Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence

Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.     

ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

Zinc is a trace element that is essential for immune responses. Therefore, changes in cellular zinc levels in specific immune cells may influence inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). However, the regulation of zinc mobilization in immune cells and its role in the pathogenesis of RA are not fully understood. Thus, we investigated the roles of zinc transporters in RA pathogenesis. We demonstrated that ZIP8 was specifically upregulated in CD4⁺ T cells that infiltrated the inflamed joint and that ZIP8 deficiency in CD4⁺ T cells abrogated collagen-induced arthritis. ZIP8 deficiency dramatically affected zinc influx in effector T cells and profoundly reduced T cell receptor (TCR)-mediated signaling, including NF-κB and MAPK signaling, which are pathways that are involved in T helper (Th) 17 cell differentiation. Taken together, our findings suggest that ZIP8 depletion in CD4⁺ T cells attenuates TCR signaling due to insufficient cellular zinc, thereby reducing the function of effector CD4⁺ T cells, including Th17 cells. Our results also suggest that targeting ZIP8 may be a useful strategy to inhibit RA development and pathogenesis.Subject terms: Autoimmunity, Immunological disorders     

Catalytic carbonylation for the synthesis of chemical intermediates

Chemistry related to three catalytic carbonylation reactions is discussed. Synthesis of diphenylurea from nitrobenzene, aniline, and CO gives isolated yields above 98% at 100–120 °C and 15–60 bar of CO in the presence of a palladium (II) complex, PPh₃ and NEt₄Cl. Experimental evidence was provided to prove a new reaction stoichiometry and involvement of a carbamoyl intermediate. In carbonylation of HCHO over ion exchange resin catalysts, reaction temperature, time, pressure, and solvent were important variables to obtain high yields of methyl glycolate. Carbonylation of isobutylphenylethanol at 120°C and 40 bar of CO in the presence of PdCl₂−PPh₃−HCl gives 98% yield of α-(4-isobutylphenyl) propionic acid (ibuprofen). Each catalyst component had a definite role that is indispensable for an efficient overall reaction.     

Redox behavior of V/MgO catalyst in H2S wet oxidation at room temperature

The room temperature wet catalytic oxidation was conducted in a batch reactor with V/MgO catalyst. The XRD study of the catalyst used indicated that V/MgO could not only oxidize H2S to sulfur selectively, but also prevent the sulfidation of metal oxide effectively at the room temperature. The XPS study indicated that the H2S oxidation with V/MgO could proceed by a redox mechanism (V⁵⁺↔ V⁴⁺) and that V³⁺ formation (V⁴⁺→ V³⁺), was responsible for the deactivation of V/MgO. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.Subject terms: Fluorescence imaging, Super-resolution microscopy, Single-molecule biophysics     

Concise syntheses of (+)-macrosphelides A and B

[reaction: see text]. Unified and highly convergent total syntheses of (+)-macrosphelides A and B are described. Key features of the syntheses include (1) concise synthesis of the optically active delta-hydroxy-gamma-keto alpha,beta-unsaturated acid fragment via the direct addition of a trans-vinylogous ester anion equivalent to the readily available Weinreb amide and (2) facile construction of the 16-membered macrolide core of the macrosphelide series via an intramolecular nitrile-oxide cycloaddition (INOC).     

Radiocatalytic H2 production with gamma-irradiation and TiO2 catalysts

The radioactive concentrations of radionuclides were measured in the soil and groundwater below the 12 GeV proton beam-line tunnel at KEK. Various long-lived radionuclides, (⁷Be, ²²Na, ⁴⁶Sc, ⁵⁴Mn, ⁶⁰Co, ¹³⁴Cs, ¹⁵²Eu and ¹⁵⁴Eu) were observed in the soil samples by -ray spectra measurements, and ³H was also detected by liquid scintillation counting. On the other hand, ³H, ²²Na and ⁵⁴Mn, which were leached from the soil were measured in the groundwater below the EP2 beam line. ³H and ²²Na were also found in slight amounts in groundwater collected in a well dug beside the East Counter Hall. From a comparison with the radioactive concentration in soil and groundwater, the order of the leaching percentages were ³H>²²Na>⁵⁴Mn. This tendency was in agreement with an experimental result of the RI laboratory.     

A polymeric photobase generator containing oxime–urethane groups: Crosslinking reaction and application to negative photoresist

A polymeric photobase generator containing oxime–urethane groups was prepared by copolymerization of methyl methacrylate and methacryloxyethyl benzophenoneoxime urethane, and its photo and thermal crosslinking reaction after irradiation was examined from the measurement of UV and IR absorption spectral changes, insoluble fraction, and molecular weight changes. The photo‐crosslinking reaction of the copolymer film was more efficient when irradiations were carried out with 310 nm UV light in the presence of benzophenone than with 254 nm UV light without the addition of benzophenone. The crosslinking reaction increased after postexposure baking (PEB), and this thermal crosslinking reaction mechanism was studied from the identification of the photolysis products of a model compound, benzophenoneoxime phenylurethane, by a high‐performance liquid chromatography. The results indicate that the thermal crosslinking reaction of the copolymer after PEB is due to the formation of urea‐type chemical bonds. Resist properties of the copolymer were examined from the measurement of normalized thickness and micropattern development. A negative tone image with a resolution of 2 μm was obtained with this copolymer, having a sensitivity (D) of 1200 mJ/cm² and contrast (γ_n) of 1.31, when irradiation was carried out with 310 nm UV light in the presence of benzophenone following chloroform development. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 975–984, 2004     

Metallic macrocycle with a 1,3-alternate calix[4]arene phosphorus ligand

The preparation of an 1,3-alternate calix[4]arene phosphorus ligand, 25,27-bis(2-(diphenylphosphino)ethoxy)-26,28-bis(1-propyloxy)calix[4]arene (3), is presented. Ligand 3 is obtained in three steps in 64% overall yield. Reaction of 3 with [Rh(cot)2]BF4 produced the encapsulated rhodium complex [Rh[(P,P)-diphen-calix[4]arene]]BF4 (4). As revealed by a single-crystal X-ray diffraction study, the rhodium center has a bent coordination environment with a P-Rh-P angle of 135.66(3) degrees. Palladation of 3 employing [Pd(MeCN)4](BF4)2 yielded the chelate palladium complex 7 in which the palladium center has a slightly bent configuration. Treatment of the ligand with Pd(cod)Cl2 and [Pd(eta3-C4H7)(THF)2]BF4 leads to the isolation of the monometallic complex. Full characterization includes X-ray structural studies of compounds 3, 4, and 6.     

Residual stress and deformation during rapid cooling process in an epoxy plate

Residual stress in the epoxy plate during a rapid cooling process was measured by the layer removal method and calculated by the linear thermoviscoelastic theory considering specific volume relaxation. The relaxations of the tensile modulus and specific volume were measured by an Instron thermomechanical analyzer. When the starting temperature of the cooling process was near the glass transition temperature of the cured epoxy, the residual stress in the epoxy plate was smaller than when the starting temperature was higher than the glass transition temperature. However, the transient stress in the cured epoxy plate was higher when the starting temperature was near the glass transition temperature than when the starting temperature was higher than the glass transition temperature. The quenched epoxy plate was compressed in the direction parallel to the surface and expanded in the thickness direction.