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BackgroundRetinal pigmented epithelium (RPE) has essential functions to nourish and support the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE in aging remain poorly defined.MethodsWe isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10 - 67 years). A quantitative proteomic analysis was performed to analyze their intracellular and secreted protein changes, and potential age-associtated mechanisms were validated by ARPE-19 and hRPE cells.ResultsAge-stage related subtypes and age-associtated proteins and functional alterations were revealed. Proteomic data and verifications showed that RNF123 and RNF149 related ubiquitin-mediated proteolysis might be an important clearance mechanism in elimination of oxidative damaged proteins in aged hRPE. In older hRPE cells, apoptotic signaling related pathways were up-regulated and endoplasmic reticulum organization was down-regulated both in intracellular and secreted proteome.ConclusionsThis work paints a detailed molecular picture of human RPE in aging process and provides new insights for molecular characteristics of RPE in aging and related clinical retinal conditions.

Objective To identify rheumatoid arthritis (RA)-associated susceptibility genes and pathways through integrating genome-wide association study (GWAS) and gene expression profile data. Methods A transcriptome-wide association study (TWAS) was conducted by the FUSION software for RA considering EBV-transformed lymphocytes (EL), transformed fibroblasts (TF), peripheral blood (NBL), and whole blood (YBL). GWAS summary data was driven from a large-scale GWAS, involving 5539 autoantibody-positive RA patients and 20,169 controls. The TWAS-identified genes were further validated using the mRNA expression profiles and made a functional exploration. Results TWAS identified 692 genes with PTWAS values PEL = 0.01293, PTF = 0.00038, PNBL = 0.02839, PYBL = 0.0978), MUT (PEL = 0.00377, PTF = 0.00076, PNBL = 0.00778, PYBL = 0.00096), FOXRED1 (PEL = 0.03834, PTF = 0.01120, PNBL = 0.01280, PYBL = 0.00583), and EBPL (PEL = 0.00806, PTF = 0.03761, PNBL = 0.03540, PYBL = 0.04254) were collectively expressed in all the four tissues/cells. Eighteen genes, including ANXA5, AP4B1, ATIC (PTWAS = 0.0113, downregulated expression), C12orf65, CMAH, PDHB, RUNX3 (PTWAS = 0.0346, downregulated expression), SBF1, SH2B3, STK38, TMEM43, XPNPEP1, KIAA1530, NUFIP2, PPP2R3C, RAB24, STX6, and TLR5 (PTWAS = 0.04665, upregulated expression), were validated with integrative analysis of TWAS and mRNA expression profiles. TWAS-identified genes functionally involved in endoplasmic reticulum organization, regulation of cytokine production, TNF signaling pathway, immune response-regulating signaling pathway, regulation of autophagy, etc. Conclusion We identified multiple candidate genes and pathways, providing novel clues for the genetic mechanism of RA.

The purpose of this study was to determine the effects of single and combined administrations of deoxynivalenol (DON) and zearalenone (ZEN) on the histology and ultrastructure of pig liver. The study was performed on immature gilts, which were divided into four equal groups. Animals in the experimental groups received DON at a dose of 12 &mu, g/kg body weight (BW) per day, ZEN at 40 &mu, g/kg BW per day, or a mixture of DON (12 &mu, g/kg BW per day) and ZEN (40 &mu, g/kg BW). The control group received vehicle. The animals were killed after 1, 3, and 6 weeks of experiment. Treatment with mycotoxins resulted in several changes in liver histology and ultrastructure, including: (1) an increase in the thickness of the perilobular connective tissue and its penetration to the lobules in gilts receiving DON and DON + ZEN, (2) an increase in the total microscopic liver score (histology activity index (HAI)) in pigs receiving DON and DON + ZEN, (3) dilatation of hepatic sinusoids in pigs receiving ZEN, DON and DON + ZEN, (4) temporary changes in glycogen content in all experimental groups, (5) an increase in iron accumulation in the hepatocytes of gilts treated with ZEN and DON + ZEN, (6) changes in endoplasmic reticulum organization in the hepatocytes of pigs receiving toxins, (7) changes in morphology of Browicz&ndash, Kupffer cells after treatment with ZEN, DON, and DON + ZEN. The results show that low doses of mycotoxins used in the present study, even when applied for a short period, affected liver morphology.

The roles of the heterotrimeric G-protein, Gi2, in regulating the actin cytoskeleton and the activation of store-operated Ca2+ channels in rat hepatocytes were investigated. Gαi2 was principally associated with the plasma membrane and microsomes. Both F-actin and Gαi2 were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant co-precipitated with Gαi2 using either anti-Gαi2 or anti-actin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some Gαi2 co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70–80% of Gαi2 in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca2+ inflow. It is concluded that (i) a significant portion of hepatocyte Gαi2 associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca2+ inflow.

Golgi stacks are often located near sites of “transitional ER” (tER), where COPII transport vesicles are produced. This juxtaposition may indicate that Golgi cisternae form at tER sites. To explore this idea, we examined two budding yeasts: Pichia pastoris, which has coherent Golgi stacks, and Saccharomyces cerevisiae, which has a dispersed Golgi. tER structures in the two yeasts were visualized using fusions between green fluorescent protein and COPII coat proteins. We also determined the localization of Sec12p, an ER membrane protein that initiates the COPII vesicle assembly pathway. In P. pastoris, Golgi stacks are adjacent to discrete tER sites that contain COPII coat proteins as well as Sec12p. This arrangement of the tER-Golgi system is independent of microtubules. In S. cerevisiae, COPII vesicles appear to be present throughout the cytoplasm and Sec12p is distributed throughout the ER, indicating that COPII vesicles bud from the entire ER network. We propose that P. pastoris has discrete tER sites and therefore generates coherent Golgi stacks, whereas S. cerevisiae has a delocalized tER and therefore generates a dispersed Golgi. These findings open the way for a molecular genetic analysis of tER sites.

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi -specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi . In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.

Calcium store depletion activates multiple ion channels, including calcium-selective and nonselective channels. Endothelial cells express TRPC1 and TRPC4 proteins that contribute to a calcium-selective store-operated current, I(SOC). Whereas thapsigargin activates the I(SOC) in pulmonary artery endothelial cells (PAECs), it does not activate I(SOC) in pulmonary microvascular endothelial cells (PMVECs), despite inducing a significant rise in global cytosolic calcium. Endoplasmic reticulum exhibits retrograde distribution in PMVECs when compared with PAECs. We therefore sought to determine whether endoplasmic reticulum-to-plasma membrane coupling represents an important determinant of I(SOC) activation in PAECs and PMVECs. Endoplasmic reticulum organization is controlled by microtubules, because nocodozole induced microtubule disassembly and caused retrograde endoplasmic reticulum collapse in PMVECs. In PMVECs, rolipram treatment produced anterograde endoplasmic reticulum distribution and revealed a thapsigargin-activated I(SOC) that was abolished by nocodozole and taxol. Microtubule motors control organelle distribution along microtubule tracks, with the dynein motor causing retrograde movement and the kinesin motor causing anterograde movement. Dynamitin expression reduces dynein motor function inducing anterograde endoplasmic reticulum transport, which allows for direct activation of I(SOC) by thapsigargin in PMVECs. In contrast, expression of dominant negative kinesin light chain reduces kinesin motor function and induces retrograde endoplasmic reticulum transport; dominant negative kinesin light chain expression prevented the direct activation of I(SOC) by thapsigargin in PAECs. I(SOC) activation is an important step leading to disruption of cell-cell adhesion and increased macromolecular permeability. Thus, microtubule motor function plays an essential role in activating cytosolic calcium transitions through the membrane I(SOC) channel leading to endothelial barrier disruption.

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.

Developmental changes in the Ca2+ dynamics of human zygotes and preimplantation embryos were related to changes in the distribution of endoplasmic reticulum (ER) and protein kinase C (PKC). The fertilization-induced Ca2+ oscillations were typically observed over > 5 h, were ryanodine-sensitive and showed a periphery-to-centre propagation of Ca2+ waves. At the same time, ER and PKC were accumulated in the cell periphery. After the appearance of pronuclei, ryanodine-sensitive Ca2+ oscillations of lower amplitude and frequency were observed until the pronuclear breakdown. However, Ca2+ waves then began in the perinuclear region, in the area of ER and PKC accumulation and spread towards the cell periphery. During the second to fourth cell cycle, small sinusoidal Ca2+ fluctuations were observed; sparse higher-amplitude Ca2+ spikes, superimposed on these basal fluctuations, appeared shortly before cell division. The sinusoidal Ca2+ fluctuations were asynchronous in individual blastomeres and disappeared progressively in arrested embryos. The direction of Ca2+ wave propagation and the distribution of ER and PKC were similar to the situation observed in pronuclear zygotes. In contrast to the zygotes, ryanodine did not arrest the Ca2+ oscillations but augmented their amplitude and frequency. These data suggest that human pre-embryos use different mechanisms of Ca2+ signalling in the early post-fertilization period, during the pronuclear development and during cleavage.

The biogenesis of the Golgi complex and the maintenance of its supramolecular organization has become subject to renewed interest recently. Much of this discussion has centred around the question of whether the Golgi arises by cisternal maturation of endoplasmic reticulum (ER)-derived membranes, thereby constantly changing its own identity, or whether all transport along the secretory pathway occurs by the fusion and fission of small carrier vesicles between stable preexisting organelles. Many new insights have been gained lately concerning the mechanism of intra-Golgi transport, but much less is known about an intimately related problem: how is the Golgi complex formed?A new study by Rossanese et al. tries to tackle this problem by looking at it from a new angle1xGolgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. Rossanese, O.W. et al. J. Cell Biol. 1999; 145: 69–81Crossref | PubMed | Scopus (217)See all References1. The authors have undertaken a morphological analysis of the ‘transitional ER’ (tER), a major site for COPII-coated vesicle production and the Golgi in two different yeasts, Pichia pastoris and Saccharomyces cerevisiae. These two, otherwise very similar, fungi exhibit major differences in their organization of the Golgi complex: Golgi cisternae are organized into stacks in Pichia pastoris, whereas, in Saccharomyces cerevisiae, they remain scattered throughout the cytoplasm. Rossanese et al. find that COPII vesicles arise from discrete tER sites adjacent to Golgi stacks in P. pastoris, whereas, in S. cerevisiae, COPII vesicles bud from the entire ER and are present throughout the cytoplasm. These data suggest that the biogenesis of the Golgi complex and its ultimate organization within the cell are intimately linked to the functional organization of the ER rather than preexisting Golgi stacks, and they should pave the way for the genetic analysis of tER sites.

The endoplasmic reticulum in some neurons of the rat hypothalamic arcuate nucleus forms concentric sheets of smooth cisternae which are known as whorl bodies. It has been reported that the number and size of these structures change under varying endocrine conditions. For example, the number of rat arcuate nucleus neurons containing whorl bodies increases after gonadectomy. In studying their relationship to other components of the cell, we found that neuron profiles which contain whorl bodies receive a significantly increased number of axosomatic presynaptic terminals (P less than 0.001). Whorl bodies may mark a subpopulation of endocrine sensitive cells which are characterized by a different pattern of connections and whose response to stimuli includes changes in endoplasmic reticulum organization.