Utilizing the tubular bioreactor for continuous recovery of secreted fusion protein from recombinant Escherichia coli

In order to improve the cultivation properties of a traditional continuous stirred tank reactor (CSTR), we introduced a circulation unit made of four inorganic membranes in stainless steel tubes in parallel configuration, the so-called Tubular Bioreactor (TBR). Furthermore, the TBR outlet tube, which has a restriction nozzle at the end, was installed on top of the fermentor vessel, thereby creating a strong jet flow into the reactor and thus improving the mixing and the oxygen transfer rate. The k _La could be increased by approximately 50%. This setup was used for cultivations of recombinant Escherichia coli in a minimal medium and high cell density. More than 50 g dry cell mass/dm³ was obtained. Simultaneously, we have produced an elongated form of human insulin-like growth factor II, which was a secreted fusion protein utilizing the E. coli secretion system based on staphylococcus protein A. The product could be recovered continuously through the TBR-membrane.     

Production of saikosaponins by tissue culture of Bupleurum falcatum L.

The studies for production of saikosaponins by tissue culture of Bupleurum falcatum L. were carried out to produce saikosaponins with several kinds of media and plant hormones. Among the media and plant hormones studied, Gamborg's B-5 [23] medium containing 0.5 ppm kinetin (k) and 1.0 ppm 3-indolebutyric acid (IBA) was the most effective medium and hormone for production of saikosaponins. The highest content of saikosaponin-d in the dried cells was 0.26%, which was similar to a concentration of Bupleuri Radix.Abbreviations MS medium used by Murashige and Skoog [22] - G medium used by Gamborg (B 5) [23] - W medium used by White [24] - NN medium used by Nitsch and Nitsch [25] - k Kinetin - BAP 6-Benzylaminopurine - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA -Naphtylacetic acid - IBA 3-Indolebutyric acid - IAA 3-Indoleacetic acid - ssd saikosaponin-d - PM Production medium - dw Dry weight     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Effect of rice fiber on human fecal microflora

The effects of brown rice, containing fourfold as much dietary fiber as polished rice, on the human fecal microflora were determined. Significantly increased numbers of Bifidobacterium adolescentis and Enterococcus faecalis were observed during the brown rice intake, whereas the total counts and the numbers of bacteroides, Eubacterium aerofaciens, and Escherichia coli during the intake were lower than those before and after the intake. Lower numbers of clostridia and low incidences of Clostridium paraputrificum and C. perfringens were observed during the brown rice intake.     

N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: a proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (NMR) spectra of a synthetic peptide (p25) corresponding to the amino-terminus of the yeast mitochondrial cytochrome oxidase subunit IV precursor protein have been analyzed. Sequence-specific resonance assignments of the peptide have been made in the presence of micelles of a phospholipid analog, perdeuterated dodecylphosphocholine (DPC), with the aid of such techniques as HOHAHA, DQF-COSY, and NOESY. The interresidue nuclear Overhauser effects (NOEs) indicate that the N-terminal half of p25 (S3-F11) takes a helical structure while the C-terminal half does not take a regular secondary structure. Addition of DPC to the solution of p25 induced chemical shift changes only of the resonances from the residues in the N-terminal half, suggesting that the N-terminal half of p25 is directly involved in binding to DPC. The induced helical structure in the N-terminal half at a lipid-water interface may be important in the ability of this presequence to direct a "passenger" protein into mitochondria.     

Purification and characterization of a Ca2+-dependent actin filament severing protein from bovine adrenal medulla

We describe the purification of an actin regulatory protein from bovine adrenal medulla. This protein caused a dose-dependent decrease of the specific viscosity of actin solution within 30 s of its addition in a Ca2+-sensitive way. Sedimentation assays and the observation by electron microscopy showed that this effect was ascribable to the fragmentation of actin filaments. This protein apparently promoted nucleation of actin polymerization and increased the critical concentration of actin for polymerization nearly 5-fold, suggesting its binding to the barbed end of actin filaments. The inhibitory effect of this protein on the elongation of actin from the barbed end of the myosin subfragment S1-labeled actin seeds confirmed this suggestion. These properties are similar to those of gelsolin. However, the physicochemical properties of this protein having a single polypeptide chain with a molecular weight of 74,000, a Stokes radius of 3.9 nm, a sedimentation coefficient (s0(20),w) of 4.5 S, and an immunological characterization showed that this protein is different from gelsolin.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.