Characterization of the solution properties of Pichia farinosa killer toxin using PGSE NMR diffusion measurements

The solution behaviour with respect to pH and NaCl concentration of the tertiary structure and propensity for aggregation of salt- mediated killer toxin (SMKT) from Pichia farinosa was examined using pulsed-gradient spin-echo NMR diffusion measurements. It was found that in 0.15m NaCl the tertiary structure of SMKT was constant below pH 5.0, with the native SMKT existing as an unaggregated heterodimer containing the -subunit in a compactly folded form. However, above pH 5.0 the -subunit dissociated and lost its compact structure, becoming a random coil with an 37% increase in effective hydrodynamic radius. To determine the effects of NaCl concentration on the tertiary structure of SMKT, diffusion measurements were performed at pH 3.5 and NaCl concentrations up to 2M. Both the tertiary structure and aggregation state of SMKT were found to be insensitive to the salt concentration which indicates that the activity of the toxin is not a direct result of salt–protein interactions.     

Onset of cryptic vicariance in the Japanese dormouse Glirulus japonicus (Mammalia, Rodentia) in the Late Tertiary, inferred from mitochondrial and nuclear DNA analysis

The sequences of the mitochondrial cytochrome b gene and restriction site variation in the spacer region of the nuclear ribosomal RNA gene [rDNA-restriction fragment length polymorphism (RFLP)] were analysed to determine the phylogeographic structure of the Japanese dormouse ( Glirulus japonicus ), which is threatened by deforestation and has been designated an endangered species in Japan. The phylogenetic tree of cytochrome b grouped G. japonicus into six geographical populations: north-eastern Honshu (I), central Honshu (II), west-central Honshu/Kii Peninsula (III), western Honshu (IV), Shikoku (V), and westernmost Honshu/Kyushu (VI); the genetic distances among these groups suggest divergence in the Late Tertiary. The lineage of group VI was located at the basal position in the phylogenetic tree, followed by the radiation of the other lineages. An rDNA-RFLP analysis of 15 restriction sites roughly supported such genetic isolation; groups I, II, III, IV, V and VI have five, two, one, one, one and four unique restriction sites, respectively, revealing four geographic groups as cryptic species: I, II, III + IV + V and VI. Our results reveal the ancient divergences of the local population, which has a complicated evolutionary history, and should be useful in developing a framework for the conservation of this species.     

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.     

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.     

Characterization of a novel acid phosphatase from embryonic axes of kidney bean exhibiting vanadate-dependent chloroperoxidase activity

A novel colorless acid phosphatase (KeACP), which was distinct from the kidney bean purple acid phosphatase, was purified to apparent homogeneity and cloned from embryonic axes of kidney bean (Phaseolus vulgaris L. Ohfuku) during germination. When orthovanadate (VO(4)(-3)) is added to the apo form of the enzyme, KeACP uniquely exhibits the chloroperoxidase activity with loss of phosphatase activity. This is the first demonstration that KeACP is a vanadate-dependent chloroperoxidase in plants to be characterized and suggests that KeACP may play a role in modifying a wide variety of chlorinated compounds that are present in higher plants. The enzyme is a dimer that presents three forms made up of the combination of the dominant 56-kDa and the minor 45-kDa subunits, and both subunits contain carbohydrate. The full-length cDNA of the KeACP gene is 1641 nucleotides, and this sequence is predicted to encode a protein having 457 amino acid residues (52,865 Da), including a signal peptide. The complete nucleotide sequence of the genomic DNA (3228 bp) of KeACP consists of seven exons and six introns. Northern blot analysis demonstrated that the KeACP gene was expressed specifically in embryonic axes of the kidney bean, and its expression coincided with elongation of the embryonic axis during germination.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Involvement of ERK, a MAP kinase, in the production of TGF-beta by macrophages treated with liposomes composed of phosphatidylserine

We have already reported that TGF-beta could be involved in the inhibitory effects of negatively charged liposomes composed of phosphatidylserine (PS-liposome) on the production of nitric oxide (NO) by mouse peritoneal macrophages stimulated with LPS [Biochem. Biophys. Res. Commun. 281 (2001) 614]. In this paper, we explored the mechanism by which PS-liposomes promote the production of TGF-beta and the involvement of MAP kinases. When macrophages were treated with PS-liposomes, extracellular signal-regulated kinase (ERK), a member of MAP kinase superfamily, was activated quickly and potently. However, no activation was observed with p38 MAP kinase. TGF-beta production was completely inhibited by U0126, a specific inhibitor for ERK. Furthermore, TGF-beta neutralizing antibody and U0126 decreased the inhibitory effect of PS-liposomes on NO production by macrophages. These findings suggested that TGF-beta is the factor produced by PS-liposomes that suppresses production of NO, and the ERK signaling pathway is intimately involved in TGF-beta production by macrophages following treatment with PS-liposomes.     

Genetic polymorphims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients

BACKGROUND AND AIMS: Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV. METHODS: Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30IU/L (low-activity group), between 30 and 80IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80IU/L irrespective of hepatoprotective treatment (high-activity group). RESULTS: There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt -155, -443, -616, and -1748) were detected in the promoter region of OPN. Among them, the SNP at nt -443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D(') and r(2) greater than 0.937 to each other without correlation to disease activity. CONCLUSIONS. OPN promoter region SNP at nt -433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.