Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus]

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.     

Stabilization of ribose-5-phosphate isomerase from tobacco

The highly purified ribose phosphate isomerase from tobaccoleaves is heat labile. 0.2% of various kinds of proteins stabilizedisomerase activity when Mg⁺⁺ was present. 1x10^–3% polyethyleneglycol2,000 showed the same effect as the proteins did. Smaller polyediyleneglycolswere less effective. Polyhydroxyl compounds showed litde effect.Mn⁺⁺ or Sr⁺⁺ was also effective as a stabilizer instead of Mg⁺⁺. (Received March 8, 1976; )     

Structure-activity relationships in human interleukin-1 alpha: identification of key residues for expression of biological activities.

To identify the sites important for the different biological activities of human interleukin-1 alpha (hIL-1 alpha), 56 single-amino acid-substituted mutants of hIL-1 alpha were produced in Escherichia coli using site-directed mutagenesis, and were examined for their biological activities such as mouse lymphocyte activating factor activity (LAF activity), cytostatic activity against human melanoma cells A-375 (A375 activity) and prostaglandin E2 (PGE2) inducing activity in human osteosarcoma cells MG-63 (PEI activity). Two amino acid residues, Asp26 and Asp151, were found to be important for these activities. The replacement of Asp26 by Val caused a decrease in LAF and PEI activities by one or two orders of magnitude and a slight decrease in A375 activity. The Tyr or Phe substitution for Asp151 caused decreases in LAF and A375 activities by one or two orders of magnitude and complete loss of PEI activity. The change from Asp151 to Lys or Arg resulted in marked decrease in LAF activity and complete loss of A375 and PEI activities. Since Asp26 and Asp151 are close to each other in the three-dimensional structure, the region involving these amino acids seems to be important for the biological activities of hIL-1 alpha.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Production of 10-ketostearic acid from oleic acid by a strain of Flavobacterium sp. 12-4A

Summary A new Flavobacterium sp., strain 12-4A, produces solely 10-ketostearic acid (KSA) from oleic acid (OA) in growing cultures. Under optimized conditions 14 mg/ml of KSA was produced from 25 mg/ml of OA after 3 days of cultivation at 30°C.     

Recombinant human IL-6 expressed inE. coli undergoes selective N-terminal degradation: Evidence that the protein consists of a stable core and a nonessential flexible N-terminal

A synthetic gene for human interleukin-6 has been expressed inE. coli. The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay. The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15. The truncated proteins retain full biological activity. The degradation results in the loss of sharp amide resonances in the¹H-NMR spectrum, and little change to the ultraviolet CD spectrum. Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment. The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.