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.     

Purification of monomeric agmatine iminohydrolase from soybean

Agmatine iminohydrolase (EC 3.5.3.12) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by chromatographic separations on Sephadex G-25, Bio-rex 70, and agmatine-affinity columns. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weights estimated by Sephadex G-100 gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis were 70,000, indicating that the soybean axes enzyme is a monomer, in contrast to the dimeric enzymes from corn and rice. The isoelectric point determined by gel electrofocusing was 7.5, higher than that of the corn enzyme (4.7). The optimal pH and temperature for activity were 6.5 and 50 degrees C, respectively. The enzyme has high specificity for agmatine, and the Km for agmatine was 2.5 x 10(-3) molar. The enzyme was sensitive to Cu2+ and also was inhibited by p-hydroxymercuribenzoate.     

Immobilization of enzymes on activated carbon: selection and preparation of the carbon support.

Based upon its superior catalytic activity for H2O2 decomposition, a bituminous coal-based activated carbon was selected for investigations of pretreatment and enzyme immobilization methods. Pretreatments considered include acid washing, exposure to strong oxidizing agents, contact with concentrated peroxide solutions, nitration and amination, isothiocyanate derivatization, silanization, and stearic acid coating. Effects of these pretreatments on morphology and trace-metal content of the carbon pellets have been studied using scanning electron microscopy and dispersive analysis of x rays. Immobilization of glucoamylase by adsorption, glutaraldehyde crosslinking, and covalent attachment to carbon activated by water-soluble diimide or diazotization have been examined. These different enzyme-carbon catalysts have been characterized by their enzyme loading, enzyme activity, catalytic activity for H2O2 decomposition, or combinations of these measures of performance.     

Current state and perspectives on erythropoietin production

Erythropoietin is a major regulator of erythropoiesis which maintains the body's red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life. In order to obtain an optimal and consistent glycoform profile of rhEPO and concurrently maintain a high production yield, various approaches in drug development and cell culture technology have been attempted. Recent advances in rhEPO production are classified into three types: the development of improved rhEPO molecules by protein engineering; improvement of production host cells by genetic engineering; and culture condition optimization by fine control of the production mode/system, process parameters, and culture media. In this review, we focus on rhEPO production strategies as they have progressed thus far. Furthermore, the current status of the market and outlook on rhEPO and its derivatives are discussed.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Differentiation-dependent suppression of platelet-derived growth factor signaling in cultured adipocytes.

A critical component of vertebrate cellular differentiation is the acquisition of sensitivity to a restricted subset of peptide hormones and growth factors. This accounts for the unique capability of insulin (and possibly insulin-like growth factor-1), but not other growth factors, to stimulate glucose uptake and anabolic metabolism in heart, skeletal muscle, and adipose tissue. This selectivity is faithfully recapitulated in the cultured adipocyte line, 3T3-L1, which responds to insulin, but not platelet-derived growth factor (PDGF), with increased hexose uptake. The serine/threonine protein kinases Akt1 and Akt2, which have been implicated as mediators of insulin-stimulated glucose uptake, as well as glycogen, lipid, and protein synthesis, were shown to mirror this selectivity in this tissue culture system. This was particularly apparent in 3T3-L1 adipocytes overexpressing an epitope-tagged form of Akt2 in which insulin activated Akt2 10-fold better than PDGF. Similarly, in 3T3-L1 adipocytes, only insulin stimulated phosphorylation of Akt's endogenous substrate, GSK-3beta. Other signaling molecules, including phosphatidylinositol 3-kinase, pp70 S6-kinase, mitogen-activated protein kinase, and PHAS-1/4EBP-1, did not demonstrate this selective responsiveness to insulin but were instead activated comparably by both insulin and PDGF. Moreover, concurrent treatment with PDGF and insulin did not diminish activation of phosphatidylinositol 3-kinase, Akt, or glucose transport, indicating that PDGF did not simultaneously activate an inhibitory mechanism. Interestingly, PDGF and insulin comparably stimulated both Akt isoforms, as well as numerous other signaling molecules, in undifferentiated 3T3-L1 preadipocytes. Collectively, these data suggest that differential activation of Akt in adipocytes may contribute to insulin's exclusive mediation of the metabolic events involved in glucose metabolism. Moreover, they suggest a novel mechanism by which differentiation-dependent hormone selectivity is conferred through the suppression of specific signaling pathways operational in undifferentiated cell types.