Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125(FAK)) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130(Cas) and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.     

Plasma polymerized membranes and gas permeability. II

Thin films were deposited onto porous substrates by plasma polymerization using three kinds of organosilicic compounds, tetramethylsilane (TMS), hexamethyldisiloxane (M₂), and octamethylcyclotetrasiloxane (D₄). Those composite membranes showed different characteristics of gas permeability. When D₄ was plasma-deposited onto a porous substrate, the composites membrane showed the highest oxygen permeability and the lowest oxygen-to-nitrogen permeability ratio. The composite membrane prepared from TMS showed the permeability characteristics opposite to the membrane obtained from D₄. Infrared spectrum of the polymer from D₄ resembles that of dimethylpolysiloxane. The plasma polymers from TMS and M₂ showed different profiles in Si? O absorption bands in the range 1100–1000 cm^-1 or in absorption bands of SiCH₃ groups in the range 850–750 cm^-1 from respective monomers. X-ray photoelectron spectroscopic observation indicated that all the plasma polymers contained more than two species of Si atom with different oxidation states. The greater part of Si atoms in plasma polymers took the same oxidation states in corresponding monomer. The gas permeability characteristics were closely related to the oxidation states of Si atom in the plasma polymers.     

Development of Mo(Si,Al)<Subscript>2</Subscript>-base oxidation-resistant coating on Nb-base structural materials

Mo(Si,Al)₂-base oxidation-resistant coatings for Nb-base structural materials have been studied. The coating is composed of a Mo(Si,Al)₂-base Al reservoir and Al₂O₃ interlayer to suppress interface reactions between the Al reservoir and the substrate. To develop a suitable Al-reservoir material, some Mo(Si_0.6,Al_0.4)₂-HfB₂ composites were prepared. Their oxidation resistance and coefficients of thermal expansion were investigated, in addition to their chemical reactivity with the Nb substrate at high temperatures. As a result, Mo(Si_0.6,Al_0.4)₂-20 vol pct HfB₂ was selected as one of the satisfactory Al reservoirs. The introduction of a stable Al₂O₃ interlayer was attempted using a novel powder metallurgical process to overlay the Nb substrates with the Al reservoir, where the Nb substrates were subjected to a slight surface oxidation prior to the coating process. The Nb specimens, which are thoroughly coated with the Al reservoir and Al₂O₃ interlayer, can be successfully fabricated by this method. The coated Nb specimens are not damaged at all after prolonged exposure in flowing Ar-20 pct O₂ at 1673 K for 120 hours. Furthermore, the Al₂O₃ interlayer is very effective and no reactions occur at the interface. Thus, this Mo(Si,Al)₂-base oxidation-resistant coating is applicable to Nb. The utility of the coating system is also confirmed for a Nb_SS/Nb₅Si₃ composite. This article is based on a presentation made in the symposium entitled “Beyond Nickel-Base Superalloys,” which took place March 14–18, 2004, at the TMS Spring meeting in Charlotte, NC, under the auspices of the SMD-Corrosion and Environmental Effects Committee, the SMD-High Temperature Alloys Committee, the SMD-Mechanical Behavior of Materials Committee, and the SMD-Refractory Metals Committee.     

Prism-pair interferometry by homodyne interferometers with a common light source for high-accuracy measurement of the absolute refractive index of glasses

A prism-pair interferometer comprising two homodyne interferometers with a common light source was developed for high-precision measurements of the refractive index of optical glasses with an uncertainty of the order of 10(-6). The two interferometers measure changes in the optical path length in the glass sample and in air, respectively. Uncertainties in the absolute wavelength of the common light source are cancelled out by calculating a ratio between the results from the interferometers. Uncertainties in phase measurement are suppressed by a quadrature detection system. The combined standard uncertainty of the developed system is evaluated as 1.1×10(-6).     

Prevalence of Salmonella isolates and antimicrobial resistance patterns in chicken meat throughout Japan

We investigated the prevalence of Salmonella in chicken meat from northern, central, and southern Japan. Between 2006 and 2008, 821 samples from these three regions were collected and examined. Salmonella isolates were detected in 164 (20.0%) of these samples, with 15 (10.0%) of 150, 113 (27.5%) of 411, and 36 (13.8%) of 260 recovered from the northern, central, and southern regions, respectively. We recovered 452 Salmonella isolates. From the isolates, 27 serovars were identified; the predominant serovars isolated were Salmonella Infantis (n=81), Salmonella Kalamu (n=56), and Salmonella Schwarzengrund (n=43). Of the 452 isolates, 443 (98.0%) were resistant to one or more antibiotics, and 221 (48.9%) showed multiple-antibiotic resistance, thereby implying that multiple-antibiotic resistant Salmonella organisms are widespread in chicken meat in Japan. Resistance to oxytetracycline was most common (72.6%), followed by dihydrostreptomycin (69.2%) and bicozamycin (49.1%). This study, the first to report Salmonella prevalence in chicken meat throughout Japan, could provide valuable data for monitoring and controlling Salmonella infection in the future.     

Thermoelectric Properties of NdGd<Subscript>1+<Emphasis Type="Italic">x</Emphasis></Subscript>S<Subscript>3</Subscript> Prepared by CS<Subscript>2</Subscript> Sulfurization

Ternary rare-earth sulfides NdGd_1+x S₃, where 0 ≤ x ≤ 0.08, were prepared by sulfurizing Ln₂O₃ (Ln = Nd, Gd) with CS₂ gas, followed by reaction sintering. The sintered samples have full density and homogeneous compositions. The Seebeck coefficient, electrical resistivity, and thermal conductivity were measured over the temperature range of 300 K to 950 K. All the sintered samples exhibit a negative Seebeck coefficient. The magnitude of the Seebeck coefficient and the electrical resistivity decrease systematically with increasing Gd content. The thermal conductivity of all the sintered samples is less than 1.9 W K⁻¹ m⁻¹. The highest figure of merit ZT of 0.51 was found in NdGd_1.02S₃ at 950 K.     

Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.     

A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.