Distribution of alkaline phosphatase in vegetative dictyostelium cells in relation to the contractile vacuole complex

The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae the membranes of which are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model.     

Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.     

网柄菌属的2个中国新记录种和2个中国大陆新记录种

从采自中国西南部分省区(云南、贵州)的土壤样品中分离出网柄菌属的2个中国新记录种,即粗茎网柄菌(Dictyostelium crassicaule)和交织网柄菌(Dictyostelium implicatum);2个中国大陆新记录种,即大头网柄菌(Dictyostelium macrocephalum)和大网柄菌(Dictyostelium magnum)。文中对该4个种进行了详细的形态学描述,并附其部分生活史的图片。     

Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling

Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.     

Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus

Aflatoxin biosynthesis in Aspergillus parasiticus requires at least 17 enzyme activities (from acetate). Although the activities of most aflatoxin biosynthetic enzymes have been established, the mechanisms that govern transport and sub-cellular localization of these enzymes are not clear. We developed plasmid constructs that express Nor-1 fused to a green fluorescent protein reporter (EGFP) to monitor transport and localization of this early pathway enzyme in real time in Aspergillus parasiticus. Plasmids expressing EGFP fused to Nor-1 were introduced into A. parasiticus B62 (carries non-functional Nor-1). Transformants were screened for increased aflatoxin accumulation (restored Nor-1 activity) on coconut agar medium and for EGFP expression using fluorescence microscopy. Increased aflatoxin accumulation was confirmed by TLC and ELISA. Nor-1 fused to EGFP at either the N- or C- terminus functionally complemented non-functional Nor-1 in B62 and increased aflatoxin synthesis to wild-type (N-terminus) or lower levels (C-terminus). We detected full-length Nor-1 fusion proteins in transformants with increased aflatoxin accumulation (Western blot) and determined that the expression plasmid integrated at the nor-1 locus in these cells (Southern blot). Confocal laser scanning microscopy (CLSM) demonstrated that Nor-1 fusion proteins localized in the cytoplasm and vacuoles of fungal hyphae grown on aflatoxin-inducing solid media for 48 h; control EGFP (no Nor-1) did not localize to vacuoles until 72 h. The highest rate of aflatoxin synthesis coincided with the highest rate of transport of Nor-1 fusion proteins to the vacuole strongly suggesting that Nor-1 is synthesized in the cytoplasm and transported to the vacuole to carry out an early step in aflatoxin synthesis.     

Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P-glycoprotein (P-gp), a 170kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na⁺/H⁺ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a selective inhibitor of the Na⁺/H⁺ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 μM was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose-dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.     

A Type IV Translocated Legionella Cysteine Phytase Counteracts Intracellular Growth Restriction by Phytate

The causative agent of Legionnaires'' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique “Legionella-containing vacuole.” The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different “effector” proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys²³¹) and arginine (Arg²³⁷) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.     

Inhibition by CIPC of mitosis and development in Dictyostelium discoideum and the isolation of CIPC-resistant mutants

The anti-mitotic herbicide isopropyl N-(3-chlorophenyl) carbamate (CIPC) prevents the growth of amoebae of Dictyostelium discoideum without killing the cells for a period of time equivalent to one generation. During in-hibition, amoebae accumulate in prophase and metaphase of mitosis. After removal of CIPC, they continue through mitosis and then divide. The addition of CIPC to amoebae under starvation conditions prevents aggregation and concomitant cell elongation. The cells, however, do not lose their ability to adhere to a surface, and they remain viable. When CIPC is added to amoebae which have formed streams, it leads to the disintegration of streams into small clusters of cells and to a loss of cell elongation. Post-aggregation stages of development can be inhibited by CIPC at the mound, slug, or Mexican hat stages. Slugs break apart into distinct aggregates. Mutants resistant to CIPC can be obtained easily. Among these mutants, many become temperature sensititive for growth (27°C) or development (27°C or 15.5°C). Others show various abnormalities at the normal temperature (22°C). Most mutants are cross resistant to the microtubule inhibitors nocodazole and thiabendazole, and some are also resistant to CIPC during development.     

烟草叶片液泡内氨基酸成份研究

液泡是植物细胞中特有的大型细胞器,具有重要的生理功能。本文报导了用双酶直接酶解法从烟草叶肉细胞中分离原生质体和完整液泡。在最适保存条件下,原生质体和液泡分别在３６和１２小时后,尚有一半保持活力。液泡内含有大量游离氨基酸,液泡膜ＡＴＰａｓｅ的最适ｐＨ为７．０,受Ｃｌ－激活,受ＮＯ３－抑制。     

Inhibition of Both Na/H and Bicarbonate-Dependent Exchange is Required to Prevent Recovery of Intracellular pH in Single Cardiomyocytes Exposed to Metabolic Stress

Although tight regulation of intracellular pH (pHi) is critical for the survival under stress, paradoxically a slowed recovery of pHi under hypoxic injury may be cardioprotective. In this study, we investigated the recovery of pHi after hypoxia-induced intracellular acidosis in cardiomyocytes loaded with the H⁺-sensitive dye SNARF-1. Exposure of single cardiomyocytes to 2,4-dinitrophenol (DNP), an inhibitor of mitochondrial oxidative phosphorylation, induced significant intracellular acidification. However, within 10–12 min upon removal of DNP, cardiomyocytes restituted their intracellular H⁺ concentration. The presence either of 5-N-ethyl-N-isopropylamiloride (EIPA) an inhibitor of Na/H antiporter, or 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), an inhibitor of bicarbonate-dependent exchange, did not modify the cellular response to DNP. But, combined use of EIPA and DIDS prevented the restitution of intracellular pH following removal of DNP. This study, thus, demonstrated, for the first time, that blockade of both Na/H and bicarbonate-dependent exchange is necessary and sufficient to maintain the hypoxia-induced intracellular acidification. Therefore, concomitant blockade of both pH-regulating mechanisms deserves to be further considered as a novel strategy against hypoxia-reoxygenation injury in the heart.     

Transport of primary metabolites across the plant vacuolar membrane

Mesophyll cells and most types of storage cells harbor large central vacuoles representing the main cellular store for sugars and other primary metabolites like carboxylic- or and amino acids. The general biochemical characteristics of sugar transport across the vacuolar membrane are already known since a couple of years but only recently the first tonoplast sugar carriers have been identified on the molecular level. A candidate sucrose carrier has been identified in a proteomic approach. In Arabidopsis, the tonoplast monosaccharide transporters (TMT) represent a small protein family comprising only three members, which reside in the vacuolar membrane. Two of three tmt genes are induced upon cold, drought or salt stress and tmt knock out mutants exhibit altered monosaccharide levels upon cold induction. These observations indicate that TMT proteins represent the first examples of tonoplast sugar carriers involved in the cellular response upon osmotic stress stimuli.     

无机盐诱导蓝藻细胞液泡化

选用NaCl、KNO3、(NH4)2SO4、MgSO4、CaCl2五种常用无机盐对分属三科的六种蓝藻进行液泡化诱导,不同蓝藻其盐敏感性不同。鱼腥藻sp.595最为敏感,五种盐均诱导其液泡化;颤藻284和极大螺旋藻438最不敏感,所试验盐类均不能诱导其液光化;念珠藻sp.96、织线藻246和伪枝藻248液泡化程度居中,少数盐类如NaC1、KNO3、(NH4)2SO4对其有诱导作用。采用压片法观察到诱导形成的液泡,液泡在相差显微镜下显示为圆球形,基本透明。     

Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum tRNA genes

Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream, and Tdd-3 elements downstream, of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are major targets for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a process of coevolution, allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome which carries only short intergenic DNA sequences. A new nomenclature is introduced to designate all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5′ and 3′ of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C. Received: 27 May 1999 / Accepted: 23 July 1999     

Visualization of vacuoplasts in isolated vacuole preparations from mesophyll protoplasts of periwinkle [Catharanthus roseus (L.) G. Don]

A procedure was developed for the rapid detection of vacuoplasts in vacuole preparations isolated from mesophyll protoplasts of Catharanthus roseus (L.) G. Don (periwinkle). The procedure relies on the staining of surface carbohydrates on the plasma membrane surrounding vacuoplasts with fluorescein-labeled lectins. When isolated under conditions of constant osmotic strength, approximately 15–20% of the vacuoles isolated showed surface labeling with FITC-agglutinin from Abrus precatorius. Isolation of vacuoles after an initial osmotic shock showed much lower (<5%) surface labeling. This lower level of surface labeling correlated well with a lower level of other non-vacuolar marker enzyme activities. A thin layer of cytoplasm was visible in a small number of these stained structures, indicating that they were vacuoplasts.Abbreviations FITC fluorescein isothiocyanate