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Vacuoles
Kinds of Vacuoles Terms modified by Vacuoles Selected AbstractsVaccine-associated granulomatous inflammation and melanin accumulation in Atlantic salmon, Salmo salar L., white muscleJOURNAL OF FISH DISEASES, Issue 1 2005E O Koppang Abstract The purpose of this study was to investigate the nature of variably sized pigmented foci encountered in fillets of farmed Atlantic salmon, Salmo salar L. The material was sampled on the fillet production line and on salmon farms from fish with an average size of 3 kg from various producers. The fish had been routinely vaccinated by injection. Gross pathology, histology, immunohistochemistry using antisera against major histocompatibility complex (MHC) class II , chain and transmission electron microscopy (TEM) were used to characterize the changes. Macroscopically, melanized foci were seen penetrating from the peritoneum deep into the abdominal wall, sometimes right through to the skin, and also embedded in the caudal musculature. Histological investigation revealed muscle degeneration and necrosis, fibrosis and granulomatous inflammation containing varying numbers of melano-macrophages. Vacuoles, either empty or containing heterogeneous material, were frequently seen. The presence of abundant MHC class II+ cells indicated an active inflammatory condition. TEM showed large extracellular vacuoles and leucocytes containing homogeneous material of lipid-like appearance. The results showed that the melanized foci in Atlantic salmon fillet resulted from an inflammatory condition probably induced by vaccination. The described condition is not known in wild salmon and in farmed salmon where injection vaccination is not applied. [source] Autophagic vacuolar myopathy in twin girlsNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 3 2006J. L. Holton Hereditary autophagic vacuolar myopathy (AVM) may occur in several diseases including the rimmed vacuolar myopathies, acid maltase deficiency, Danon disease, infantile autophagic vacuolar myopathy and X-linked myopathy with excessive autophagy (XMEA). In the latter three conditions the vacuoles are lined by membranes with sarcolemmal features. We present two unusual cases of autophagic vacuolar myopathy in twin girls born at term with no family history of neurological disease. After initial normal developmental milestones they developed progressive leg weakness and wasting with contractures from the age of 12 years. Investigations showed raised CK, normal female karyotype, normal acid maltase activity, normal nerve conduction and myopathic EMG features. Frozen sections of skeletal muscle were stained using routine tinctorial and histochemical methods. Immunohistochemical staining for spectrin, merosin, dystrophin, complement membrane attack complex and sarcoglycans was performed and ultrastructural examination undertaken. Direct sequence analysis of the lamp-2 gene using genomic DNA extracted from lymphocytes was performed. Histological analysis of the muscle biopsies demonstrated myofibres with vacuoles lacking glycogen and lipid many of which were delineated using immunohistochemistry for merosin, dystrophin and sarcoglycans. Ultrastructural examination showed duplication of the myofibre basal lamina with associated autophagic material. Vacuoles within myofibres were either membrane bound containing autophagic material or lined by plasma membrane and basal lamina. Intermyofibrillar glycogen was increased. Sequence analysis of the coding region and intron/exon boundaries of the lamp-2 gene was normal. This is the first report of female cases of AVM with sarcolemmal features. We suggest that these patients may represent manifesting carriers of XMEA, or alternatively, a new form of disease with a similar phenotype having autosomal recessive inheritance. [source] Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cellsNEW PHYTOLOGIST, Issue 4 2006Sandra Tuszy Summary ,,Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. ,,Vacuoles, mitochondria and microtubules were labelled with Oregon Green® 488 carboxylic acid diacetate, 3,3,-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-,-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0,1 mmol l,1 and examined microscopically. ,,Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. ,,These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion. [source] The Nature of Exocytosis in the Yolk Trophoblastic Layer of Silver Arowana (Osteoglossum bicirrhosum) Juvenile, the Representative of Ancient Teleost FishesTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 11 2009Marta Jaroszewska Abstract We have chosen the silver arowana (Osteoglossum bicirrhosum), a representative of the most ancient teleost family Osteoglossidae, to address the question of yolk nutrients utilization. Silver arowana have particularly large eggs (1,1.5 cm of diameter) and a unique morphology of the yolk. We present evidence that the yolk cytoplasmic zone (ycz) in the "yolksac juveniles" is a very complex structure involved in sequential processes of yolk hydrolysis, lipoprotein particles synthesis, their transport, and exocytosis. Vacuoles filled with yolk granules in different stages of digestion move from the vitellolysis zone through the ycz to be emptied into the microvillar interspace in the process of exocytosis. The area of the ycz with the abundance of the mitochondria must play an important role in providing energy for both the transport of vacuoles and the release of their contents. Therefore, we postulate that the function of yolk syncytial layer (ysl) as the "early embryonic patterning center" transforms in fish larvae or yolksac juveniles into a predominantly specialized role as the yolk trophoblastic layer (ytl) involved in yolk nutrients utilization. In addition to discovering the mechanism of transformation of the ysl function into ytl function, we suggest that the machinery involved in nutrient mobilization and exocytosis in yolk of arowana yolksac juveniles can be very attractive system for studies of regulatory processes in almost all secretory pathways in animal cells. Anat Rec, 2009. © 2009 Wiley-Liss, Inc. [source] Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotesCELLULAR MICROBIOLOGY, Issue 12 2009Norikiyo Ueno Summary The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not (P < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways. [source] The roles of actin cytoskeleton and microtubules for membrane recycling of a food vacuole in Tetrahymena thermophilaCYTOSKELETON, Issue 7 2009Maki Sugita Abstract Phagocytosis is a fundamental cellular event for the uptake of nutrients from the environment in several kinds of eukaryote. Most ciliates egest waste and undigested materials in food vacuoles (FVs) through a cytoproct, which is a specific organelle for defecation. It is considered that FV egestion is initiated by fusion between the FV membrane and plasma membrane in a cytoproct and completed with retrieval of the membrane into a cytoplasmic space. In addition, electron microscopy indicated that microfilaments might be involved in the recycling process of the FV membrane in ciliates over 30 years ago; however, there is no conclusive evidence. Here we demonstrated actin organization on FV near a cytoproct in Tetrahymena thermophila by using a marker for a cytoproct. Moreover, it was revealed that cells treated with actin cytoskeletal inhibitor, Latrunculin B, might be suppressed for membrane retrieval in a cytoproct following FV egestion. On the other hand, the actin structures, likely to be the site of membrane retrieval, were frequently observed in the cells treated with cytoplasmic microtubules inhibitor, Nocodazole. We concluded that actin filaments were probably required for recycling of the FV membrane in a cytoproct although the role was not essential for FV egestion. In addition, it was possible that microtubules might be involved in transportation of recycling vesicles of FV coated with F-actin. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Fine structure of unusual spermatozoa and spermiogenesis of the mite Megisthanus floridanus Banks, 1904 (Acari: Gamasida: Antennophorina)ACTA ZOOLOGICA, Issue 4 2002Gerd Alberti Abstract The aflagellate spermatozoa of the gamasid mite Megisthanus floridanus are characterized by a large vacuole which contains a cytoplasmic column protruding into the vacuole from the region defined as the posterior part of the cell. The membrane of the column and the inner membrane of the posterior part of the cytoplasmic mantle (outer sheath) surrounding the vacuole bear numerous so-called cellular processes. However, most of the outer sheath is reduced and represented solely by a very thin membrane-like envelope. The posterior part of the cell bears extensive folds. The cell, or, more precisely, the column, shows a deep posterior invagination. This invagination contains extracellular material composed of thin filaments or strands. Peripheral folds emerging from the posterior rim of the cell form a thin-walled tube that contains the same material as the invagination. The elongated nucleus is attached to a peculiar acrosomal complex consisting of a flat acrosomal cisterna that parallels most of the cell membrane, an attachment cone, and a short acrosomal filament which is embedded in a narrow canal within the nucleus. The spermatozoa of M. floridanus represent a peculiar version of the vacuolated type of sperm known to be plesiomorphic within the anactinotrichid Acari. Some details of spermiogenesis are described and consequences for phylogenetic and systematic considerations are discussed. [source] Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. KuntzeENVIRONMENTAL TOXICOLOGY, Issue 4 2007Prashant Mohanpuria Abstract Glutathione, a tripeptide with sulfhydryl (-SH) group is a very crucial compound primarily involved in redox balance maintenance of the cellular environment. In this study, we monitored the influence of Cd exposure on the transcript levels of glutathione metabolic genes in bud tissues, the youngest leaf, of Camellia sinensis L. In addition, some physiochemical parameters were also studied. Cd exposure decreased chlorophyll and protein contents, while increase was observed in lipid peroxidation upon Cd treatments. These changes were found to be concentration and duration dependent, indicating the occurrence of oxidative stress upon Cd exposure. The transcript levels of glutathione biosynthetic genes viz. ,-glutamylcysteine synthetase (,-ECS) and glutathione synthetase (GSHS) increased upon Cd exposure. Furthermore, transcript levels of glutathione reductase (GR), an enzyme involved in reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), also showed upregulation on Cd exposure. However, the transcript levels of glutathione-S-transferase (GST), an enzyme involved in forming metal,GSH complex and help in sequestration of high levels of metal ions to vacuole, did not show any change on Cd treatment. This study document that Cd exposure induces oxidative stress in Camellia sinensis and the upregulation in transcript levels of glutathione metabolic genes except GST have suggested the role of these enzymes in the protection of plants from high level Cd exposure. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 368,374, 2007. [source] Characterization of Lead Precipitate Following Uptake by Roots of Brassica junceaENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2009Donald E. R. Meyers Abstract Seedlings of Brassica juncea (L.) Czern. were grown in solution culture for 14 d prior to exposure to Pb2+ at an activity of 31 ,M for 72 h. Electron-dense deposits found within the apoplast and symplast were analyzed using scanning transmission electron microscopy/energy dispersive spectroscopy to determine the chemical identity of the deposits and potential toxicity resistance mechanisms. Irrespective of the cellular compartment in which they were found, the deposits contained Pb, O, P, and Cl. For the extracellular deposits, the average Pb:P:O atomic ratio was 1:0.54:3.0, which together with the hexagonal crystal system suggests that Pb is present as chloropyromorphite (Pb5(PO4)3Cl). A weak Ca signal also was detected in approximately half of the spectra, possibly indicating the presence of small concentrations of phosphohedyphane (Pb3Ca2(PO4)3Cl). The evidence suggests that B. juncea resists Pb toxicity by storing precipitated Pb in the vacuole. [source] Uncompromised generation of a specific H-2DM-dependent peptide-MHC class,II complex from exogenous antigen in Leishmania mexicana -infected dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2003Clare Abstract Leishmania infection inhibits the capacity of macrophages (M,) to present antigens to CD4+ T cells. Relocation of MHC class,II and H-2DM to the parasitophorous vacuole (PV) and their subsequent degradation by the parasite may contribute to this defect. Dendritic cells (DC) are critical for initiation of primary T cell responses. DC can process Leishmania antigen and elicit Leishmania -specific T cells, but it is unknown whether exposure to Leishmania impairs this capacity. In particular, it is not clear whether DC containing live parasites efficiently process and present antigens. We investigated the ability of mouse bone marrow-derived DC infected with L. mexicana to generate pigeon cytochrome,c (PCC) peptide-MHC class II complexes, using the mAb D4, which recognizes PCC89,104 H-2Ek, and the PCC-specific T cell hybridoma 2B4. We show that H-2DM-dependent complex generation is not compromised by infection and that complexes are fully recognized by specific T cells. We further show that in contrast to infected M,, in infected DC cytoplasmic H-2DM is not down-regulated and not relocated to the parasite-containing vacuole. This observation may explain the continued ability of infected DC to present PCC, and also indicates differences in the habitat of these intracellular parasites in DC compared to M,. [source] Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 11 2003Olivier Gueldry In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using ,-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury,glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury. [source] The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl, across the plasma membraneFEMS YEAST RESEARCH, Issue 8 2007Angélica López-Rodríguez Abstract Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl, channel that remained permanently open and was blocked by nitro-2-(3-phenylpropylamino) benzoic acid and niflumic acid. NPPB induced petite -like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl, through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined. [source] Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genesFEMS YEAST RESEARCH, Issue 1 2001Anna Rita Bellu Abstract In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes. [source] The mechanisms of resistance to antimalarial drugs in Plasmodium falciparumFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 2 2003Jacques Le Bras Abstract Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs. Plasmodium falciparum drug-resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i) impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii) one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii) the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv) treatment with sulphadoxine,pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino-alcohols (quinine, mefloquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug-resistance encountered in the forests of South-east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance. [source] Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiaeGENES TO CELLS, Issue 6 2008Keisuke Obara Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3)P. The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3)P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3)P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3)P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3)P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3)P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3)P in autophagy. [source] Vacuolar membrane dynamics revealed by GFP-AtVam3 fusion proteinGENES TO CELLS, Issue 7 2002Tomohiro Uemura Background: The plant vacuole is a multifunctional organelle that has various physiological functions. The vacuole dynamically changes its function and shape, dependent on developmental and physiological conditions. Our current understanding of the dynamic processes of vacuolar morphogenesis has suffered from the lack of a marker for observing these processes in living cells. Results: We have developed transgenic Arabidopsis thaliana expressing a vacuolar syntaxin-related molecule (AtVam3/SYP22) fused with green fluorescent protein (GFP). Observations using confocal laser scanning microscopy demonstrated that the plant vacuole contained a dynamic membrane system that underwent a complex architectural remodelling. Three-dimensional reconstitution and time-lapse analysis of GFP-fluorescence images revealed that cylindrical and sheet-like structures were present in the vacuolar lumen and were moving dynamically. The movement, but not the structure itself, was abolished by cytochalasin D, an inhibitor of actin polymerization. This moving structure, which sometimes penetrated through the vacuolar lumen, possessed a dynamic membrane architecture similar to the previously recognized ,transvacuolar strand.' Conclusion: We propose two possible models for the formation of the vacuolar lumenal structure. Membrane structures including protruding tubules and reticular networks have recently been recognized in many other organelles, and may be actively involved in intra- and/or inter-organelle signalling. [source] Microinjected neutrophils retain the ability to take up bacteriaJOURNAL OF ANATOMY, Issue 5 2002M. M. Bird It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 × 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 µL FITC-anti rat IgG and 1 µL TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 µL of PBS, washed 6 times in PBS and mounted on slides in 5 µL Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source] Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoitesJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2009S. Ferro Abstract Aims:, To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments. Methods and Results:, We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment. Conclusions:, The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage. Significance and Impact of the Study:, Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis, opening new perspectives for the disinfection of microbiologically polluted waters. [source] The effect of superoxide dismutase deficiency on cadmium stressJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2004Paula D. B. Adamis Abstract Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:12,17, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20000 [source] Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiaeAGING CELL, Issue 4 2009Ashley L. Alvers Summary Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast. [source] Effects of chromium stress on the subcellular distribution and chemical form of Ca, Mg, Fe, and Zn in two rice genotypesJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 1 2010Fanrong Zeng Abstract A hydroponic experiment was carried out to study effects of chromium (Cr) stress on the subcellular distribution and chemical form of Ca, Mg, Fe, and Zn in two rice genotypes differing in Cr accumulation. The results showed that Ca, Mg, Fe, and Zn ions were mainly located in cell walls and vacuoles in roots. However, large amounts of metal ions were transferred from the vacuole to the nucleus and to other functional organelles in shoots. Chromium concentrations in the nutrient solution of 50 ,M and above significantly decreased Ca concentrations in the chloroplast/trophoplast, the nucleus, and in mitochondria. It further increased Mg concentrations in the nucleus and in mitochondria, as well as Zn and Fe concentrations in the chloroplast/trophoplast. These Cr-induced changes in ion concentrations were associated with a significant reduction in plant biomass. It is suggested that Cr stress interferes with the functions of mineral nutrients in rice plants, thus causing a serious inhibition of plant growth. The chemical forms of the four nutrients were determined by successive extraction. Except for Ca, which was mainly chelated with insoluble phosphate and oxalic acid, Mg, Zn, and Fe were extractable by 80% ethanol, d-H2O, and 1,M NaCl. The results indicated that these low,molecular weight compounds, such as organic acids and amino acids, may play an important role in deposition and translocation of Mg, Zn, and Fe in the xylem system of rice plants. [source] Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010Y. WU Summary.,Background and objective:,Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. Methods and results:,We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and ,v,3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of ,v,3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. Conclusions:,HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction. [source] Chloroquine resistance in the malarial parasite, Plasmodium falciparumMEDICINAL RESEARCH REVIEWS, Issue 5 2002Lyann M.B. Ursos Abstract Malarial parasites remain a health problem of staggering proportions. Worldwide, they infect about 500 million, incapacitate tens of millions, and kill approximately 2.5 million (mostly children) annually. Four species infect humans, but most deaths are caused by one particular species, Plasmodium falciparum. The rising number of malarial deaths is due in part to increased drug resistance in P. falciparum. There are many varieties of antimalarial drug resistance, and there may very well be several molecular level contributions to each variety. This is because there are a number of different drugs with different mechanisms of action in use, and more than one molecular event may sometimes be relevant for resistance to any one class of drugs. Thus, "multidrug" resistance in a clinical setting likely entails complex combinations of overlapping resistance pathways, each specific for one class of drug, that then add together to confer the particular multidrug resistance phenotype. Nonetheless, rapid progress has been made in recent years in elucidating mechanisms of resistance to specific classes of antimalarial drugs. As one example, resistance to the antimalarial drug chloroquine, which has been the mainstay therapy for decades, is becoming well understood. This article focuses on recent advances in determining the molecular mechanism of chloroquine resistance, with particular attention to the biochemistry and biophysics of the P. falciparum digestive vacuole, wherein changes in pH have recently been found to be associated with chloroquine resistance. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 5, 465,491, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10016 [source] An Integrin and Rho GTPase-Dependent Pinocytic Vacuole Mechanism Controls Capillary Lumen Formation in Collagen and Fibrin MatricesMICROCIRCULATION, Issue 1 2003GEORGE E. DAVIS ABSTRACT A major question that remains unanswered concerning endothelial cell (EC) morphogenesis is how lumens are formed in three-dimensional extracellular matrices (ECMs). Studies from many laboratories have revealed a critical role for an ECM-integrin-cytoskeletal signaling axis during EC morphogenesis. We have discovered a mechanism involving intracellular vacuole formation and coalescence that is required for lumen formation in several in vitro models of morphogenesis. In addition, a series of studies have observed vacuoles in vivo during angiogenic events. These vacuoles form through an integrin-dependent pinocytic mechanism in either collagen or fibrin matrices. In addition, we have shown that the Cdc42 and Rac1 guanosine triphosphatases (GTPases), which control actin and microtubule cytoskeletal networks, are required for vacuole and lumen formation. These GTPases are also known to regulate integrin signaling and are activated after integrin-matrix interactions. Furthermore, the expression of green fluorescent protein-Rac1 or -Cdc42 chimeric proteins in ECs results in the targeting of these fusion proteins to intracellular vacuole membranes during lumen formation. Thus, a matrix-integrin-cytoskeletal signaling axis involving both the Cdc42 and Rac1 GTPases regulates the process of EC lumen formation in three-dimensional collagen or fibrin matrices. [source] An endocytic mechanism for haemoglobin-iron acquisition in Candida albicansMOLECULAR MICROBIOLOGY, Issue 1 2008Ziva Weissman Summary The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem-iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)-anchored, haemin- and haemoglobin-binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C. albicans vacuole. We relied on recapitulation of the Rbt51-dependent haem-iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin-iron utilization and haemoglobin uptake. These mutants define a novel endocytosis-mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway. [source] PfCRT and the trans -vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IXMOLECULAR MICROBIOLOGY, Issue 1 2006Patrick G. Bray Summary It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt -modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV. [source] Root cadmium desorption methods and their evaluation with compartmental modelingNEW PHYTOLOGIST, Issue 1 2010Wayne T. Buckley Summary ,Desorption of plant roots is often employed in studies of plant physiology and nutrition; however, there have been few studies on the validity of desorption procedures. ,Branched and in-line kinetic models with five compartments , cadmium (Cd)-chelate, Cd2+, root apoplast, root symplast and vacuole , were developed to evaluate the efficacy of diethylenetriaminepentaacetic acid (DTPA) and CaCl2 methods for the desorption of Cd from roots of durum wheat seedlings. Solution Cd2+ could exchange with apoplast and symplast Cd simultaneously in the branched model and sequentially in the in-line model. ,A 10-min desorption with 1 × 10,6 M DTPA at room temperature or cold (0°C) 5 × 10,3 M CaCl2 was required to achieve 99% recovery of apoplast-bound 109Cd when experimental results were interpreted with the branched model. However, when the same data sets were analysed with the in-line model, only partial desorption was achieved. Arguments are presented that suggest that the branched model is correct. ,It is suggested that compartmental modeling is a suitable tool for the study of plant root uptake and desorption kinetics, and that there are advantages over more commonly used calculation procedures. [source] pH regulation in an acidophilic green alga , a quantitative analysisNEW PHYTOLOGIST, Issue 2 2009Birgit Bethmann Summary ,,Short-term cytosolic pH regulation has three components: H+ binding by buffering groups; H+ transport out of the cytosol; and H+ transport into the vacuole. To understand the large differences plants show in their tolerance to acidic environments, these three components were quantified in the acidophilic unicellular green alga Eremosphaera viridis. ,,Intracellular pH was recorded using ion-selective microelectrodes, whereas constant doses of weak acid were applied over different time intervals. A mathematical model was developed that describes the recorded cytosolic pH changes. Recordings of cytosolic K+ and Na+ activities, and application of anion channel inhibitors, revealed which ion fluxes electrically compensate H+ transport. ,,The cytosolic buffer capacity was , = 30 mM. Acidification resulted in a substantial and constant H+ efflux that was probably driven by the plasmalemma H+ -ATPase, and a proportional pH regulation caused by H+ pumped into the vacuole. Under severe cytosolic acidification (, 1 pH) more than 50% of the ATP synthesized was used for H+ pumping. While H+ influx into the vacuole was compensated by cation release, H+ efflux out of the cell was compensated by anion efflux. ,,The data presented here give a complete and quantitative picture of the ion fluxes during acid loading in an acidophilic green plant cell. [source] Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cellsNEW PHYTOLOGIST, Issue 4 2006Sandra Tuszy Summary ,,Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. ,,Vacuoles, mitochondria and microtubules were labelled with Oregon Green® 488 carboxylic acid diacetate, 3,3,-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-,-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0,1 mmol l,1 and examined microscopically. ,,Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. ,,These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion. [source] Managing the manganese: molecular mechanisms of manganese transport and homeostasisNEW PHYTOLOGIST, Issue 3 2005Jon K. Pittman Summary Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis. [source] | ||||||||||||||||||||||||||||||||||||||||