Absolute side-chain structure at position 13 is required for the inhibitory activity of bromein

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition.     

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k_on value and/or a low k_off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen (k_on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen (k_off′), the effective antigen concentration (C), the time for the binding process (t_b) and the time for the washing process (t_w). An optimum set of operating parameters (C, t_b and t_w) for isolating phages displaying an antibody with a high k_on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k_on value and one displayed an antibody with a 30-fold greater k_on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.     

Induction of cytotoxicity of peritoneal exudate cells by agrimoniin,a novel immunomodulatory tannin of Agrimonia pilosa Ledeb

Summary The cytotoxic activities of the PEC after an i.p. injection of agrimoniin, a tannin contained in Agrimonia pilosa Ledeb. were studied. The plastic nonadherent PEC had significantly higher NK cell activity than the untreated control, and the adherent PEC were cytostatic toward MM2 and MH134 cells. The adherent PEC did not cause tumor cell lysis by themselves, but were cytolytic against MM2 cells in the presence of anti-MM2 sera. In the course of these effects of PEC after the i.p. injection of agrimoniin, the augmentation of NK cell activity was the earliest reaction, reaching a peak at 2 days after the injection; then, cytostatic activity increased. The induction of antibody-dependent cell lytic activity was a later reaction, which reached a peak at 6 days after the injection. Abbreviations used: PEC, peritoneal exudate cells; NK cell, natural killer cell; ADCC, antibody-dependent cell-mediated cytotoxicity; PMN, polymorphonuclear leukocytes     

Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.     

Naloxone inhibits prolactin and growth hormone release induced by intracellular glucopenia in the rats

Intraventricular administration of 2-deoxy-D-glucose (2DG), which causes intracellular glucopenia in the central nervous system, increased plasma prolactin and growth hormone levels in the urethane anesthetized male rats. Naloxone, an opiate antagonist, inhibited the 2DG-induced prolactin and growth hormone release. Apomorphine, a dopaminergic agonist, also inhibited the release of these hormones induced by 2DG. These results suggest that endorphins play a role in hypoglycemia-induced prolactin and growth hormone release and that the dopaminergic mechanism may be involved in this phenomenon.     

Murine Endothelial Cell Line Cells,F-2: Interaction with Leukocytes and Cytokines Production

Flowcytometry demonstrated that murine endothelial cell line F-2 expresses MHC class I antigen, FcR II, Mac-1 and vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1 (ICAM-1) and class II antigen. However, co-culturing with TNF-α for 24 hr resulted in the increased expression of ICAM-1, and the decreased expression of VCAM-1. IL-1α and IFN-γ exerted this regulatory effect on VCAM-1 but not on ICAM-1. T (Con A blast) and B (LPS blast) cells adhered to F-2 cells at almost equal levels, and the adhesion was enhanced 20 to 50% when the cells were precultured with TNF-α for 24 hr. The inhibition assay using either (anti-ICAM-1 + anti-LFA-1, lymphocyte function-associated antigen-1) or (anti-VCAM-1 + anti-VLA-4, very late antigen-4) mAbs demonstrated that the ICAM-1 system was utilized more preferentially by T than B blasts when F-2 cells were stimulated with TNF-α, and the VCAM-1 system was vice versa under the unstimulated and stimulated conditions. Granulocytes also adhered to F-2 cells, but no mAbs could inhibit the adhesion. Although F-2 cells produced a considerable amount of IL-6, GM-CSF and neutrophil chemotactic activity, a 24 hr incubation with TNF-α resulted in an increase of 12 fold in IL-6 and 3 fold in neutrophil chemotactic activity production.