Size Of Vacuoles Research Articles

Environment-Responsive Alkanol-Based Supramolecular Solvents: Characterization and Potential as Restricted Access Property and Mixed-Mode Extractants

Self-assembly, the process by which supramolecular solvents (SUPRAS) with an ordered structure are produced, provides unique opportunities to obtain tailored solvents with advanced functional features. In this work, environment-responsive (C(7)-C(14)) alkanol-based SUPRAS were synthesized and their potential for analytical extractions was assessed. The global composition of the solvent, the size of the coacervate droplets that form it, and the aqueous cavities of the inverted hexagonal arrangement of the alkanols can be tailored by controlling the environment [specifically, the tetrahydrofuran (THF):water ratio in the bulk solution] for alkanol self-assembly. Interestingly, supramolecular solvents are highly adaptive and the previous features can all be reversed by modifying the environment. The spontaneous self-assembly of these solvents followed predictable routes, and their composition and volume can be accurately predicted from equations derived in this work. The solvents were structurally elucidated by light and cryo-scanning electron microscopy. Extractive applications exploiting the molecular size-based restricted access properties of SUPRAS were developed and their ability to engage in mixed-mode mechanisms for solute solubilization was established. Thus, solutes of increasing molecular weight were extracted from food and environmental samples with recoveries dependent on vacuole size in the SUPRAS, while macromolecules such as proteins, carbohydrates, and humic acids were excluded. The ability of SUPRAS to establish hydrogen-bonding and dispersion interactions was exploited to extract carcinogenic chlorophenols (CCPs) from environmental waters, and a simple and fast method was developed with quantitation limits (e.g., 0.21-0.23 μg·L(-1)) low enough to comply with legislation (e.g., maximum permitted levels for pentachlorophenol are in the range 0.4-1 μg·L(-1)).

PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Abstract 2628PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth.In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo.We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Effect of gold nanoparticles on adipogenic differentiation of human mesenchymal stem cells

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity after different exposure times (4–21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles.

Nuclear vacuoles and apoptosis markers after annexin V columns in patients with severe teratozoospermia

Annexin V columns (AnV-C) has been proposed as a tool to reduce levels of sperm apoptosis. The aim of this study was to determine the presence of nuclear vacuoles and apoptotic markers after using AnV-C and assess the relation between the size and number of nuclear vacuoles with sperm apoptosis.

Abstract 2984: A novel role for survivin, a potential target for cancer drugs, in vesicle trafficking

Abstract Survivin is a 16.5 kD protein that is highly expressed in most cancer cells. Although it is also expressed in embryonic tissues and proliferating adult cells, it may be a promising target for cancer therapy if it has distinct functions in normal versus malignant cells. It is well established that survivin has an essential role in dividing cells as a member of the chromosome passenger complex (CPC). We recently showed that survivin is highly expressed in orthochromatic erythroblasts, cells that have exited the cell cycle and undergoing enucleation. Based on this observation, we predicted that survivin would play a novel role in maturation of these late erythroblasts. To discover the function of survivin in these post-mitotic cells, we first analyzed the localization of survivin and other members of the CPC by immunofluorescence. We discovered that survivin is expressed in cytoplasm, but did not colocalize with aurora B kinase or INCENP in primary enucleating erythroblasts. Next, we overexpressed survivin in the murine erythroleukemia (MEL) cell line, which undergoes terminal differentiation but does not enucleate, and quantified the effect on maturation by flow cytometry. In contrast to control MEL cells, which down-regulate survivin and fail to enucleate, nearly 15% of the survivin overexpressing cells showed nuclear extrusion. To obtain insight into this novel effect of survivin, we purified survivin protein complexes from MEL cell lysates and performed mass spectrometry. We found that EPS-15 (EGFR pathway substrate clone 15) and clathrin, two proteins that are involved in endocytic vesicle trafficking, interact with survivin. We confirmed these interactions by immunoprecipitation and immunofluorescence studies in primary human erythroblasts. Next, we individually knocked down survivin and EPS-15 in primary human erythroblast cultures and demonstrated that both proteins are required for optimal enucleation. Moreover, deletion of the survivin gene in fetal liver erythroblasts isolated from survivin floxed embryos, reduced the enucleation efficiency without affecting cell survival or differentiation. In order to determine how survivin and EPS15 contribute to enucleation, we performed light microscopic and ultrastructural examination of primary murine red cells undergoing terminal maturation. We observed that loss of survivin was associated with a decrease in the number of large cellular vacuoles within erythroblasts. Furthermore, by immunogold-EM, we found that survivin co-localized with EPS-15 within LAMP1-positive vacuoles in late erythroblasts. Finally, we observed that vacuolin-1, a small molecule that increases the number and size of vacuoles, rescued the enucleation defect of survivin-/- erythroblasts. Together, these results suggest that survivin plays an important and novel role in vesicle trafficking within maturing red blood cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2011-2984

Adaptability evaluation of switchgrass ( Panicum virgatum L.) cultivars on the Loess Plateau of China

In the study, the growth traits, photosynthesis and morphology characteristics of several cultivars of switchgrass ( Panicum virgatum L.) have been assessed the yield potential and adaptability in diverse environments (Yangling, Dingbian of Shaanxi province, Guyuan of Ningxia) on the Loess Plateau of China. Alamo was the best adapted switchgrass cultivar for biomass production in Yangling with dry matter (DM) yields of 44.22 t/ha; Illinois USA and Cave-in-Rock grown at Guyuan had DM yield of 10.59 t/ha and 9.36 t/ha, respectively. Similarly, Cave-in-Rock in Dingbian performed better than others except the lowland cultivars (Alamo and Kanlow), which could not overcome cold stress at Guyuan and Dingbian. Moreover, Cave-in-Rock and Nebraska 28 has the highest photosynthesis rate which reflects its high productivity. Nebraska 28 and Pathfinder shown strong drought tolerance due to their higher WUE. It appears that the upland cultivars with high ploidy (e.g. 8 n) would have better establishment than lowland varieties there. Optimal mown management seems to enhance the growth and productivity of switchgrass. Morphological characteristics were further studied using light-and scanning electron microscopy (SEM). Silica particles, vacuole size and other traits in switchgrass tissues (stem, leaf and root), as well as trichomes (leaf) showed that Cave-in-Rock and Pathfinder had larger stoma area, up to 824.4 μm 2 and 770.1 μm 2, respectively. Silica particle length was the longest in Pathfinder and shortest in Cave-in-Rock. There was a highest density of silica particles in cv. Forestberg, and lowest in Cave-in-Rock and Pathfinder. The morphological characters seemed to be associated with their ploidy levels and the arid habitat from which they were selected. Therefore, if switchgrass is to be introduced and extended on the Loess Plateau of China, Cave-in-Rock and other upland cultivars with a high chromosome ploidy might be optimal choices for biomass plants.

An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay

The aims of this study were to establish whether individual differences exist in the frequency and size of vacuoles found in human sperm and to ascertain whether such vacuoles are involved in causing DNA damage. Morphologically normal sperm were obtained from 15 IVF and 2 ICSI patients and 3 fertile donors. (i) Sperm heads were analyzed for the presence of vacuoles under a 1000× differential interference contrast microscope. (ii) In three patients and two donor samples, structural chromosomal damage was evaluated in normal sperm containing large vacuoles and selected at 1000× magnification for injection into mouse oocytes. (iii) In 10 patients and two donor samples, confocal laser microscopy detected DNA damage in sperm-exhibiting large vacuoles and stained with an in situ cell death detection kit. (i) Vacuoles were observed in almost all normal sperm from patient and donor ejaculates and were mainly located at the tip or middle area of the sperm heads. However, average incidence of normal sperm exhibiting large vacuoles was 4.6 and 4.2% in the patient and donor groups, respectively. (ii) Sperm chromosome assays did not reveal any differences in the incidence of structural chromosome aberrations between sperm exhibiting large vacuoles and those without them (9.1 versus 4.1%). (iii) No significant difference in frequency of TUNEL-positive cells was found between normal sperm with large vacuoles and those without them in the samples examined. Among 227 sperm exhibiting large vacuoles, only 7 cells were TUNEL positive. The results showed that large vacuoles were not responsible for DNA damage, suggesting that intra-cytoplasmic injection of morphologically selected sperm may not be required for patients who produce high-quality semen.

Di(n‐butyl) Phthalate Induces Vimentin Filaments Disruption in Rat Sertoli Cells: A Possible Relation with Spermatogenic Cell Apoptosis

Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

An endoplasmic reticulum response pathway mediates programmed cell death of root tip induced by water stress in Arabidopsis

Drought induces root death in plants; however, the nature and characteristics of root cell death and its underlying mechanisms are poorly understood. Here, we provide a systematic analysis of cell death in the primary root tips in Arabidopsis during water stress. Root tip cell death occurs when high water deficit is reached. The dying cells were first detected in the apical meristem of the primary roots and underwent active programmed cell death (PCD). Transmission electron microscopic analysis shows that the cells undergoing induced death had unambiguous morphological features of autophagic cell death, including an increase in vacuole size, degradation of organelles, and collapse of the tonoplast and the plasma membrane. The results suggest that autophagic PCD occurs as a response to severe water deficit. Significant accumulation of reactive oxygen species (ROS) was detected in the stressed root tips. Expression of BAX inhibitor-1 (AtBI1) was increased in response to water stress, and atbi1-1 displayed accelerated cell death, indicating that AtBI1 and the endoplasmic reticulum (ER) stress response pathway both modulate water stress-induced PCD. These findings form the basis for further investigations into the mechanisms underlying the PCD and its role in developmental plasticity of root system architecture and subsequent adaptation to water stress.

Ouabain-Induced Vacuolar Formation in Marginal Cells in the Stria Vascularis Is Dependent on Perilymphatic Na+

In the stria vascularis (SV), it is known that the Na⁺-K⁺-ATPase is expressed abundantly and its activity in the basolateral membrane of marginal cells is high. Ouabain, an inhibitor of the Na⁺-K⁺-ATPase, causes not only a decline in the endocochlear DC potential but also acute vacuolar formation in marginal cells. We studied the ionic mechanisms underlying the ouabain-induced vacuolar formation in marginal cells using perilymphatic perfusion in guinea pigs. Perilymphatic perfusion with 1 mM ouabain dissolved in the artificial perilymph for 50 min caused many vacuoles of a wide range of sizes in the apical cytoplasm of marginal cells, the bulging of marginal cells into the scala media and strial volume increase. Removal of K⁺ from the perilymph reduced the proportion of vacuoles and strial thickening, but the bulging of marginal cells remained. In contrast, the sizes of vacuoles were drastically reduced and extrusion of marginal cells into the scala media could not be observed in the absence of perilymphatic Na⁺. Furthermore, the total volume of SV was obviously reduced in comparison with the control. These results indicate that perilymphatic Na⁺ and K⁺ are responsible for these morphological changes caused by ouabain, and that perilymphatic Na⁺ plays an important role in the cellular volume regulation in SV in the presence of ouabain. It is supposed that the transport system of perilymphatic Na⁺ and K⁺ into marginal cells may contribute to vacuolar formation when ouabain is administered. Regarding Na⁺, we hypothesize two possibilities for the perilymphatic Na⁺ transporting pathway as follows. Na⁺ in the perilymph could enter the endolymph via Reissner’s membrane or the basilar membrane; Na⁺ in the endolymph would then be taken up by marginal cells via the apical membrane and secreted into the intrastrial space by Na⁺-K⁺-ATPase in the basolateral membrane. Another, less likely, possibility is that Na⁺ in the perilymph is transported into basal cells or fibrocytes in the spiral ligament, then into intermediate cells via gap junctions, and finally secreted into the intrastrial space via Na⁺-K⁺-ATPase of intermediate cells. Regarding K⁺, it is expected that the K⁺ recycling pathway plays a role in ouabain-induced vacuolar formation in marginal cells.

Effect of Feed Liquid Temperature on the Structural Morphologies of d‐Limonene Microencapsulated Powder and Its Preservation

The influence of the feed liquid temperature of the spray drying was investigated on the particle morphology (vacuole size and shell thickness of hollow particle) and the stability of encapsulated d-limonene. The feed liquid was the mixture of d-limonene emulsified in a wall material of blending gum arabic (GA) and maltodextrin (MD) at the mass ratio of 1 : 2, and the liquid temperature was varied from 40 to 80 degrees C. The percentage of the hollow particle in the spray-dried powder was over 40% and slightly increased with the rising of the feed temperature. Hollow particles have shells of different thickness, which was estimated from the image with a confocal laser scanning microscope (CLSM). The shell thickness increased with the increase in the feed temperature. This finding was qualitatively supported by scanning electron microscopy (SEM) images of the fractured particles. In addition, release and oxidation stability of encapsulated d-limonene were measured at constant temperature and humidity. The powder at high temperature of feed has higher stability for release and oxidation of d-limonene than that at a lower feed temperature. The result may partly be attributed to the increase of the shell thickness of the particle at a higher feed temperature. The thicker shell wall possibly served as the barrier of the flavor release and the diffusion of oxygen and moisture from the surrounding environment.

Anti-aging and anti-inflammation effects of natural mineral extract on skin keratinocytes

In this study, we investigated the effects of a natural mineral extract on damaged skin keratinocytes. We have focused on the possible effects of the natural mineral extract on UV-irradiated normal human skin keratinocytes. Human keratinocytes were UV-irradiated and were treated with 0.1, 1.0, or 10% of natural mineral extract. Study controls included non-UV-irradiated cells, UV-irradiated cells (no extract), and UV-irradiated cells treated with 25 nM ascorbic acid. Cell viability following UV irradiation was significantly higher in the groups treated with the natural mineral extract at doses of 0.1, 1.0, and 10% than in the control group with UV irradiation only. The UV-irradiated cells demonstrated increased secretion of laminin relative to non-irradiated controls. We found that treating these UV-irradiated keratinocytes with natural mineral extract reduced vacuole size and number, and it dose-dependently increased laminin secretion and reduced IL-2 synthesis. Also, MMP-1 and MMP-2 activity were significantly less in cells treated with natural mineral extract than in UV-irradiated controls. The UV irradiation reduced MMP-1 levels to 45±5 ng/mL from 130±5 ng/mL. In addition, MMP-2 production in keratinocytes was significantly reduced by 11±1 ng/mL compared to the UV-irradiation control. In conclusion, the results of this work suggest that natural mineral extract has effects on keratinocytes damaged by UV exposure.

Vacuolar ATPase-Mediated Cellular Concentration and Retention of Quinacrine: A Model for the Distribution of Lipophilic Cationic Drugs to Autophagic Vacuoles

The antiprotozoal agent quinacrine is a lipophilic cationic drug highly distributed to tissues. It has been used in the present experiments to examine whether the vacuolar and autophagic cytopathology induced by organic amines is independent from the therapeutic class. Furthermore, we tested the presence of the concentrated cationic drug itself in the enlarged vacuoles by exploiting the intense green fluorescence of quinacrine. Finally, the influence of lipophilicity on the apparent affinity of amine pseudotransport has been addressed by comparing quinacrine to another substituted triethylamine, procainamide. Quinacrine was concentration-dependently taken up by human smooth muscle cells (cytosolic granular-vacuolar morphology at and above 25 nM; in cell extracts, uptake nearly maximal in 2 h, apparent K(m) of 8.7 microM). The vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine uptake by cells or released the cell-associated drug in preloaded cells. The lipidated (II) form of microtubule-associated protein light chain 3 accumulated at and above a quinacrine concentration of 2.5 microM (4 h), indicating the conserved macroautophagic nature of the vacuolar cytopathology, although vacuole size was modest. The enlarged vacuoles containing quinacrine excluded cherry fluorescent protein; many vacuoles were lined with cherry fluorescent protein-conjugated Rab7, a GTPase associated with late endosomes/lysosomes. Taken together, these results are compatible with the transition of quinacrine-concentrating vacuoles toward an autophagolysosome identity. Quinacrine is concentrated in cells via V-ATPase-mediated ion trapping with an apparent affinity approximately 500-fold higher than that of the less lipophilic drug procainamide, and, despite the small size of ensuing vacuoles, the macroautophagic signature of this cytopathology was observed.

Actin Dynamics and Rho GTPases Regulate the Size and Formation of Parasitophorous Vacuoles Containing Coxiella burnetii

Q fever is a disease caused by Coxiella burnetii. In the host cell, this pathogen generates a large parasitophorous vacuole (PV) with lysosomal characteristics. Here we show that F-actin not only is recruited to but also is involved in the formation of the typical PV. Treatment of infected cells with F-actin-depolymerizing agents alters PV development. The small PVs formed in latrunculin B-treated cells were loaded with transferrin and Lysotracker and labeled with an antibody against cathepsin D, suggesting that latrunculin B did not affect vacuole cargo and its lysosomal characteristics. Nevertheless, the vacuoles were unable to fuse with latex bead phagosomes. It is known that actin dynamics are regulated by the Rho family GTPases. To assess the role of these GTPases in PV formation, infected cells were transfected with pEGFP expressing wild-type and mutant Rac1, Cdc42, and RhoA proteins. Rac1 did not show significant PV association. In contrast, PVs were decorated by both the wild types and constitutively active mutants of Cdc42 and RhoA. This association was inhibited by treatment of infected cells with chloramphenicol, suggesting a role for bacterial protein synthesis in the recruitment of these proteins. Interestingly, a decrease in vacuole size was observed in cells expressing dominant-negative RhoA; however, these small vacuoles accumulated transferrin, Lysotracker, and DQ-BSA. In summary, these results suggest that actin, likely modulated by the GTPases RhoA and Cdc42 and by bacterial proteins, is involved in the formation of the typical PV.

Effect of feeding by two Neochetina species or infestation with Tetranychus urticae Koch on histological structure of water hyacinth leaves

Both weevils, Neochetina bruchi (Hustache) and Neochetina eichhorniae Warner feed on water hyacinth plants as a biocontrol agents. Tetranychus urticae Koch infested the plants grown under light and thermostatic controlled aquatic weed green house, and caused repellency of the weevils. Studying some histological structure, of water hyacinth leaves infested with mites , showed increase of the thickness of upper epidermis, number of palisade cell and thickness of leaf petiole, compared with the control and leaves infested with weevils which recorded decrease in the thickness of leaf petiole and increase in lower palisade tissue. The mites infested plants lost chlo rophyll, turned brownish and lost size of vacuoles or air chambers used to store the oxygen. The volume of these vacuoles was very small in case of mite infested plants compared with leaves infested with weevils as the same as control. The weevils feeding reflected in increase in both, length and width of fed upon leaves.

Role of Dexamethasone and Oncostatin M on the Formation of Vacuoles in Human Fetal Liver Cells

Combined treatment with dexamethasone and oncostatin M (DEX/OSM) or interleukin-6 (DEX/IL-6) resulted in the appearance of numerous large vacuoles in human fetal liver (HFL) cells and showed synergistic effects on the formation of vacuoles. The number of vacuoles formed by DEX, DEX/OSM, or DEX/IL-6 was significantly suppressed by RU-486, a glucocorticoid receptor antagonist. On the other hand, the size of vacuoles formed by OSM, IL-6, DEX/OSM, or DEX/IL-6 was significantly decreased to about 65% by madindoline A (MDL-A), which is a non-peptide antagonist of gp130 and an inhibitor of cytokines, such as IL-6, mediated by gp130 homodimerization, while RU-486 did not affect the size of vacuoles. Expression of IL-6 mRNA in HFL cells was markedly induced by OSM. Expression of IL-6R mRNA was induced by DEX. These results indicate that DEX contributes to the formation of vacuoles through glucocorticoid receptors and that OSM and IL-6 contribute to enlargement of these vacuoles.

The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1Delta mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1Delta mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.

Niemann-Pick C1 Functions in Regulating Lysosomal Amine Content

Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.

Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

Histopathology of the Urinary Bladders of Cynomolgus Monkeys Treated with PPAR Agonists

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, hyperlipidemias, and related complications. Consequently, a mechanistic understanding of PPAR subtypes and their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus and the metabolic syndrome. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. Sufficient data are not yet available to explain the mode(s) of action for most of these tumor types. There has been information presented by FDA that indicates there are urothelial changes in the monkey (and possibly the dog) in addition to the rat. Outstanding questions exist regarding potency, species differences, safety margins, and other issues. In 2005, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established to advance research on the modes of action and potential human relevance of emerging rodent tumor data. Additionally, the HESI PPAR Agonist Project Committee authorized a Pathology Working Group (PWG) to examine the urinary bladder from cynomolgus monkeys. The focus of this PWG was to establish consistent diagnostic criteria for urothelial changes and to assess the potential relationship of these changes to treatment. Specific diagnostic criteria and nomenclature were recommended for the diagnosis of urothelial granules, vacuolation, hypertrophy, and hyperplasia in studies conducted with PPARgamma and dual alpha/gamma agonists in cynomolgus monkeys, which will assist investigators performing toxicity studies to provide data in a consistent manner between studies and laboratories. In this review of selected tissues, treatment with PPAR agonists was not associated with urothelial hypertrophy or hyperplasia, but there was an increased incidence in the size and frequency of vacuoles within the superficial urothelial and adjacent intermediate cell layers.