Search results for: vacuole

Authors: Muhammad Arifur Rahman, Talukdar M. Waliullah, Takashi Ushimaru

Abstract:

Nucleophagy selectively degrades nuclear materials, especially nucleolus after nutrient starvation or inactivation of TORC1 kinase in budding yeast. Budding yeast phosphatidate (PA) phosphatase Pah1 that converts PA to diacylglycerol is essential for partitioning of lipid precursors between membrane and storage that is crucial for many aspects of cell growth and development. Pah1 is required for nuclear/ER membrane biogenesis and vacuole function, but whether Pah1 and its activator Nem1/Spo7 protein phosphatase complex are involved in autophagy is largely unknown. Loss of Pah1 causes expansion of the nucleus and fragmentation of the vacuole. Here we show that Pah1 is required for bulk autophagy and nucleophagy after TORC1 inactivation. Loss of Pah1 impaired nucleophagy severely and bulk autophagy to a lesser extent. Loss of the Pah1 activator Nem1-Spo7 protein phosphatase exhibited similar features.

Keywords: autophagy, Nem1/Spo7 phosphatase, Pah1, nucleophagy, TORC1

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Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

Procedia PDF Downloads 127

Authors: Muccifora S., Rinallo C., Bellani L.

Abstract:

Calcium (Ca²+) is an element essential to the plant being involved in plant growth and development. At high concentrations, it is toxic and can influence every stage, process and cellular activity of plant life. Given its toxicity, cells implement mechanisms to compartmentalize calcium in a vacuole, endoplasmic reticulum, mitochondria, plastids and cell wall. One of the most effective mechanisms to reduce the excess of calcium, thus avoiding cellular damage, is its complexation with oxalic acid to form calcium oxalate crystals that are no longer osmotically or physiologically active. However, the sequestered calcium can be mobilized when the plant needs it. Calcium crystals can be accumulated in the vacuole of specialized sink-cells called idioblasts, with different crystalline forms (druse, raphyde and styloid) of diverse physiological meanings. Actinidia deliciosa cv. Hayward presents raphydes and styloid localized in idioblasts in cells of photosynthetic and non-photosynthetic tissues. The purpose of this work was to understand if there is a relationship between the age of Actinidia leaves and the presence, distribution, dimension and shape of oxalate crystals by means of light, fluorescent, polarized and transmission electron microscopy. Three vines from female plants were chosen at the beginning of the season and used throughout the study. The leaves with petioles were collected at various stages of development from the bottom to the shoot of the plants monthly from April to July. The samples were taken in corresponding areas of the central and lateral parts of the leaves and of the basal portion of the petiole. The results showed that in the leaves, the number of raphyde idioblasts decreased with the progress of the growing season, while the styloid idioblasts increased progressively, becoming very numerous in the upper nodes of July. In June and in July samples, in the vacuoles of the highest nodes, a portion regular in shape strongly stained with rubeanic acid was present. Moreover, the chlortetracycline (CTC) staining for localization of free calcium marked the wall of the idioblasts and the wall of the cells near vascular bundles. In April petiole samples, moving towards the youngest nodes, the raphydes idioblast decreased in number and in the length of the single raphydes. Besides, crystals stained with rubeanic acid appeared in the vacuoles of some cells. In June samples, numerous raphyde idioblasts oriented parallel to vascular bundles were evident. Under the electron microscope, numerous idioblasts presented not homogeneous electrondense aggregates of material, in which a few crystals (styloids) in the form of regular holes were scattered. In July samples, an increase in the number of styloid idioblasts in the youngest nodes and little masses stained with CTC near styloids were observed. Peculiar cells stained with rubeanic acid were detected and hypothesized to be involved in the formation of the idioblasts. In conclusion, in Actinidia leaves and petioles, it seems to confirm the hypothesis that the formation of styloid idioblasts can be correlated to increasing calcium levels in growing tissues.

Keywords: calcium oxalate crystals, actinidia deliciosa, light and electron microscopy, idioblasts

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Authors: Nthatisi I. Molefe-Nyembe, Keisuke Suganuma, Oriel M. M. Thekisoe, Xuan Xuenan, Noboru Inoue

Abstract:

African trypanosomosis is a devastating disease of animals caused by parasites of the genus Trypanosoma negatively affecting the economic status of more than 36 African countries. Few available drugs for the treatment of trypanosomosis remain inaccessible in remote areas, are associated with severe toxicity and most importantly, resistance has widely developed against their usage. Therefore, safe, effective and easily administrable drugs are urgently in need. The objective of the current study was to determine efficacy of azithromycin (AZM), on T. congolense, T. brucei brucei in vitro and in vivo. A 96 well luciferase assay was conducted to determine the trypanocidal effect of AZM on T. congolense, T. b. brucei and T. evansi as well as the cytotoxicity effect on the MDBK and NIH 3T3 cells. Additionally, TEM analysis was conducted to determine the morphological alteration on the AZM treated samples. Mice were infected with T. congolense and T. b. brucei and orally treated with AZM for 7 and 28 days referred to as the short and the long-term treatment. The in vitro IC50 values of AZM on T. congolense, T. b. brucei and T. evansi was 0.19 ± 0.17; 3.69 ± 2.26 and 1.81 ± 1.82 μg/mL, respectively, while the cytotoxicity effects values were greater than 25 μg/mL. A vacuole-like structure was observed in the TEM imaging of AZM treated T. congolense, while the presence of glycosomes and acidocalcisome-like structured were detected in T. b. brucei samples. In vivo, AZM was more effective against T. congolense infected mice than T. b. brucei. In conclusion, AZM exhibited the trypanocidal effects on T. congolense and T. b. brucei infected mice. However, further studies are necessary to determine the metabolic pathway responsible for the observed efficacy.

Keywords: animal trypanosomosis, azithromycin, oral administration, trypanosoma congolense

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Authors: Alina Goldberg Cavalleri, Sara Franco Ortega, Nawaporn Onkokesung, Richard Dale, Melissa Brazier-Hicks, Robert Edwards

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Non-target site based resistance (NTSR) to herbicides in weeds is a polygenic trait associated with the upregulation of proteins involved in xenobiotic detoxification and translocation we have termed the xenome. Among the xenome proteins, ABC transporters play a key role in enhancing herbicide metabolism by effluxing conjugated xenobiotics from the cytoplasm into the vacuole. The importance of ABC transporters is emphasized by the fact that they often contribute to multidrug resistance in human cells and antibiotic resistance in bacteria. They also play a key role in insecticide resistance in major vectors of human diseases and crop pests. By surveying available databases, transcripts encoding ABCs have been identified as being enhanced in populations exhibiting NTSR in several weed species. Based on a transcriptomics data in black grass (Alopecurus myosuroides, Am), we have identified three proteins from the ABC-C subfamily that are upregulated in NTSR populations. ABC-C transporters are poorly characterized proteins in plants, but in Arabidopsis localize to the vacuolar membrane and have functional roles in transporting glutathionylated (GSH)-xenobiotic conjugates. We found that the up-regulation of AmABCs strongly correlates with the up-regulation of a glutathione transferase termed AmGSTU2, which can conjugate GSH to herbicides. The expression profile of the ABC transcripts was profiled in populations of black grass showing different degree of resistance to herbicides. This, together with a phylogenetic analysis, revealed that AmABCs cluster in different groups which might indicate different substrate and roles in the herbicide resistance phenotype in the different populations

Keywords: black grass, herbicide, resistance, transporters

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Authors: Sara Franco Ortega, Alina Goldberg Cavalleri, Nawaporn Onkokesung, Richard Dale, Melissa Brazier-Hicks, Robert Edwards

Abstract:

Safeners are agrochemicals that enhance the selective chemical control of wild grasses by increasing the ability of the crop to metabolise the herbicide. Although these compounds are widely used, their mode of action is not well understood. It is known that safeners enhance the metabolism of herbicides, by up-regulating the associated detoxification system we have termed the xenome. The xenome proteins involved in herbicide metabolism have been previously divided into four different phases, with cytochrome P450s (CYPs) playing a key role in phase I metabolism by catalysing hydroxylation and dealkylation reactions. Subsequently, glutathione S-transferases (GSTs) and UDP-glucosyltransferases lead to the formation of Phase II conjugates prior to their transport into the vacuole by ABCs transporters (Phase III). Maize (Zea mays), was been treated with different safeners to explore the selective induction of xenome proteins, with a special interest in the regulation of the CYP superfamily. Transcriptome analysis enabled the identification of key safener-inducible CYPs that were then functionally assessed to determine their role in herbicide detoxification. In order to do that, CYP’s were codon optimised, synthesised and inserted into the yeast expression vector pYES3 using in-fusion cloning. CYP’s expressed as recombinant proteins in a strain of yeast engineered to contain the P450 co-enzyme (cytochrome P450 reductase) from Arabidopsis. Microsomes were extracted and treated with herbicides of different chemical classes in the presence of the cofactor NADPH. The reaction products were then analysed by LCMS to identify any herbicide metabolites. The results of these studies will be presented with the key CYPs identified in maize used as the starting point to find orthologs in other crops and weeds to better understand their roles in herbicide selectivity and safening.

Keywords: CYPs, herbicide detoxification, LCMS, RNA-Seq, safeners

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Authors: Prerna Kalra, Surender Singh

Abstract:

Cisplatin is the most common chemotherapeutic agent used in different solid tumors, but its main limiting factor is dose-dependent nephrotoxicity by generating reactive oxygen species, by stimulating inflammatory and apoptotic pathways. Additional adjuvant therapies to decrease the toxicity of this chemotherapeutic drug are essential. This study was designed to evaluate the protective role of Emblica officinalis Geartn (Indian gooseberry) against cisplatin induced nephrotoxicity. Emblica officinalis was orally administered to Wistar rats (n=6) for 10 days in 50, 100 and 200mg/kg body weight. On day 7, 8mg/kg of cisplatin was administered intra-peritoneally to rats in all groups. Serum creatinine, blood urea nitrogen and antioxidant levels were measured on day10. The renal damage was evaluated by histopathological and transmission electron microscopy. We found that 200mg/kg dose of Emblica officinalis significantly inhibited the elevation of biochemical parameters i.e. serum creatinine, blood urea nitrogen, oxidant stress marker (malondialdehyde) and increased the reduced levels of antioxidant marker (endogenous glutathione and superoxide dismutase). Cisplatin treated rats have shown acute tubular necrosis and infiltration of inflammatory cells in rat kidney which was reversed after treating the animals with Emblica officinalis in the treatment group. In ultrastructural changes cisplatin treated group showed the damaged mitochondria (M) with dissolved cristae and large number of lysosomes (L) and vacuole (V) formation in tubular epithelial cells. EOE administered group showed visible cristae formation and sign of autophagy vacuoles at a dose of 200mg/kg. Further in-silico studies revealed that ellagic acid is responsible for its nephroprotective effect. The above findings conclude that the Emblica officinalis may be used as an adjuvant therapy in cisplatin induced nephrotoxicity.

Keywords: antioxidant, cisplatin, Emblica officinalis, in silico, nephrotoxicity

Procedia PDF Downloads 291