer dense layers, a less dense central layer, and often 1-3 faint layers on either side of the latter. Outer layers showed periodicities at 3-4 densities/100 nm of width, while layers on either side of the central layer showed 5-7 densities/100 nm of width. RER membranes were ribosome-free when parallel to these layers, but showed four ribosomes per motif at the growing ends: one ribosome at each outer dense layer, and one on either side of the less dense central layer. Granules appeared as single or as multiple motifs, stacked, curved, folded, or branching together within the same RER profile. Hexagons of around 30 nm in diameter with central densities were seen in tangential cuts of outer dense layers. Granule incidence varied: guinea pig > ferret > dog. Possible homologous structures occurred in rabbit and cat, but not in rat or mouse. Surfactant protein A (SP-A), a C-type lectin produced in Type II cells, forms trimers and bouquet-like 18-mer and can oligomerize further. Two pairs of SP-A 18-mers with carbohydrate recognition domains pointing inwardly and outwardly, stacked vertically as a column of four molecules, then repeated side by side in rows, approximated the size and layering patterns observed in these granules. Sequence analyses of SP-A from these species showed phylogenetic distances consistent with the observed occurrence and frequency of patterned granules. Anat Rec, 301:1290-1302, 2018. © 2018 Wiley Periodicals, Inc.

Hepatic subcellular distribution of squalene changes according to the experimental setting.

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies.

Effect of Cyanophycin Metabolism in Recombinant Sinorhizobium (Ensifer) meliloti 1021 on the Symbiosis with Alfalfa (Medicago sativa)

The aim of this study was to investigate the effect of the synthesis and degradation of the nitrogen-rich polymer cyanophycin in rhizobia on symbiotic nitrogen fixation and crop yield in legumes. For this, cyanophycin synthetase from Anabaena sp. PCC7120 was expressed in the bacteroids of the symbiont S. meliloti 1021 either alone or together with an intracellular cyanophycinase from the same bacterium, either in the wild type or in a polyhydroxybutyratenegative (PHB-) mutant, and the effect on the growth of alfalfa host plants was studied. All strains induced the formation of nitrogen-fixing nodules in the host, but clear differences in various parameters were noticeable. Alfalfa plants infected with the wild type expressing only cyanophycin synthetase showed significantly lower shoot nitrogen contents and higher nitrogen fixation rates than plants inoculated with wild type, but the wild type phenotype was exceeded in S. meliloti 1021 expressing cyanophycinase in addition to cyanophycin synthetase. Growth of plants infected with the PHB- mutant expressing only cyanophycin synthetase was severely impaired in comparison to growth of plants infected with the PHB- mutant expressing both or neither of the two proteins. Transmission electron micrographs of sections of nodules induced by wild type S. meliloti 1021 producing cyanophycin synthetase and cyanophycinase showed that the rough endoplasmic reticulum and Golgi membranes were extended further in comparison to the nodules induced by the wild type, the PHB- mutant, or all other recombinant S. meliloti strains, indicating higher metabolic activities in these nodules.

Identification and characterization of transcriptional control region of the human beta 1,4-mannosyltransferase gene.

All asparagine-linked glycans (N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT-1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (β1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT-1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1kb region upstream from HMT-1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from -33 to -1 positions might act in HMT-1 transcription at basal level and that the region from -200 to -42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200bp region upstream from HMT-1 ORF might significantly upregulate HMT-1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at -148 to -134 positions should serve as a strong silencer. The control of the HMT-1 transcription by these motifs resided within the 200bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT-1 expression.

MRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells

MicroRNA (miRNA) binds to the 3'-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.

Evidence for a KATP Channel in Rough Endoplasmic Reticulum (rerKATP Channel) of Rat Hepatocytes.

We report in a previous study the presence of a large conductance K+ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a KATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to KATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to KATP, suggesting that opening of this channel may enhance Ca2+ releases, alter the dynamics of the Ca2+ transient and prevent accumulation of Ca2+ in the ER during Ca2+ overload.

Structural differences in epiphyseal and physeal hypertrophic chondrocytes.

We have observed that epiphyseal and physeal hypertrophic chondrocytes in BALB/c mice show considerable differences of light microscopic and ultrastructural appearance, even when the cells are at the same stage of differentiation. In addition, cell structure maintenance improved with tissue preparation controlled for osmolarity and for membrane stabilization using 0.5% ruthenium hexammine trichloride (RHT) for both light microscopy (LM) and electron microscopy (EM) or 0.5% lanthanum nitrate for LM. Physeal hypertrophic chondrocytes showed a gradual increase in size closer to the metaphysis and a change in shape as cells elongated along the long axis. The nucleus remained central, with uniformly dispersed chromatin, and the rough endoplasmic reticulum (RER) was randomly dispersed throughout cytoplasm with little to no presence against the cell membrane. Even the lowermost cells showed thin elongated or dilated cisternae of RER and intact cell membranes. Epiphyseal chondrocytes remained circular to oval with no elongation. Nucleus and RER were positioned as a complete transcellular central nucleocytoplasmic column or as an incomplete bud with RER of the column/bud always continuous with RER peripherally against the intact cell membrane. RER was densely packed with parallel cisternae with adjacent cytoplasm empty of organelles but often filled with circular deposits of moderately electron-dense material consistent with fat. Optimal technique for LM involved fixation using glutaraldehyde (GA) 1.3%, paraformaldehyde (PFA) 1% and RHT 0.5% (mOsm 606) embedded in JB-4 plastic and stained with 0.5% toluidine blue. Optimal technique for EM used fixation with GA 1.3%, PFA 1%, RHT 0.5% and cacodylate buffer 0.03 M (mOsm 511) and post-fixation including 1% osmium tetroxide. These observations lead to the possibility that the same basic cell, the hypertrophic chondrocyte, has differing functional mechanisms at different regions of the developing bone.

Mutations in theSPTLC1Protein Cause Mitochondrial Structural Abnormalities and Endoplasmic Reticulum Stress in Lymphoblasts

Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems

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

Ultrastructure and hydrolase activity in tobacco leaves exposed to chitosan

The mesophyll ultrastructure and hydrolase activity in leaves of tobacco cv. Samsun exposed to chitosan were studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus size and amount of both mitochondria and membranes of rough endoplasmic reticulum increased). At the same time, we observed activation of the lytic compartment displayed as stimulated production of dictyosomes, smooth ER elements, and cytoplasmic vacuoles, which are prominent constituents of this compartment. Biochemical experiments showed that, in the leaves, chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) as compared to untreated leaves. In some cells, chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm, etc.) that can be classified as development of programmed cell death.

Exocyst Sec10 Is Involved in Basolateral Protein Translation and Translocation in the Endoplasmic Reticulum

Background: Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -β, and -γ. Sec61β facilitates translocation, and we and others have shown that the highly conserved eight-protein exocyst complex interacts with Sec61β. We have also shown that the exocyst is involved in basolateral, not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery has been reported. Furthermore, we have shown that the exocyst is necessary for formation of primary cilia, organelles found on the apical surface. Methods: GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic endoplasmic reticulum (ER) microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and ³⁵S-methionine for protein detection were used to investigate the role of the exocyst in synthesis of polarized proteins. P³²-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Results: Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced exocyst recruitment to endoplasmic reticulum membranes following exocyst depletion and basolateral G protein of vesicular stomatitis virus protein translation, compared to apical hemagglutinin of influenza virus protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. Conclusion: These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β.

Endoplasmic Reticulum-Derived Multilamellar Bodies in Oocytes of Mouse Follicle Cultures under Oxidized Low-Density Lipoprotein Treatment

Objective: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL). Method: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12–48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy. Results: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress. Conclusion: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy.

Protection Effect of GDNF and Neurturin on Photosensitized Crayfish Neurons and Glial Cells

Neurons and glial cells can protect each other from stress and following death by mutual exchange with neurotrophins. In order to examine involvement of different neurotrophic factors in neuroglial interactions in a photosensitized crayfish stretch receptor, a simple model object consisting of only two sensory neurons enveloped by glial cells, we studied the influence of glial cell line-derived neurotrophic factor (GDNF), neurturin, and ciliary neurotrophic factor (CNTF) on its photodynamic injury. Photodynamic treatment, which causes strong oxidative stress, induced firing abolition and necrosis of neurons, necrosis, and apoptosis of glial cells. GDNF significantly reduced photoinduced neuronal necrosis and neurturin but not CNTF showed a similar tendency. Both of them significantly reduced necrosis and apoptosis of glial cells. At the ultrastructural level, neurons and glial cells treated with GDNF in the darkness contained large mitochondria with well-developed cristae, numerous ribosomes, polysomes, rough endoplasmic reticulum (ER), and dictyosomes. This indicated the high level of bioenergetic, biosynthetic, and transport processes. Photodynamic treatment caused swelling and vacuolization of mitochondria, dictyosomes, and ER. It also impaired formation of glial protrusions and double membrane vesicles that transfer glial material into the neuron. GDNF prevented photoinduced mitochondria swelling that disturbed the cellular bioenergetics and cytoplasm vacuolization associated with injury of intracellular organelles. It also preserved the structures involved in protein synthesis and transport: rough ER, dictyosomes, polysomes, microtubule bundles, submembrane cisterns, and double membrane vesicles. GDNF-mediated maintenance of metabolism and ultrastructure of photosensitized neurons and glial cells may be the basis of its neuro- and glia protective effects.

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.

Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.