Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Abstract Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.     

Gd(-) Gifu and Gd(-) Fukuoka. Two new variants of glucose-6-phosphate dehydrogenase found in Japan

Summary Two new glucose-6-phosphate dehydrogenase (G6PD) variants were discovered in Japan. The first, found in a 9-year-old male, was associated with chronic hemolysis and hemolytic crises after upper respiratory infections. The enzyme activity of the variant was 2.9% of normal. The patient's G6PD showed an increased utilization of substrate analogue, deamino-NADP, and thermal instability. The second variant occurred in a 7-year-old male with druginduced hemolysis. The main enzymatic characteristics were reduced enzyme activity, being 6.4% of normal, faster-thannormal anodal electrophoretic mobility, slightly high Michaelis constant for glucose-6-phosphate, thermal instability, and biphasic pH optima. Enzymatic properties of these variants allowed each to be distinguished from previously reported variants. The first variant was designated Gd (-) Gifu and the other, Gd (-) Fukuoka.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

Microbial Production of Ursodeoxycholic Acid from Lithocholic Acid by Fusarium equiseti M41

A fungus identified as Fusarium equiseti was isolated from soil and found to carry out 7β-hydroxylation of lithocholic acid to ursodeoxycholic acid (35% yield; 350 mg/liter) in 112 h.     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.