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.     

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.     

Cardiac and muscle fatigue due to relative functional overload induced by excessive stimulation, hypersensitive excitation-contraction coupling, or diminished performance capacity correlates with sarcoplasmic reticulum failure

The development of muscle fatigue due to exhaustive exercise is associated with impaired sarcoplasmic reticulum (SR) Ca-transport activity. This study tested the hypothesis that SR failure is a consistent feature of cardiac and skeletal muscle fatigue owing to relative functional overload regardless of the method of induction: excessive stimulation, diminished performance capacity, or excessive excitation-contraction coupling. The Ca-transport activity was determined using three unique models of muscle fatigue: chronic and rapid ventricular pacing in dogs; metabolic inhibition caused by global cardiac ischemia in swine; and the hypermetabolic syndrome of porcine malignant hyperthermia (MH). Both pacing- and ischemia-induced fatigue resulted in reduction of SR Ca-transport ATPase activity: from 275 +/- 58 to 159 +/- 57 nmol.min-1.mg-1 (mU/mg) and from 577 +/- 82 to 177 +/- 133 mU/mg, respectively. Both pacing-induced fatigue and halothane-induced MH resulted in reduction of Ca-sequestration activity of muscle homogenates from 5.95 +/- 2.4 to 3.11 +/- 0.67 nM/s at 300 nM Ca and 38.7 +/- 10.5 to 16.3 +/- 8.0 nM/s at 1500 nM Ca, respectively (all p less than 0.01). The isolated SR Ca-ATPase activity correlated with Ca-sequestration activity of myocardial homogenates (r = 0.76; p less than 0.005). Different models were used to study the relationship of Ca-transport activity with relaxation function, degree of acidosis, and ionized Ca concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.     

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”.