Expression of the Longin domain of TI-VAMP impairs lysosomal secretion and epithelial cell migration

BACKGROUND INFORMATION: TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v-SNAREs (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). The regulatory N-terminal extension, called the Longin domain, of TI-VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI-VAMP to form SNARE complexes and it binds to the delta subunit of the AP-3 (adaptor protein 3) complex in early endosomes, thereby targeting TI-VAMP to late endosomes. RESULTS: We have generated MDCK (Madin-Darby canine kidney) cell lines expressing the Longin domain of TI-VAMP coupled to GFP (green fluorescent protein) in a doxycycline-dependent manner. As expected, AP-3delta (AP-3 delta subunit) is not properly localized in Longin-expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre-internalized fluorescent fluid-phase marker and by electron microscopy of the membrane-associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited. CONCLUSIONS: The results of the present study suggest that the expression of the Longin domain of TI-VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.     

Helicobacter pylori VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.     

Phenotypic diversity and altered environmental plasticity in Arabidopsis thaliana with reduced Hsp90 levels

The molecular chaperone HSP90 aids the maturation of a diverse but select set of metastable protein clients, many of which are key to a variety of signal transduction pathways. HSP90 function has been best investigated in animal and fungal systems, where inhibition of the chaperone has exceptionally diverse effects, ranging from reversing oncogenic transformation to preventing the acquisition of drug resistance. Inhibition of HSP90 in the model plant Arabidopsis thaliana uncovers novel morphologies dependent on normally cryptic genetic variation and increases stochastic variation inherent to developmental processes. The biochemical activity of HSP90 is strictly conserved between animals and plants. However, the substrates and pathways dependent on HSP90 in plants are poorly understood. Progress has been impeded by the necessity of reliance on light-sensitive HSP90 inhibitors due to redundancy in the A. thaliana HSP90 gene family. Here we present phenotypic and genome-wide expression analyses of A. thaliana with constitutively reduced HSP90 levels achieved by RNAi targeting. HSP90 reduction affects a variety of quantitative life-history traits, including flowering time and total seed set, increases morphological diversity, and decreases the developmental stability of repeated characters. Several morphologies are synergistically affected by HSP90 and growth temperature. Genome-wide expression analyses also suggest a central role for HSP90 in the genesis and maintenance of plastic responses. The expression results are substantiated by examination of the response of HSP90-reduced plants to attack by caterpillars of the generalist herbivore Trichoplusia ni. HSP90 reduction potentiates a more robust herbivore defense response. In sum, we propose that HSP90 exerts global effects on the environmental responsiveness of plants to many different stimuli. The comprehensive set of HSP90-reduced lines described here is a vital instrument to further examine the role of HSP90 as a central interface between organism, development, and environment.     

Evolutionary and biogeographical approach on Australoheros angiru (Cichlidae) from lagoons in a dividing plateau between the basins of the Iguassu River and the Uruguay River,Brazil

Australoheros is represented by species of Cichlidae spread over different Brazilian river systems. Individuals of Australoheros angiru were collected in isolated lagoons at 950 m of altitude in a dividing plateau between the basins of the Iguassu River and the Uruguay River, located in the São Lourenço do Oeste region (Santa Catarina State), and subject to basic and molecular cytogenetic analyzes. Moreover, a review about the occurrence and collections sites of A. angiru was used to the clarification of biogeographical issues of Australoheros and other species of fish that are common between the basins of the Uruguay River and Iguassu River. The analysis by conventional staining with Giemsa found a diploid number of 48 chromosomes (18 submetacentric + 30 subtelocentric/acrocentric) for males and females, without sex chromosome differentiation. The nucleolar organizing regions identified by silver impregnation and by fluorescence in situ hybridization with 18S rDNA probes showed to be simple, located on the end of the short arm of subtelocentric chromosome pair 15. Heterochromatin was observed in the centromeric region of most of the chromosomes of the complement, in addition to coincide with the nucleolar organizing regions. The data presented in this study are the first reports on the location of sites of 5S rDNA for Australoheros, being located in interstitial position on the long arm of subtelocentric chromosome pair 11. The basic and molecular cytogenetic data presented here contribute to the better understanding of chromosomal evolution of Cichlidae, act as interesting tools for taxonomy and phylogeny of the group, in addition to help in understanding the dispersion of A. angiru between the two hydrographic basins.     

云南常见牛肝菌属真菌中汞含量及食用安全评价

汞(Hg)是危害人类健康的主要重金属元素之一,多数食用菌对Hg有很强的富集能力,测定食用菌中Hg含量,并对其进行食用安全性评价具有重要意义。采用冷原子吸收直接测汞仪系统测定85份云南常见牛肝菌属真菌菌盖、菌柄中总Hg含量;以同一牛肝菌子实体菌盖与菌柄总Hg含量比(Q(C/S))分析牛肝菌属真菌对Hg的富集特征;根据联合国粮农组织和世界卫生组织(FAO/WHO)现行每周Hg允许摄入量(provisional tolerable weekly intake,PTWI)标准和中国GB 2762—2012规定的食用菌中Hg限量标准评价样品的食用安全性。结果表明,菌盖和菌柄中总Hg含量分别在0.13~22.00、0.20~8.40 mg·kg-1DW,不同产地、种类及不同采集年份的样品中总Hg含量存在明显差异;同一牛肝菌菌盖、菌柄总Hg含量比(Q(C/S))在0.28~4.08,92%的样品Q(C/S)1,表明多数样品中菌盖对Hg的富集能力强于菌柄。根据GB 2762—2012规定的食用菌及其制品中总Hg含量限量标准(≤0.1 mg·kg-1),发现所有测试样品的总Hg含量均超标;根据PTWI标准,假设成年人(60 kg)每周食用300 g新鲜牛肝菌,则多数样品Hg摄入量低于允许摄入量,无安全风险,而少数样品Hg摄入量高于允许摄入量,食用有一定的潜在风险。同时,牛肝菌总Hg含量与种类、产地、采集时间等因素密切相关,采食及安全评价需综合考虑这些因素。     

Manganese accumulation in rice: implications for photosynthetic functioning

In order to gain fundamental insights into the nature of the adaptation to Mn excess, the characterisation of the photosynthetic apparatus in Mn-treated rice was carried out in 21-day-old plants. We found 17- and 11-fold increases in Mn in the leaf tissues and in thylakoid, respectively, when the plants were grown hydroponically in nutrient solutions with Mn concentrations between 0.125 and 32 mg l(-1) (2.3 and 582.5 microM). Net photosynthesis and the photosynthetic capacity decreased after the 0.5 and 2 mg l(-1) (9.1 and 36.4 microM) Mn treatment, respectively. The stomatal conductance displayed a similar trend to that of photosynthetic capacity. The levels of basal chlorophyll fluorescence and the ratio between variable and maximum chlorophyll fluorescence did not vary significantly among treatments, but the photochemical quenching and the quantum yield of non-cyclic electron transport increased until the 2 mg l(-1) (36.4 microM) Mn treatment. The lipid matrix of thylakoids revealed a global increase in the proportions of phospholipids, relative to galactolipids. This pattern was coupled with diminishing levels of monogalactosyldiacylglycerol. The relative ratio between total carotenoids and total chlorophylls decreased until the last Mn treatment, yet the levels of carotenes, zeaxanthin, and violaxanthin plus antheraxanthin displayed different patterns. It was further found that the de-epoxidation state involving the components of the xanthophylls cycle increased until the 8 mg l(-1) (145.6 microM) Mn treatment. The levels of the photosynthetic electron carriers displayed different patterns, with plastocyanin and the high and low forms of cytochrome b559 remaining steady, whereas cytochromes b563 and f increased until the 8 mg l(-1) (145.6 microM) Mn treatment and the quinone pool increased until the highest Mn treatment. It was concluded that Mn-mediated inhibition of rice photosynthesis barely implicates stomatal conductance, as well as the distribution of energy within the photosystems. In this context, alterations to the relative proportions of the different acyl lipids and isoprenoids, as well as to the accumulations of the photosynthetic electron carriers, seem to play a major role.     

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.     

Endosomes, exosomes and Trojan viruses

Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of infected cells. Recently it has become apparent that some of these viruses use the endocytic pathway to coordinate their assembly and release. In addition, these and some other enveloped viruses exploit the machinery that generates the internal membranes of multivesicular bodies (MVB). These observations and others have led to the suggestion that retroviruses be regarded as "viral exosomes". Here we discuss this concept and the emerging evidence that compartments of the endocytic pathway play important roles in the biogenesis of both the internal vesicles of MVB and viruses.     

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.