A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells

Purpose In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems.Methods We have produced an anti-epidermal growth-factor receptor (EGFR) × anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E. coli) expression system with refolding method. The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro. This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen.Results When T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-, GM-CSF, and TNF- than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37°C for 48 h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice. When Ex3 diabody (20 g/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth.Conclusion The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.     

Enhancement of insulin sensitivity in adipocytes by ginger

Antidiabetic and hypoglycemic drugs have been reported to enhance adipocyte differentiation of 3T3-L1 preadipocytes. We previously reported that ginseng (active constituents: ginsenosides) enhanced the differentiation [1]. In this experiment, effect of some ginger group food extracts on the adipocyte differentiation was investigated using cultured mouse 3T3-L1 preadipocytes. 3T3-L1 cells were grown as monolayer cultures at 37 degrees C in DMEM supplemented by 10% FBS under the atmosphere of 5% CO(2)-95% air. Ginger extracts were found to enhance the adipocyte differentiation. Active constituent was purified and identified as gingerol. In the gingerol-treated cells, insulin-sensitive glucose uptake was increased. It is expected that ginger enhance the insulin-sensitivity, and improve chronic disease, such as diabetes.     

Periovulatory changes in the local release of vasoactive peptides, prostaglandin f(2alpha), and steroid hormones from bovine mature follicles in vivo

We previously proposed that an endothelin-angiotensin-atrial natriuretic peptide system may contribute to inducing ovulation of mature bovine follicles by modulating follicular secretion of steroids and prostaglandins (PGs). Thus, this study aimed to determine the real-time changes in the local release of angiotensin II (Ang II), endothelin (ET), atrial natriuretic peptide (ANP), PGF(2alpha), and steroid hormones from bovine mature follicles during the periovulatory period in vivo. Seven cows were treated for superovulation using FSH and PGF(2alpha) injections. Two dialysis capillary membranes per follicle were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system (MDS). Fractions of the perfusate were collected from Day -1 (Day 0 = LH surge) to Day 3. Five out of seven treated cows were normally ovulated, and the newly formed corpora lutea were observed at the end of the experiment. In these five ovulated cows, the release of estradiol, androstenedione, and progesterone in the theca layer increased (P < 0.05) synchronously with the LH surge. Acute increases in PGF(2alpha) and Ang II concentrations in the ovarian venous plasma (OVP) were observed at 24-48 h after the peak of the LH surge, when multiple ovulations were expected to occur. The follicular Ang II release was low during the pre-LH surge period and rose (P < 0.05) at the beginning of the increase in the LH surge. On the other hand, ET-1 release dropped (P < 0.05) when plasma LH started to increase. However, no clear changes in ANP concentration in the MDS perfusate and plasma were observed. The above local changes in Ang II, PGF(2alpha), as well as steroid hormones were not observed in cows (n = 2) that did not show an LH surge and ovulation. The present results demonstrate for the first time the local release of Ang II, ET-1, and ANP from the bovine mature follicle in real-time in vivo and show that Ang II and PGF(2alpha) concentrations in the OVP acutely increase around the time of ovulation. The overall results support the concept of a local functional ET-Ang-ANP system in the bovine mature follicle that may be involved in the ovulatory process.     

Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form

Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.     

Cell type-specific E2F activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I

The transactivator protein Tax of human T-cell leukemia virus type I plays an important role in the development of adult T-cell leukemia probably through modulation of growth regulatory molecules including p16(INK4a). The molecular mechanism of leukemogenesis induced by Tax has yet to be elucidated. We analyzed Tax function in the cell cycle using an interleukin-2 (IL-2)-dependent human T-cell line (Kit 225) that can undergo cell cycle arrest at G(0)/G(1) phase by deprivation of IL-2. Tax activated endogenous E2F activity in IL-2-starved Kit 225 cells, resulting in activation of E2F site-carrying promoters of genes involved in G(1) to S phase transition in a cell type-dependent and p16(INK4a)-independent manner. The ability of Tax mutants to activate E2F coincided with that to activate nuclear factors kappaB and AT, sole expression of which, however, did not activate E2F, suggesting involvement of another pathway in activation of E2F. Introduction of Tax by a recombinant adenovirus induced cell cycle progression to G(2)/M phase in resting Kit 225 cells accompanied by endogenous cyclin D2 gene expression. Similarly, Tax-induced cell cycle progression was seen with peripheral blood lymphocytes prestimulated with phytohemagglutinin. Analyses with Tax mutants did not allow Tax-induced cell cycle progression to be differentiated from Tax-dependent activation of E2F, suggesting that Tax induces cell cycle progression presumably through activation of E2F. Nevertheless, infection with an E2F1-expressing virus, which is sufficient for induction of S phase in serum-starved fibroblasts, was not sufficient for either E2F activation or cell cycle progression in IL-2-starved Kit 225 cells, implying differential regulation of E2F activation and cell cycle progression in T-cells that is activated by Tax.     

Chalcanol glucosides from seeds of Trifolium alexandrinum

Three new chalcanol glucosides have been isolated from the seeds of Trifolium alexandrinum, of which the first two are alpha'-chalcanol-alpha, beta-epoxides and the third one is an alpha, beta-dihydroxy-alpha'-chalcanol. The structures of the isolated compounds were verified by means of MS and 2D NMR spectral analyses.     

Involvement of suppressor of cytokine signalling-1-mediated degradation of MyD88-adaptor-like protein in the suppression of Toll-like receptor 2-mediated signalling by the murine C-type lectin SIGNR1-mediated signalling

Dendritic cells recognize pathogens through pattern recognition receptors such as Toll-like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross-talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C-type lectin SIGNR1 ligand lipoarabinomannan and the Toll-like receptor 2 ligand FSL-1. The co-stimulation significantly suppressed FSL-1-mediated activation of NF-κB as well as production of TNF-α, IL-6 and IL-12p40 in a dose-dependent manner. The suppression was significantly but not completely recovered by knock-down of SIGNR1. SIGNR1 was associated with Toll-like receptor 2 in XS106 cells. The co-stimulation upregulated the expression of suppressor of cytokine signalling-1 in XS106 cells, the knock-down of which almost completely recovered the suppression of the FSL-1-mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88-adaptor-like protein in XS106 cells was degraded by co-stimulation with FSL-1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock-down of suppressor of cytokine signalling-1. This study suggests that Toll-like receptor 2-mediated signalling is negatively regulated by SIGNR1-mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling-1-mediated degradation of the MyD88-adaptor-like protein.     

CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.