Red cell membrane elasticity as determined by flow channel technique.

The elasticity of red cell membrane was determined in a rectangular flow channel under controlled shear flow. The relation between shear stress and cell extension ratio (lambda) has been analyzed with the use of Evans' two-dimensional model. The deformed cell shapes observed experimentally agreed well with the model with lambda up to 1.4. The best correlation was found at lambda = 1.2. The analysis suggests a nonlinear extensional membrane modulus in the low stress range encountered in the flow channel. In terms of an appropriate strain parameter, the elastic modulus is shown to rise toward the level encountered in micropipette aspiration experiments. The implications of the present findings in modeling of cell mechanics and in cell hemolysis are discussed.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.     

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.     

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.     

Cytochrome c peroxidase catalyzed oxidation of ferrocytochrome c by hydrogen peroxide: ionic strength dependence of the steady-state rate parameters

The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.     

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.     

Cycloheximide resistance in carrot culture: a differentiated function

Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues.