GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA

The RhoA GTPase plays a vital role in assembly of contractile actin-myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced cell contraction. Furthermore, the nocodazole-induced activation of RhoA and Rho-associated kinase (ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1–depleted cells. Conversely, RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole-induced cell contractility.     

Vinculin controls PTEN protein level by maintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.     

Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility

The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling. We report here facile syntheses (four steps, >50% overall yield) of iodoacetamide, sulfhydryl-reactive derivatives of the Cy3 and Cy5 fluorophores. These novel derivatives have good water solubility (>2.5 mM) and bear only one reactive side chain, reducing possible protein cross-linking encountered with previous derivatives.     

Efficacy of a quaternary ammonium compound against planktonic and sessile populations of differentLegionella pneumophila strains

Efficacy of Gemacide PN-50^TM (a quaternary ammonium compound) as a commercial formulation recommended for disinfecting heat exchangers was determined for both planktonic and sessile populations of variousLegionella pneumophila strains. The quaternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. PlanktonicL. pneumophila strains were suspended in tap water while sessile ones were grown on stainless steel that is used in construction of the cooling towers, then both group of strains were exposed to the biocide. The sensitivity of both planktonic and sessile populations ofL. pneumophila strains to the biocide was different. The biocide was found effective below recommended dosages (1000–2000 mg/L) against planktonic populations ofL. pneumophila, whereas it was determined that higher than the recommended dosages were required for sessile populations. The environmental isolates were more resistant to the biocide than the ATCC isolate was. The results indicated that studying only the planktonic populations ofL. pneumophila for biocide tests might not be sufficient to provide the optimum dosage and contact time information for field trials. Therefore, biocidal activity of a water treatment chemical must be evaluated in terms of dosage and contact times on both planktonic and sessile bacteria.     

A case of suicide attempt with long-acting methylphenidate (Concerta)

The prescribed use of methylphenidate in the treatment of attention deficit hyperactivity disorder (ADHD) is widespread. The intranasal and parenteral abuse of methylphenidate (Ritalin) among teenagers is becoming increasingly more common, and deaths have been reported. Newer medical treatment options of long-acting stimulants offer effective treatment with a lower risk of abuse potential. We describe a case of a 17-year-old girl who had attempted suicide by ingesting 270 mg of Concerta. During the third years of treatment with Concerta, parents of patient reported that the patient had a depressive mood in the last week, and had attempted suicide with five tablets of Concerta 54 mg. She was sent to a local hospital with a diagnosis of long-acting methylphenidate overdose. All of vital and laboratory findings were normal except heart rate, which was 132 beats/min. Since more than 3 h have elapsed after the time of ingestion, activated charcoal administration was not carried out at the hospital. She was only observed for 12 h at the emergency department and later discharged from the hospital. While long-acting stimulants offer lower risk of abuse, their greater availability increases the likelihood of ingestion of this nature. Education of clinicians and families to be aware of this risk should reduce the frequency of this complication of treatment.     

In vitro labeling of hydroxyapatite minerals by an engineered protein

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.     

Global regulatory systems operating in Bacilysin biosynthesis in Bacillus subtilis

In Bacillus subtilis, bacilysin is a nonribosomally synthesized dipeptide antibiotic composed of L-alanine and L-anticapsin. The biosynthesis of bacilysin depends on the bacABCDEywfG operon (bac operon)and the adjacent ywfH gene. To elucidate the effects of global regulatory genes on the expression of bac operon, we used the combination of lacZ fusion analysis and the gel mobility shift assays. The cell density-dependent transition state induction of the bac operon was clearly shown. The basal expression level of the bac operon as well as transition state induction of bac is directly ComA dependent. Three Phr peptides, PhrC, PhrF and PhrK, are required for full-level expression of ComA-dependent bac operon expression, but the most important role seemed to be played by PhrC in stimulating bac expression through a RapC-independent manner. Spo0A is another positive regulator which participates in the transition state induction of bac both directly by interacting with the bac promoter and indirectly by repressing abrB expression. AbrB and CodY proteins do not only directly repress the bac promoter, but they also mutually stimulate the transition state induction of bac indirectly, most likely by antagonizing their repressive effects without preventing each other's binding since both proteins can bind to the bac promoter simultaneously.     

GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.     

Sunflower seed lipase: extraction, purification, and characterization

A simple procedure for the extraction of the lipolytic activity from sunflower seed has been developed. Various conditions of extraction have been optimized in order to obtain maximum yield of lipase. A new lipase enzyme was purified by the fractional salt precipitation from the supernatant, dialysis on a cellulose membrane, and gel column chromatography on Sephadex G-75. The lipase was monomeric, with an apparent Mr of 22 kDa and a pI of 8, with the electrophoretic analysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with Km and Vmax, values of 1.33 mM and 555 U/mg. All triglycerides were efficiently hydrolyzed by the enzyme, but this showed a preference towards triglycerides of natural mono unsaturated fatty acids. The optimum temperature, pH, and incubation time for lipolytic activity were 50 degrees C, 7.5, and 5 min, respectively. The stability of the sunflower lipase was investigated under operational and storage conditions. It was found that this enzyme preserved its lipolytic activity at temperatures between at 35-50 degrees C, alkaline pH, and for a period of about four months.