Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E . coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E . coli channel, and a C-terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.     

Solvent effects on the cooperative order-disorder transition of aqueous solutions of schizophyllan, a triple-helical polysaccharide

A triple helical polysaccharide schizophyllan in aqueous solution exhibited a highly cooperative transition between ordered and disordered states associated with the conformation of its side chains and nearby water molecules. The transition was followed by optical rotation and calorimetry using water containing additives such as NaOH and DMSO as solvents. The ordered state was stabilized or destabilized depending on the kind and amount of the additive employed; in particular, the addition of DMSO had a remarkable stabilizing effect. This effect was analyzed by means of a statistical mechanical theory of linear cooperative transitions, where DMSO was assumed to interact favorably with the ordered side chains. A small amount of NaOH in a solvent mixture stabilized the ordered state and made the transition curve very gradual. No molecular mechanism was elucidated to account for the role of NaOH.     

A new record of Amphitetranychus quercivorus (Acari: Tetranychidae) in Korea and molecular comparison with A. viennensis

Amphitetranychus quercivorus (Ehara and Gotoh, 1990) was first identified in Mongolian oak tree Quercus mongolica in Daegu, Korea. In comparison to A. viennensis (Zacher), A. quercivorus was much shorter in the distal, dorsally directed portion of the male aedeagus, as well as smaller and less folded in the distal anastomosed portion of the female peritreme. Comparison of the nucleotide sequences of ITS2 and COI revealed 90% and 89% similarities, respectively, between the two species. In addition, species-specific primer sets for each species were designed using unique ITS2 sequences and then used to diagnose these two species under plant quarantine. We report the Korean name of this species as “Sin-gal-na-mu-eung-ae”.     

Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.     

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.     

Antibacterial activity of disodium succinoyl glycyrrhetinate,a derivative of glycyrrhetinic acid against Streptococcus mutans

Streptococcus mutans is a cariogenic bacterium that localizes in the oral cavity. Glycyrrhetinic acid (GRA) is a major component of licorice extract. GRA and several derivatives, including disodium succinoyl glycyrrhetinate (GR‐SU), are known to have anti‐inflammatory effects in humans. In this study, the antimicrobial effect of GRA and its derivatives against the S. mutans UA159 strain were investigated. Minimum inhibitory concentrations (MICs) of GRA and GR‐SU showed antibacterial activity against the S. mutans strain, whereas other tested derivatives did not. Because GR‐SU is more soluble than GRA, GR‐SU was used for further experiments. The antibacterial activity of GR‐SU against 100 S. mutans strains was evaluated and it was found that all strains are susceptible to GR‐SU, with MIC values below 256 µg/mL. A cell viability assay showed that GR‐SU has a bacteriostatic effect on S. mutans cells. As to growth kinetics, sub‐MICs of GR‐SU inhibited growth. The effect of GR‐SU on S. mutans virulence was then investigated. GR‐SU at sub‐MICs suppresses biofilm formation. Additionally, GR‐SU greatly suppresses the pH drop caused by the addition of glucose and glucose‐induced expression of the genes responsible for acid production (ldh and pykF) and tolerance (aguD and atpD). Additionally, expression of enolase, which is responsible for the carbohydrate phosphotransferase system, was not increased in the presence of GR‐SU, indicating that GR‐SU suppresses incorporation of sugars into S. mutans. In conclusion, GR‐SU has antibacterial activity against S. mutans and also decreases S. mutans virulence.