Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Optimization of dose of collagen hydrolysate to prevent UVB-irradiated skin damage

Collagen hydrolysate (CH) was orally administered to UVB-irradiated hairless mice at doses of 20, 200–2000 mg/kg BW/day. The low dose of CH increased the skin hydration and reduced the transepidermal water loss on damaged skin. These results suggested the optimal dose of collagen to improve the UV-damaged skin condition.     

Eosinophilic globule cells in mouse MFH-like sarcomas

Light and electron microscopic observations were made on eosinophilic globule (EG) cells found in 27 subcutaneous malignant fibrous histiocytoma (MFH)-like sarcomas in aged ICR mice. These tumors, which were composed of fibroblast-like cells as the major component, with small numbers of histiocyte-like cells and undifferentiated cells, showed one or more of five growth patterns: storiform, pleomorphic, fascicular, myxoid, and hemangiopericytoma-like. EG cells were interspersed among the tumor cells and were also present in metastatic lesions. They were pleomorphic in shape and contained various numbers of cytoplasmic globules, which were positive with the periodic acid-Schiff reaction and resistant to diastase digestion. Electron microscopic observation revealed that these cells had small finger-like and pseudopodia-like projections, and contained varied numbers of characteristic osmiophilic globules and glycogen particles in the cytoplasm which seemed to become more abundant as the cells differentiated. The osmiophilic globules consisted of a dense homogeneous core and a marginal area rich in small vesicular membranous structures. A small population of EG cells exhibited features suggesting differentiation to fibroblasts; these were characterized by an increased amount of rough endoplasmic reticulum. It is suggested that the EG cell is a neoplastic cell, perhaps derived from a primitive mesenchymal cell, although an inflammatory cell origin is also possible.     

Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue

A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)     

The primary structure of skeletal muscle myosin heavy chain: III. Sequence of the 22 kDa fragment and the alignment of the 23 kDa, 50 kDa, and 22 kDa fragments

The amino acid sequence of the 197-residue 22 kDa fragment from chicken pectoralis muscle was determined to be as follows: K-K-G-S-S-F-Q-T-V-S-A-L-F-R-E-N-L-N-K-L- M-A-N-L-R-S-T-H-P-H-F-V-R-C-I-I-P-N-E-T-K-T-P-G-A-M-E-H-E-L-V-L-H-Q-L-R- C-N-G-V- L-E-G-I-R-I-C-R-K-G-F-P-S-R-V-L-Y-A-D-F-K-Q-R-Y-R-V-L-N-A-S-A-I-P-E-G-Q- F-M-D-S- K-K-A-S-E-K-L-L-G-S-I-D-V-D-h-T-Q-Y-R-F-G-H-T-K-V-F-F-K-A-G-L-L-G-L-L-E- E-M-R-D- D-K-L-A-E-I-I-T-R-T-Q-A-R-C-R-G-F-L-M-R-V-E-Y-R-R-M-V-E-R-R-E-S-I-F-C-I- Q-Y-N-V-R-S-F-M-N-V-K-H-W-P-W-M-K-L-F-F-K, where h stands for 3-N-methylhistidine. The amino acid sequences of the 22 kDa fragment and its equivalent fragment from chicken ventricle and gizzard muscle myosins were also determined by our group. Predicted secondary structures of these 22 kDa fragment regions and of the reported chicken embryo myosin revealed some possible structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbodiimide-catalyzed cross-linking sites in the heads of gizzard heavy meromyosin attached to F-actin

In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of mitogen activated protein kinase (MAPK) during D-galactosamine intoxication in the rat liver

A significant increase in plasma glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase was observed 6 h after intraperitoneal administration of D-galactosamine (D-Galn). Three hours after administration of D-Galn, the vitamin C concentration in the liver decreased significantly compared to that in a control group and thereafter the hepatic vitamin C concentration remained at a significantly lower level. Phosphorylated JNK (c-Jun NH2-terminal kinase) and phosphorylated ERK (extracellular signal-regulated kinase) started increasing 3 h after D-Galn treatment and remained at a high level for 6-12 h after the treatment, while phosphorylated p38 MAPK increased significantly 6 h after D-Galn administration. These results indicated that oxidative stress and the activation of JNK and ERK took place almost simultaneously, followed by the activation of p38 MAPK.     

Intravesical administration of γδ T cells successfully prevents the growth of bladder cancer in the murine model

Background  Superficial bladder cancers are usually managed with transurethral resection followed by the intravesical administration of Bacillus Calmette-Guerin which requires major histocompatibility complex (MHC) class I expression on cancer cells. Since cancer cells often loose MHC expression, a novel immunotherapy such as MHC-unrestricted γδ T cell therapy is desired. Objective  To clarify the relationship between the expression of MHC class I and clinicopathological features in bladder cancer patients, and investigate the effects of the administration of intravesical γδ T cells on bladder cancer. Methods  Samples from 123 patients who had undergone either transurethral resection or radical cystectomies were examined for MHC expression and the relationship between this and the clinicopathological features was analyzed statistically. The in vitro and in vivo effects of γδ T cells expanded by zoledronic acid (ZOL) against several types of cancer cell line and an orthotopic bladder cancer murine model which was pretreated with ZOL were investigated. Results  MHC-diminished superficial bladder cancer was significantly more progressive than MHC-conservative bladder cancer (P = 0.047). In addition, there was a significant association between diminished MHC expression and poor disease free survival (P = 0.041) and overall survival (P = 0.018) after radical cystectomy. In vitro, all of the cell lines pretreated with 5-μM ZOL showed a marked increase in sensitivity to lysis by γδ T cells. Moreover, intravesical administration of γδ T cells with 5-μM ZOL significantly demonstrated antitumor activity against bladder cancer cells in the orthotopic murine model (P < 0.001), resulting in prolonged survival. Conclusion  The present murine model provides a potentially interesting option to develop immunotherapy using γδ T cells for bladder cancer in human. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Yuasa and K. Sato contributed equally to the study.     

A single amino acid substitution converts gamma-glutamyltranspeptidase to a class IV cephalosporin acylase (glutaryl-7-aminocephalosporanic acid acylase)

The aspartyl residue at position 433 of gamma-glutamyltranspeptidase of Escherichia coli K-12 was replaced by an asparaginyl residue. This substitution enabled gamma-glutamyltranspeptidase to deacylate glutaryl-7-aminocephalosporanic acid, producing 7-aminocephalosporanic acid, which is a starting material for the synthesis of semisynthetic cephalosporins.