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Shigeharu Harada

Publications and source records attributed to Shigeharu Harada.

15 recordsLinked to original sources
PubMed2006

Simple colorimetric inhibition assay of heme crystallization for high-throughput screening of antimalarial compounds.

Current assays for screening new antimalarials need initiators of beta-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in beta-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of beta-hematin formation.

Animals↗
PubMed2006

Expression, purification and crystallization of L-methionine gamma-lyase 2 from Entamoeba histolytica.

L-Methionine gamma-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 A. The crystal diffracted to a resolution of 2.0 A. The presence of a tetramer in the asymmetric unit (4 x 43.1 kDa) gives a Matthews coefficient of 2.2 A(3) Da(-1). The structure was solved by the molecular-replacement method and structure refinement is now in progress.

Animals↗
PubMed2006

Interaction of subtilisin BPN' and recombinant fungal protease inhibitor F from silkworm with substituted P1 site residues.

Fungal protease inhibitor F (FPI-F) from silkworm inhibits subtilisin and fungal proteases. FPI-F mutants P(1) residues of which, Thr(29), were replaced with Glu, Phe, Gly, Leu, Met, and Arg, were prepared. The inhibitory activities of mutated FPI-F against subtilisin and other mammalian proteases indicated that FPI-F might be a specific inhibitor toward subtilisin-type protease.

Amino Acid Sequence↗
PubMed2006

Serine proteinase inhibitor from wax gourd (Benincasa hispida [Thunb] Cogn.) seeds.

Two squash family protease inhibitors were obtained from wax gourd (Benicasa hispida [Thunb] Cogn.). Even though they were distinctly separated by reversed-phase chromatography, the amino acid sequences of two inhibitors were identical. Both inhibitors were converted into each other, perhaps due to cis-trans isomerization of characteristic Pro in the C-terminal region.

Amino Acid Sequence↗
PubMed2005

Inhibition assay of beta-hematin formation initiated by lecithin for screening new antimalarial drugs.

Measurement of heme crystallization provides a tool for screening new antimalarial drugs. Current assays for heme crystallization have employed initiators such as thermo, histidine-rich proteins, and lipids extracted from parasites and infected plasma. These initiators are unnatural or require laborious steps to prepare. In this study, we used a commercially available lipid, lecithin, a kind of phospholipid containing about 50% unsaturated fatty acids, as an initiator for heme crystal (beta-hematin) formation. We demonstrated that the inhibition of lecithin-based beta-hematin formation by antimalarial drugs is highly correlated with the preformed beta-hematin-based method. In addition, the lecithin-based assay is sensitive and convenient for large-scale screening of new novel antimalarials. We also indicated that dimethyl sulfoxide is an ideal solvent for preparation of heme stock solution, which is stable and can be used for 1 month.

Antimalarials↗
PubMed2005

Leukocyte activation by malarial pigment.

Malarial pigment, a unique hemozoin crystal composed of unit cells of heme dimers, is present in large amounts in circulating monocytes and neutrophils and can persist unchanged in macrophages for several months. In the present study, we investigated the effect of hemozoin not only on macrophages, but also on neutrophils. We used beta-hematin (BH), a chemically synthetic crystal structurally identical to hemozoin, for these studies. In vitro, BH up-regulated the expression of tumor necrosis factor-alpha in whole blood and in isolated peritoneal macrophages, indicating that hemozoin is able to stimulate monocytes. BH stimulated murine peritoneal neutrophils to express macrophage inflammatory protein-2 (MIP-2), a homologue of human interleukin-8 that is used as a marker of neutrophil activation. Injecting BH into the peritoneal cavity resulted in a dose-dependent migration of neutrophils and a high level of myeloperoxidase activity of peritoneal cells. Finally, BH directly induced neutrophil chemotaxis in vitro. Taken together, these results suggest that the malarial pigment hemozoin can activate leukocytes and may participate in the pathology of severe malaria.

Animals↗
PubMed2005

Chondroitin sulfate proteoglycan phosphacan associates with parallel fibers and modulates axonal extension and fasciculation of cerebellar granule cells.

Phosphacan is a nervous system-specific chondroitin sulfate proteoglycan and one of the major components of extracellular matrix in the brain. In the present study, we examined its spatiotemporal expression, ultrastructural localization, binding manner, and in vitro analysis on cell adhesion, axonal extension, and fasciculation in rat cerebellum. The present light microscopic immunohistochemistry showed that phosphacan immunoreactivity was localized mainly at the molecular layer in the cerebellum, but not at the external granular layer. Further double labeling immunohistochemical and immunoelectron microscopic studies revealed that phosphacan was localized around parallel fibers, but not at synapses. The binding of phosphacan to membrane and/or extracellular matrix partly required Ca2+ and was mediated through its core glycoprotein. Phosphacan inhibited adhesion and axonal extension of cerebellar granule cells in dissociated culture, while it promoted axonal fasciculation of their aggregated culture. These results indicate that phosphacan around parallel fibers may be the repulsive substratum for adhesion and extension of granule cells and promote the fasciculation of parallel fibers.

Animals↗
PubMed2005

Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

Aminomethyltransferase↗
PubMed2005

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries.

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

Combinatorial Chemistry Techniques↗
PubMed2003

Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex II (succinate-ubiquinone reductase).

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.

Adolescent↗
PubMed2002

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Animals↗
PubMed2002

Expression, purification, and characterization of AknX anthrone oxygenase, which is involved in aklavinone biosynthesis in Streptomyces galilaeus.

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.

Amino Acid Sequence↗