Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.     

Detection of in vivo protein tyrosine nitration in petite mutant of Saccharomyces cerevisiae: Consequence of its formation and significance

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with physiological and pathophysiological conditions. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group. In our previous study we first time showed that PTN occurs in vivo in Saccharomyces cerevisiae. In the present study we observed occurrence of PTN in petite mutant of S. cerevisiae which indicated that PTN is not absolutely dependent on functional mitochondria. Nitration of proteins in S. cerevisiae was also first time confirmed in immunohistochemical study using spheroplasts. Using proteosomal mutants Rpn10Δ, Pre9Δ, we first time showed that the fate of protein nitration in S. cerevisiae was not dependent on proteosomal clearing and probably played vital role in modulating signaling cascades. From our study it is evident that protein tyrosine nitration is a normal physiological event of S. cerevisiae.     

Studies on solvatochromic properties of aminophenylstyryl-quinolinum dye, LDS 798, and its application in studying submicron lipid based structure

The styryl group of dyes has been used in cellular studies for over 20 years because of their solvatochromic and/or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red styryl dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles and lipid based nanoparticles and found that solvatochromic properties of this dye used in tandem with fluorescence correlation spectroscopy can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This technique has the potential to provide essential information about liposomal and vesicular structures and their movement in vitro and in situ.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Inactivation and injury of <Emphasis Type="Italic">Escherichia coli</Emphasis> in a copper water storage vessel: effects of temperature and pH

Copper has been used as a disinfectant since ancient times and recent research has demonstrated that antimicrobial copper surfaces may have practical applications in healthcare and related areas. The present study was carried out to establish the effects of temperature and pH on inactivation and sub-lethal injury of Escherichia coli in water stored in a copper vessel, to determine the operational limits of the process in terms of these variables. To investigate the effects of temperature, a bacterial suspension at pH 7.0 was stored for up to 48 h in copper vessels at 5, 15, 25 and 35°C. For pH, a bacterial suspension was stored at 30°C for up to 48 h in copper vessels at pH 6.0, 7.0, 8.0 and 9.0. Both temperature and pH had substantial effects on inactivation and injury, with the fastest inactivation observed at elevated temperature and at pH values furthest from neutrality, while the greatest amount of sub-lethal injury, manifest as sensitivity to conventional aerobic enumeration, was observed at a temperature of 35°C. These findings have important implications for the practical application of copper-based water disinfection methods, in terms of their likely efficacy under environmental conditions.     

Antennal regulation of migratory flight in the neotropical moth Urania fulgens

Migrating insects use their sensory systems to acquire local and global cues about their surroundings. Previous research on tethered insects suggests that, in addition to vision and cephalic bristles, insects use antennal mechanosensory feedback to maintain their airspeeds. Owing to the large displacements of migratory insects and difficulties inherent in tracking single individuals, the roles of these sensory inputs have never been tested in freely migrating insects. We tracked individual uraniid moths (Urania fulgens) as they migrated diurnally over the Panama Canal, and measured airspeeds and orientation for individuals with either intact or amputated flagella. Consistent with prior observations that antennal input is necessary for flight control, 59 per cent of the experimental moths could not fly after flagella amputation. The remaining fraction (41%) was flight-capable and maintained its prior airspeeds despite severe reduction in antennal input. Thus, maintenance of airspeeds may not involve antennal input alone, and is probably mediated by other modalities. Moths with amputated flagella could not recover their proper migratory orientations, suggesting that antennal integrity is necessary for long-distance navigation.     

Alpha-santalol,a chemopreventive agent against skin cancer,causes G<Subscript>2</Subscript>/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Background

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

Methods

MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.

Results

α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G₂/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G₂/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.

Conclusions

This study for the first time identifies effects of α-santalol in G₂/M phase arrest and describes detailed mechanisms of G₂/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.