Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma... more
Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation—sUC) and size- (size exclusion chromatography—SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MA...
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... ANA PLEMENITA AND NINA GUNDE-CIMERMAN 464 ... halotolerant yeast species like D. hansenii (Almagro, 2000; Andre et al., 1988; Larsson et al., 1990; Prista et al., 1997; Ramos, 1999), C. versatilis (Silva-Graça et al., 2003), R.... more
... ANA PLEMENITA AND NINA GUNDE-CIMERMAN 464 ... halotolerant yeast species like D. hansenii (Almagro, 2000; Andre et al., 1988; Larsson et al., 1990; Prista et al., 1997; Ramos, 1999), C. versatilis (Silva-Graça et al., 2003), R. mucilaginosa, P. guillermondii (Lahav et al ...
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Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative... more
Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1. To this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, ...
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Research Interests: Biology, Pheromones, Membrane Proteins, Cell Biology, Medicine, and 15 moreCytoskeleton, HIV, Biological Sciences, Humans, Mitogen Activated Protein Kinase, Human immunodeficiency virus, CDC, Acquired Immune Deficiency Syndrome, Protein Kinase, Amino Acid Sequence, Cell Cycle Proteins, Gtp Binding Proteins, Molecular Sequence Data, Medical and Health Sciences, and Gene products nef
HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral... more
HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.
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Research Interests: Biology, Cell Biology, Medicine, HIV, Biological Sciences, and 15 moreCell line, Humans, Mice, Animals, CDC, Rac, Cell Cycle Proteins, Rab, DNA binding proteins, Gtp Binding Proteins, Gene Expression Regulation, Cell Growth, Psychology and Cognitive Sciences, Medical and Health Sciences, and Gene products nef
Research Interests: Biophysics, Materials Science, Scanning Electron Microscopy, Medicine, Anisotropy, and 15 moreBiological Sciences, Mathematical Sciences, Animals, Peptides, Glycoproteins, Lung, Membrane, Theoretical, Mechanical Stress, Phase Separation, Lipid bilayers, Human Fibroblasts, Cell Membrane, fibroblasts, and Cricetinae
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Ostreolysin, a 15 kDa pore‐forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their... more
Ostreolysin, a 15 kDa pore‐forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono‐ or di‐unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin's activity. It appears that the protein recognizes specifically a cholesterol‐rich lipid phase, probably the liquid‐ordered phase.
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In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B,... more
In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.
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We have investigated regulation of HMG‐CoA reductase (HMGR) in one of the most salt‐tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified,... more
We have investigated regulation of HMG‐CoA reductase (HMGR) in one of the most salt‐tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo‐saline and extremely hyper‐saline conditions, and down‐regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation‐directed fate for mevalonate utilization in H. werneckii.
Research Interests: Biochemistry, Biology, Enzyme Inhibitors, Mitochondria, Extremophiles, and 14 moreMedicine, Ubiquitin, Endoplasmic Reticulum, Osmotic pressure, Yeast, SALT TOLERANCE, Isoenzymes, Protein Degradation, Optimal Growth, Ascomycota, Protein Binding, Biochemistry and cell biology, Ubiquitination, and HMG CoA Reductase
Research Interests: Biology, Salt Stress, Medicine, Gene expression, Signal Transduction, and 15 moreBiological Sciences, Salinity, Research article, Osmotic pressure, Gene, Mitogen Activated Protein Kinase, Yeasts, MAP Kinase, Sodium Chloride, Differential Gene Expression, Differential expression, High Salt, Signaling pathway, Mitogen- Activated Protein Kinases, and Medical and Health Sciences
Research Interests: Microbiology, Plant Biology, Biology, Medicine, EPR Spectroscopy, and 15 moreSaccharomyces cerevisiae, Stress tolerance, Temperature, Yeast, Membrane, Fungal Biology, Electron Paramagnetic Resonance Spectroscopy, Low Temperature, Yeasts, Sodium Chloride, Plasma Membrane, Membrane Fluidity, Cell Membrane, Stress Physiological, and Cold Temperature
Research Interests: Engineering, Technology, Biology, Salt Stress, Magnetic Resonance Spectroscopy, and 14 moreMedicine, Biological Sciences, Salinity, Yeast, Ice, Water Microbiology, Yeasts, Aureobasidium Pullulans, Sodium Chloride, Plasma Membrane, Membrane Fluidity, Adaptation in extreme environment, Extremophile, and Cell Membrane
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This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of... more
This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Deplet...
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To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects. We... more
To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects. We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort. This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects. Here, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA level...
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Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small... more
Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small number are amenable to laboratory cultivation and manipulation. A detailed characterisation of the genome architecture of such organisms is important to illuminate how they adapt to environmental stresses. One excellent example of a fungal extremophile is the halophile Hortaea werneckii (Pezizomycotina; Dothideomycetes, Capnodiales), a yeast-like fungus able to thrive at near-saturating concentrations of sodium chloride and which is also tolerant to both UV irradiation and desiccation. Given its unique lifestyle and its remarkably recent whole genome duplication, H. werneckii provides opportunities for testing the role of genome duplications and adaptability to extreme environments. We previously assembled the genome of H. werneckii using short-read...
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HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte... more
HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte concentrations, and regulates expression of a similar set of genes independent of osmolyte type. To understand how HwHog1 kinases activity is regulated in H. werneckii, we studied HwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activities of recombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and soluble recombinant Hog1 homologues were isolated from insect cells. Our results demonstrate that HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl and KCl concentrations. Importantly, phosphorylation profiles differ depending on the osmolyte type. Add...
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The discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. H. werneckii is also a... more
The discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. H. werneckii is also a promising source of transgenes to improve osmotolerance of industrially important yeasts, as well as crops. This chapter describes the physiological and molecular
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... Page 426. 410 NINA GUNDE-CIMERMAN ET AL. ... The latter was obtained from sea water, sea and glacier ice. In hypersaline waste water ponds in Israel it represeted one of the two dominant microorganisms (Lahav et al., 2002). ...
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Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats... more
Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats with increasing salinities, from moderate to hypersaline. These present one of the most extreme environments, because of the low levels of biologically available water and the toxic concentrations of ions. Up to the year 2000, hypersaline environments were considered to be populated almost exclusively by prokaryotic microorganisms till fungi were reported to be active inhabitants of solar salterns. Since then, numerous fungal species have been described in hypersaline waters around the world. The mycobiota of salterns is represented by different species of the genus Cladosporium and the related meristematic melanized black yeasts, of non-melanized yeasts, of the filamentous genera Penicillium and Aspergillus and their teleomorphic forms (Eurotium and Emericella), and of the basidiomycetous genus Wallemia. Among these, two species became new model organisms for studying the mechanisms of extreme salt tolerance: the extremely halotolerant ascomycetous black yeast Hortaea werneckii and the obligate halophilic basidiomycete Wallemia ichthyophaga.
Research Interests: Botany, Biology, Fungi, Medicine, Salinity, and 7 moreSeawater, SALT TOLERANCE, Ecosystem, Yeasts, Ascomycota, Sodium Chloride, and Mycobiota
Hypersaline environments support substantial microbial communities of selected halotolerant and halophilic organisms, including fungi from various orders. In hypersaline water of solar salterns, the black yeast Hortaea werneckii is by far... more
Hypersaline environments support substantial microbial communities of selected halotolerant and halophilic organisms, including fungi from various orders. In hypersaline water of solar salterns, the black yeast Hortaea werneckii is by far the most successful fungal representative. It has an outstanding ability to overcome the turgor loss and sodium toxicity that are typical for hypersaline environments, which facilitates its growth even in solutions that are almost saturated with NaCl. We propose a model of cellular responses to high salt concentrations that integrates the current knowledge of H. werneckii adaptations. The negative impact of a hyperosmolar environment is counteracted by an increase in the energy supply that is needed to drive the energy-demanding export of ions and synthesis of compatible solutes. Changes in membrane lipid composition and cell-wall structure maintain the integrity and functioning of the stressed cells. Understanding the salt responses of H. werneckii and other fungi (e.g., the halophilic Wallemia ichthyophaga) will extend our knowledge of fungal stress tolerance and promote the use of the currently unexploited biotechnological potential of fungi that live in hypersaline environments.
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Salterns provide special living conditions for microorganisms. They are extreme environments because of high concentrations of NaCl and other salts, occasional rapid changes in water activity, low oxygen concentration, and high UV... more
Salterns provide special living conditions for microorganisms. They are extreme environments because of high concentrations of NaCl and other salts, occasional rapid changes in water activity, low oxygen concentration, and high UV radiation (Brock 1979). It is generally assumed that microbial life in concentrated seawater at the highest salinities is mainly composed of Archaea and Bacteria and one eukaryotic species, the alga Dunaliella salina. Other eukaryotic microorganisms usually appear at lower salinities and are represented by different species of algae and protozoa (Ramos-Cormenzana 1991; Pedros-Alio et al. 2000). Surprisingly, until recently, fungi have not been isolated from natural hypersaline environments (Buchalo et al.1998; GundeCimerman et al. 2000), although xerophilic fungi able to grow on media with low water activities are frequently isolated from food preserved with high concentrations of salt or sugar (Filtenborg et al. 2000). It seems that growth of the few known xerophilic species of food-borne fungi in the presence of high concentrations of the solute is determined primarily by the water activities of the medium and not by the chemical nature of the solute. This explains why only as late as 1975 the term halophilic fungi was introduced for those few xerophilic food-borne species that exhibit superior growth on media with NaCl as controlling solute (Pitt and Hocking 1985). Only a few reports describe the isolation of fungi from natural moderately saline environments such as salt marshes (Newell 1996), saline soil (Guiraud et al.1995) and seawater (Kohlmeyer and Volkmann-Kohlmeyer 1991). Recently, however, we made a novel observation that fungi, representing the only kingdom so far not known to sustain extremely saline conditions, populate salterns nearly saturated with NaCl (Gunde-Cimerman et al. 2000).
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By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that... more
By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.