Tandem electric field gradient focusing system for isolation and concentration of target proteins

Two electric field gradient focusing (EFGF) systems, one based on a hollow dialysis fiber and the other based on a shaped ionically conductive polymer were serially integrated to trap and concentrate selected proteins while simultaneously desalting and removing other unwanted proteins from the sample. A series of experiments were performed to test the EFGF systems individually and after integration. Online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng mL(-1) (200 pM) from a sample volume of 100 microL. Concentration of human alpha1-acid glycoprotein with simultaneous removal of human serum albumin was also demonstrated. Elimination of small buffer components while concentrating trypsin inhibitor, and selective concentration and separation of myoglobin from a mixture were performed using the integrated EFGF system.     

Nuclear spin-lattice and nuclear spin-spin relaxation time measurements in EuB6 at low temperatures using the spin-echo technique

Experimental results on the nuclear spin-lattice and nuclear spin-spin relaxation times in the ferromagnetic EuB₆ at temperatures below 4·2 K are presented using the external magnetic field,H _ext, in the range of 0 ⩽H _ext ⩽ 10 kG. Nuclear spin-spin relaxation time computed on the basis of the Suhl-Nakamura process turns out to be 3·2μs, which compares well with the experimental value 11·1μs obtained with the 10 kG magnetic field at 1·7 K. It is found that in the ferromagnetic EuB₆,T ₁ is approximately 5 × 10³ times larger thanT ₂ at 1·7 K with the 10 kG magnetic field. Thus the effect ofT ₁ onT ₂ can be neglected. From the experimental value ofT ₂, the value of the homogeneous line broadening is found to be 14 kHz. The corresponding value obtained from the cw method is 175 kHz. This evidently shows the presence of the inhomogeneous line broadening in the cw NMR.     

Pion exchange and the universal impact parameter hypothesis

In order to describe pion exchange reactions a modification to the $\text{“b-}\text{universality}\text{”}$ hypothesis is proposed which simply relates the impact parameter profiles of helicity amplitudes for different values of net helicity-flip. This proposal is shown to give an excellent quantitative fit to the π^?p → ?⁰n and π^?p → f⁰ data at 17.2 Ge V/c. It also provides a natural explanation for the presence and size of the necessary absorptive cut corrections.     

The importance of arginyl residues for phosphorylation of rat liver cell sap proteins

Arginyl residues in phosvitin, histone and cell sap protein were blocked by 1,2-cyclohexanedione, resulting in markedly impaired phosphorylation of histone and cell sap. Interestingly, the phosphate incorporation into phosvitin was not changed by this treatment. Intact arginyl residues in the protein kinase substrates seemed to be essential for more than half of the cell sap phosphorylation at 5 mM ATP. Furthermore both phosvitin kinase and histone kinase activities in cell sap were inhibited by arginyl residue blockade, indicating that these enzymes had functional arginyl residues.     

Pomeron dominance in a triple-Regge representation of inclusive Δ++ production in K−p and π−p reactions

Data are presented on the reactions K^?p → Δ⁺⁺ + anything at 10 and 16 GeV/c and π^?p → Δ⁺⁺ + anything at 16 GeV/c. In the K^?p reaction, scaling is observed between 10 and 16 GeV/c in the variable M²/s. The scaling occurs at relatively low values of M in marked contrast to the reaction π⁺p → Δ⁺⁺ + anything. This result can be explained by duality arguments on a triple Regge picture, since the exchanged reggeon-incident particle scattering is exotic. Comparison of the π^?p reaction, which is similarly exotic, with the K^?p reaction at the same energy gives further evidence for factorization in pomeron-dominated inclusive reactions.     

Influence of transport properties in electric field gradient focusing

Miniaturized devices for electric field gradient focusing (EFGF) were developed that consist of a cylindrical separation channel surrounded by an acrylic-based polymer hydrogel. The ionic transport properties of the hydrogel enable the manipulation of the electric field inside the separation channel. A changing cross-section design was used in which the hydrogel is shaped such that an electric field gradient is established in the separation channel. One of the challenges with this type of EFGF device has been that experimental resolution between protein analytes is lower than theoretically predicted. In order to investigate this phenomenon, a mathematical transport model was developed using FEMLAB. Model results and experimental observations showed that the reduced performance was caused by concentration gradients formed in the EFGF channel, and that these concentration gradients were the result of an imbalance in cation transport between the open separation channel and the hydrogel. Removing acidic impurities from the monomers that form the hydrogel reduced this tendency and improved the resolution. These transport-induced concentration gradients can be used to establish electric field gradients that may be useful for sample pre-concentration. Both the results of simulation and experiments demonstrate how transport-induced concentration gradients lead to the establishment of electric field gradients.     

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown.Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309-382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.