Regulated conformation of myosin V

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.     

The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs

Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery. Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA). GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein. Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes. Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin. These motifs are found within different helices within the three-helix GAT subdomain. When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.     

Traction Force Screening Enabled by Compliant PDMS Elastomers

Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening. Critically, rather than the frequently employed hydrogel polyacrylamide, we fabricate these plates using polydimethylsiloxane rubber. Key to this approach is that the polydimethylsiloxane used is very compliant, with a lower-bound Young’s modulus of ～0.4 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells. By directly quantifying the endpoint of therapeutic intent, airway-smooth-muscle contractile force, this approach fills an important methodological void in current screening approaches for bronchodilator drug discovery, and, more generally, in measuring contractile response for a broad range of cell types and pathologies.     

Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AimEthanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated.Main methodsChronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis.Key findingsInhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2 mM pyrazole or ADH and CYP2E1 with 5 mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury.SignificanceCYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.     

Transmission of seed-borne infection of chilli by Burkholderia solanacearum and effect of biological seed treatment on disease incidence

Abstract

A survey of chilli fields in the state of Karnataka, India, showed the presence of bacterial wilt disease in important chilli growing regions. The disease incidence ranged from 26?–?32%. The pathogen was isolated from infected plant material and seeds. Infected plant material showed the release of milky white bacterial ooze. Burkholderia solanacearum was detected from chilli seeds by liquid assay and its identity was confirmed by biochemical tests, hypersensitive reaction and pathogenicity tests. Seed transmission of the pathogen up to 45% was observed in seeds artificially infested with the pathogen. Among different tissues of the seed, endosperm showed the presence of the pathogen. Biological seed treatment with antagonistic Pseudomonas fluorescens significantly (p?=?0.05) improved the seed quality parameters under laboratory conditions and drastically reduced the bacterial wilt incidence under field conditions. Seed-borne nature, transmission and effect of Pseudomonas fluorescens in both the forms of pure culture and formulation on seed quality parameters and bacterial wilt incidence are discussed in the present work.     

Rhizobacterial-mediated induction of defense enzymes to enhance the resistance of turmeric (Curcuma longa L) to Pythium aphanidermatum causing rhizome rot

Rhizobacteria isolated from the rhizosphere soil were evaluated for their ability to control rhizome rot in turmeric (Curcuma longa L). These isolates were characterised as Pseudomonas fluorescens and Bacillus subtilis. Under in vitro condition, two isolates, namely P. chlororaphis (PcPA23) and B. subtilis (BsCBE4), showed maximum inhibition of mycelial growth of Pythium aphanidermatum, were found effective in reducing rhizome rot of turmeric both under greenhouse and field conditions and increased the plant growth and rhizome yield. Both the isolates were further tested for its ability to induce production of defense-related enzymes and chemicals in plants. Increased activities of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, chitinase and β-1,3-glucanase were observed in PcPA23 and BsCBE4 pre-treated turmeric plants challenged with P. aphanidermatum. Moreover, higher accumulation of phenolics was noticed in plants pre-treated with PcPA23 and BsCBE4 challenged with P. aphanidermatum. Thus, the present study shows that in addition to direct antagonism and plant growth promotion, induction of defense-related enzymes involved in the phenyl propanoid pathway collectively contributed to enhance resistance against invasion of Pythium in turmeric.     

Comparing antibiotic resistance in commensal and pathogenic bacteria isolated from wild-caught South Carolina shrimps vs. farm-raised imported shrimps

The objective of this study was to assess and differentiate wild-caught South Carolina (SC) shrimps from imported shrimps on the basis of microbiological analysis. Seven wild-caught SC shrimp and 13 farm-raised imported shrimp samples were analyzed. Total plate counts from wild-caught shrimp samples ranged from 4.3 to 7.0 log10 CFU/g, whereas counts from imported shrimp samples ranged from 3.2 to 5.7 log10 CFU/g. There was no difference (P > 0.05) between total bacterial counts of wild-caught SC shrimp and farm-raised imported shrimp. However, the percentages of bacteria with reduced susceptibility towards ceftriaxone and tetracycline were higher (P < 0.05) for farm-raised shrimp than for wild-caught samples. Salmonella spp. detected only in one farm-raised sample was resistant to ampicillin, ceftriaxone, gentamicin, streptomycin, and trimethoprim. Vibrio vulnificus was detected in both wild-caught and farm-raised shrimp samples; however, only the isolate from farm-raised shrimp was resistant to nalidixic acid and trimethoprim. Escherichia coli detected in one wild-caught sample was resistant to ampicillin. Both Listeria spp. and Salmonella spp. were absent with wild-caught SC samples. Therefore, the presence of more ceftriaxone- and tetracycline-resistant bacteria and the observed antimicrobial resistance phenotypes of isolates from the imported shrimp may reflect the possible use of antibiotics in raising shrimp in those countries.     

Spectral Investigations on the Fluorescence Quenching of 1,4-dihydroxy-2,3-dimethylanthracene-9,10-dione by Plasmonic Silver Nanoparticles

Silver nanoparticles (Ag NPs) of different sizes have been prepared by Lee and Meisel’s method using trisodium citrate as reducing agent under ultra sonication. Optical absorption and fluorescence emission techniques were employed to investigate the interaction of 1,4-dihydroxy-2,3-dimethyl anthracene-9,10-dione (DHDMAD) with silver nanoparticles. In fluorescence spectroscopic study, we used the DHDMAD and Ag NPs as component molecules for construction of Förster Resonance Energy Transfer (FRET), whereas DHDMAD serve as donor and Ag NPs as acceptor. The surface plasmon resonance (SPR) peak of the prepared silver colloidal solution was observed from 419 nm to 437 nm. The synthesized silver nanoparticles at different heating time intervals were spherical in shape about the size of 25 nm and 55 nm. The fluorescence interaction between silver nanoparticles and DHDMAD confirms the FRET mechanism. According to Förster theory, the distance between silver nanoparticles and DHDMAD and the critical energy transfer distance were calculated and it is increase with heating time.     

The RAB5‐GEF Function of RIN1 Regulates Multiple Steps During Listeria monocytogenes Infection

Listeria monocytogenes is a food‐borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome‐mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5‐directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1‐mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.