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.     

冬小麦春化过程中低温诱导的与花芽分化相关的mRNA和蛋白质的合成

应用SDS电泳及高分辨双向电泳技术,分析了冬小麦经春化、脱春化及超期春化处理后茎类蛋白质组分的变化。发现52.5,38kD和16.2kD蛋白质与冬小麦开花诱导密切相关。mRNA体外翻译结果表明,春化作用中低温诱导与开花紧密相关的mRNA的出现有一定的顺序性。因此推测:春化诱导开花是低温导致某些特定基因表达的结果,而表达的调节主要发生在转录水平上。     

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.     

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.     

Effect of heating on properties of some soils from Southern Nigeria and growth of rice

Summary The effect of heating on the properties of Apomu (Psammentic Usthorthent), Egbeda (Oxic Paleustalf) and Gambari (Typic Plinthustalf) surface soils were studied under laboratory conditions. Heating at low temperatures (100°C) have no detrimental effects on soil properties, on the contrary it increased the soil extractable P, Mg, Fe, Mn and Zn levels. Pronounced reductions in total N, Org. C, Org. P and extractable Ca and Mg levels and marked increases in extractable P, Zn, Mn and Fe were observed by heating to 200°C. Heating to 500° had an adverse effect on soil chemical and physical properties.Plant height and dry matter yeild of rice plants were higher when grown on Egbeda soil previously heated to 100°C. With addition of N, P and K there was no observed beneficial effect of the heating treatment. Rice plants grown on Egbeda soil previously heated to 200°C showed high uptake of Mn. Plants grew badly in soil previously heated to 500°C.     

Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.