Makoto Kida

Production of human monoclonal antibodies by heterohybridomas

SummaryMouse human-human heterohybridomas secreting human monoclonal antibodies (MoAb) against tetanus toxoid and hepatitis B virus surface antigen were effectively cultivated in a medium containing a serum substitute called GFS, a 55% to 70% ammonium sulphate fraction of serum from adult cattle. A perfusion culture system using a jar fermentor equipped with a cell sedimentation column with a double jacket was developed and applied to produce human MoAb. In this fermentor, maximum cell density of a heterohybridoma reached 1.2×107 cells/ml and MoAb was continuously accumulated at a constant rate for at least 40 days; this led to the production of more than one gram of human MoAb using a culture vessel with a 1-1 working volume.

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.

SCE-963, a New Broad-Spectrum Cephalosporin: In Vitro and In Vivo Antibacterial Activities

SCE-963 {7β-[2-(2-aminothiazol-4-yl)acetamido]-3-[({1-(2-dimethylaminoethyl)- 1H-tetrazol-5-yl}thio)methyl]-ceph-3-em-4-carboxylic acid}, a new semisynthetic cephalosporin, showed excellent antibacterial activity against gram-positive and gram-negative bacteria, including Haemophilus influenzae, indole-positive Proteus, Enterobacter species, and Citrobacter freundii. The minimum inhibitory concentrations of SCE-963 against most strains of clinically isolated Escherichia coli, Klebsiella pneumoniae, H. influenzae, and Proteus mirabilis were within the range of 0.2 to 0.78 μg/ml. These activities were about 10 times more potent than those of cefazolin, cephaloridine, and cephalothin. Variations in pH, addition of horse serum, and type of growth medium had no significant effect on the activity of the cephalosporin, but the inoculum size elicited a considerable effect on the activity of β-lactamase-producing strains of bacteria. SCE-963 exerted bactericidal and bacteriolytic effects on Staphylococcus aureus and E. coli. The pronounced in vitro activity was reflected in the remarkable protection in mice infected with a wide range of gram-negative bacteria, such as E. coli, K. pneumoniae, P. mirabilis, Proteus vulgaris, Proteus morganii, and Proteus rettgeri. The protective effects of SCE-963 in mice infected with E. coli, K. pneumoniae, and P. vulgaris varied according to the challenge dose. The activity of SCE-963 was far more potent when the drug was administered parenterally rather than orally.

Cefmenoxime (SCE-1365), a novel broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities.

The activity of cefmenoxime (SCE-1365), 7 beta-[2-(2-aminothiazol-4-yl)-(Z)-2-methoxyiminoacetamido]-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]ceph-3-em-4-carboxylic acid, was compared with that of other cephalosporins. Cefmenoxime exhibited high activity against a wide variety of gram-positive and gram-negative bacteria. The in vitro activity of cefmenoxime against Streptococcus pyogenes, Haemophilus influenzae, and Enterobacteriaceae, including indole-positive Proteus, Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii, was 10 to 1,000 times greater than that of several other cephalosporins. Against Pseudomonas aeruginosa, cefmenoxime showed activity two to four times that of sulbenicillin and carbenicillin but less than that of cefsulodin. Variation in pH, addition of horse serum, and type of growth medium had definite effects on the activity of cefmenoxime, and the inoculum size affected the activity against bacterial species. In Escherichia coli cefmenoxime showed marked affinity for penicillin-binding protein 3 (PBP-3), followed by PBP-1 (1A and 1B). This affinity profile was well correlated with its filamentous cell-forming activity under extremely low drug concentrations and with its bactericidal activity against microorganisms. The high in vitro activity of cefmenoxime was reflected in the degree of protection observed in mice infected intraperitoneally with a wide variety of gram-positive and gram-negative bacteria. Furthermore, cefmenoxime showed good therapeutic activity against infection models in mice such as respiratory tract infection caused by Klebsiella pneumoniae and urinary tract infection caused by Proteus mirabilis. Images

Accumulation of Tripeptide Derivatives by Mutants of Cephalosporium acremonium

The two β-lactam antibiotics produced by Cephalosporium acremonium ATCC 14553, cephalosporin C and penicillin N, have been proposed to be biosynthesized through the peptide, δ-(l-α-aminoadipyl)-l-cystemyl-d-valine.Many β-lactam negative mutants were derived from the strain No. 52, which was a more potent producer of the β-lactam antibiotics than the parent strain, C. acremonium ATCC 14553. Some of them were found to accumulate two sulfur-containing peptides. These compounds were isolated from the culture filtrate of one of the mutants, N-2 and determined to be the dimer of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine and the S-methylthio derivative of the tripeptide.

Induction of interferon and host resistance in vivo by double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid

Several double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid (poly IG∶C) were found to possess interferon inducing activity stronger than poly I∶Cin vivo, Their activity increased in parallel with increase in the ratio of guanine base to hypoxanthine base in these copolymers as far as double-strand formation was observed with polyribocytidylic acid. Many other combinations of copolyribonucleotide with homopolyribonucleotide were also investigated, and several of them were found to induce interferon. However, the interferon inducing effects of these combinations including complementary base-pairings of hypoxanthine and cytosine increased in parallel with the length of the base-pairings, thus approaching to that of poly I∶C. It is, therefore, supposed that the activity of poly IG∶ C is somewhat different from poly I∶C and that those of other combinations owe to the essential structure of poly I∶C. Furthermore, kinetics of interferon induction, cross tolerance to reinduction, and antiviral effectsin vivo of poly IG∶C and poly I∶C were studied.

Selective Phosphorylation of the 5′′-Hydroxy Group of Ribostamycin by a New Enzyme from Pseudomonas aeruginosa

It was found that the 5′′-hydroxy group of ribostamycin was preferentially phosphorylated by a cell-free extract from Pseudomonas aeruginosa GN 573, a clinical isolate.

Studies on the Guanosine Degrading System in Bacterial Cell:Part III. Purification and Properties of Guanosine Deaminase

A highly purified preparation of the enzyme, guanosine deaminase, has been obtained by a four-step purification procedure from the cells of Pseudomonas convexa No. 149. The enzyme deaminates guanos...