Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.     

Gene therapy for pancreatic cancer targeting the genomic alterations of tumor suppressor genes using replication-selective oncolytic adenovirus

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we examined the genetic alternations of pancreatic cancer. Based on these results, we are developing a new gene therapy targeting the genetic character of pancreatic cancer using mutant adenoviruses selectively replication-competent in tumor cells. Loss of heterozygosity (LOH) of 30% or more were observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed the significant association with poor prognosis. These data strongly suggest that mutation of the putative suppressor genes, TP53 and SMAD4 play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy targeting tumors without normal TP53 function. E1B-55kDa-deleted adenovirus (AxE1AdB) can selectively replicate in TP53-deficient human tumor cells but not cells with functional TP53. We evaluated the therapeutic effect of this AxE1AdB on pancreatic cancer without normal TP53 function. The growth of human pancreatic tumor in SCID mice model was markedly inhibited by the consecutive injection of AxE1AdB. Furthermore, AxE1AdB is not only the strong weapon but also useful carrier of genes possessing anti-tumor activities as a virus vector specific to tumors without normal TP53 function. It was reported that uracil phosphoribosyl transferase (UPRT) overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits Thymidylate Synthetase (TS). The therapeutic advantage of restricted replication competent adenovirus that expresses UPRT (AxE1AdB-UPRT) was evaluatedin an intra-peritoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the adenovirus, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. These results revealed thatthe AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.     

Spectroscopic evidence that salicylic acid converts a temporally inactivated form of horseradish peroxidase (compound III) to the irreversibly inactivated verdohemoprotein (P-670)

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-catalyzed reactions are discussed.     

Domain-specific olivocerebellar projection regulated by the EphA-ephrin-A interaction

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. Here we show that in the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains.     

MotX and MotY,specific components of the sodium-driven flagellar motor,colocalize to the outer membrane in Vibrio alginolyticus

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery

An a-Amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipitation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and 60 degrees C. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had alpha-anomeric forms, as determined by 1H-NMR. This maltose-forming alpha-Amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62% respectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.     

Construction of BAC libraries derived from the ascidian Ciona intestinalis

Large insert genomic bacterial artificial chromosome (BAC) libraries were constructed from a basal chordate, the ascidian Ciona intestinalis. Insert analyses of randomly selected clones indicated that in the first library the mean insert size was 135 kb and predicted a 15-fold coverage of the Ciona genome, and in the second library the mean insert size was 165 kb and predicted a 5-fold coverage of the genome. These first large insert genomic libraries of the ascidian should increase the speed of genomic analyses of basal chordates.