Browsing by Subject "Osmolar Concentration"

Now showing 1 - 4 of 4
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    Bacterial pathogens deliver water- and solute-permeable channels to plant cells.
    (Nature, 2023-09) Nomura, Kinya; Andreazza, Felipe; Cheng, Jie; Dong, Ke; Zhou, Pei; He, Sheng Yang
    Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells1,2. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants3-5. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria6. These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.
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    L form bacteria growth in low-osmolality medium.
    (Microbiology (Reading, England), 2019-08) Osawa, Masaki; Erickson, Harold P
    L form bacteria do not have a cell wall and are thought to require medium of high osmolality for survival and growth. In this study we tested whether L forms can adapt to growth in lower osmolality medium. We first tested the Escherichia coli L form NC-7, generated in 1987 by Onoda following heavy mutagenesis. We started with growth in osmoprotective medium (~ 764 mOsm kg-1) and diluted it stepwise into medium of lower osmolality. At each step the cells were given up to 10 days to adapt and begin growing, during which they apparently acquired multiple new mutations. We eventually obtained a strain that could grow in LB containing only 34 mM NaCl, 137 mOsm kg-1 total. NC-7 showed a variety of morphologies including spherical, angular and cylindrical cells. Some cells extruded a bud that appeared to be the outer membrane enclosing an enlarged periplasm. Additional evidence for an outer membrane was sensitivity of the cells to the compound CHIR-090, which blocks the LPS pathway, and to EDTA which chelates Mg that may stabilize and rigidify the LPS in the outer membrane. We suggest that the mechanical rigidity of the outer membrane enables the angular shapes and provides some resistance to turgor in the low-osmolality media. Interestingly, cells that had an elongated shape underwent division shortly after addition of EDTA, suggesting that reducing the rigidity of the outer membrane under some turgor pressure induces division before lysis occurs. We then tested a well-characterized L form from Bacillus subtilis. L form strain LR-2L grew well with sucrose at 1246 and 791 mOsm kg-1. It survived when diluted directly into 440 mOsm kg-1 but grew poorly, achieving only 1/10 to 1/5 the density. The B. subtilis L form apparently adapted to this direct dilution by rapidly reducing cytoplasmic osmolality.
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    Protein purification: adsorption chromatography on controlled pore glass with the use of chaotropic buffers.
    (Science (New York, N.Y.), 1976-01) Bock, HG; Skene, P; Fleischer, S; Cassidy, P; Harshman, S
    Chromatography on controlled pore glass in combination with chaotropic buffers makes possible, in a single step, protein purifications of several hundredfold. The new emphasis is on highly selective controllable adsorption. The method is useful for the purification and concentration of proteins from large volumes of complex media and for the purification of proteins that are poorly soluble or tend to aggregate in aqueous solution D-(-)-Beta-Hydroxybutyrate dehydrogenase, a mitochondrial membrane-bound protein, several soluble proteins, and staphylococcal alpha toxin, which can be purified directly from large volumes of culture medium, are used to illustrate the method.
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    The effects of osmotic stress on the structure and function of the cell nucleus.
    (2010) Finan, John Desmond
    Chondrocytes maintain cartilage by transducing joint load into appropriate biosynthetic activity, a process commonly known as mechanotransduction. Malfunctioning mechanotransduction leads to cartilage degradation and osteoarthritis. The mechanism of mechanotransduction is only partially understood but osmotic stresses are thought to play an important role. This study shows that the chondrocyte nucleus shrinks and wrinkles under hyper-osmotic stress. It shrinks because the chromatin inside the nucleus contracts as the macromolecules in the cell become more crowded. It wrinkles because the nuclear lamina buckles as the nucleus contracts. These morphological changes accelerate transport across the nuclear envelope. Many cells have organized actin caps around their nuclei that constrain the nucleus from contracting under hyper-osmotic stress. Agents exist that can reverse this loss of osmotic sensitivity in vitro without damaging the cell.