Biomedical subjects

Tohru Suzuki

Publications and source records attributed to Tohru Suzuki.

At least 19 recordsLinked to original sources

PubMed2004

Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita.

Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.

Alanine Transaminase↗

PubMed2004

Developmental roles of pufferfish Hox clusters and genome evolution in ray-fin fish.

The pufferfish skeleton lacks ribs and pelvic fins, and has fused bones in the cranium and jaw. It has been hypothesized that this secondarily simplified pufferfish morphology is due to reduced complexity of the pufferfish Hox complexes. To test this hypothesis, we determined the genomic structure of Hox clusters in the Southern pufferfish Spheroides nephelus and interrogated genomic databases for the Japanese pufferfish Takifugu rubripes (fugu). Both species have at least seven Hox clusters, including two copies of Hoxb and Hoxd clusters, a single Hoxc cluster, and at least two Hoxa clusters, with a portion of a third Hoxa cluster in fugu. Results support genome duplication before divergence of zebrafish and pufferfish lineages, followed by loss of a Hoxc cluster in the pufferfish lineage and loss of a Hoxd cluster in the zebrafish lineage. Comparative analysis shows that duplicate genes continued to be lost for hundreds of millions of years, contrary to predictions for the permanent preservation of gene duplicates. Gene expression analysis in fugu embryos by in situ hybridization revealed evolutionary change in gene expression as predicted by the duplication-degeneration-complementation model. These experiments rule out the hypothesis that the simplified pufferfish body plan is due to reduction in Hox cluster complexity, and support the notion that genome duplication contributed to the radiation of teleosts into half of all vertebrate species by increasing developmental diversification of duplicate genes in daughter lineages.

Animals↗

PubMed2004

Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) mediated PCR clamping.

Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57 degrees C and 45 degrees C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.

Base Pair Mismatch↗

PubMed2003

Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification.

The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.

Amino Acid Sequence↗

PubMed2003

Combination effects of chloroquine with the febrifugine and isofebrifugine mixture against a blood-induced infection with chloroquine-resistant Plasmodium berghei NK65 in ICR mice.

The combination effects of chloroquine with a mixture of febrifugine and isofebrifugine were evaluated against a blood-induced infection with chloroquine-resistant P. berghei NK65 in ICR mice. Mice in the untreated control showed a progressively increasing parasitemia leading to mouse death. A two-day dosage of 20 mg base/kg of chloroquine alone showed little effect against P. berghei NK65 infection, and all mice died from day 13 to 14 with an increasing parasitemia. A four-day dosage of 1 mg/kg of the febrifugine and isofebrifugine mixture alone showed a little antimalarial activity, but all mice died from day 19 to 27 with an increasing parasitemia. On the other hand, mice treated with chloroquine plus alkaloids survived during the experiment. All mice treated with chloroquine alone or the alkaloid mixture alone showed low parasitemia levels during a drug administration and following a few days, but then malaria parasites increased in the bloodstream of the treated mice until death. On the other hand, malaria parasites in the mice given chloroquine plus alkaloids decreased on day 6 and then were not detected by a microscopic examination during observation period.

Animals↗

PubMed2003

Removal of organic polyelectrolytes and their metal complexes by adsorption onto xonotlite.

Natural organic polyelectrolytes (humic and fulvic acids) and their metal complexes were removed by adsorption onto xonotlite. The removal percentages of humic and fulvic acids by xonotlite were approximately 80% and 30%, respectively. Humic acid removal from solution by adsorption onto xonotlite took place more readily than fulvic acid removal. The molecular weight distributions of the humic substances remaining in solution after adsorption with the xonotlite were measured with size exclusion chromatography. A comparison of molecular weight distributions demonstrated conclusively that large molecular weight components were adsorbed preferentially, indicating that adsorption efficiency depends on the number of functional groups of humic substances. Furthermore, the surface topography of the adsorbent was observed before and after adsorption by scanning electron microscopy. The calculated heat of adsorption was of 330 kJ mol(-1) which was evaluated from the Clapeyron-Clausius equation. Therefore, the adsorption type can be considered chemical. Since xonotlite can be easily synthesized and obtained at low cost, the adsorption method of humic and fulvic acids is superior to their precipitation.

Adsorption↗

PubMed2003

Identification of gastrin and multiple cholecystokinin genes in teleost.

To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.

Amino Acid Sequence↗

PubMed2003

Removal of heavy metals in rinsing wastewater from plating factory by adsorption with economical viable materials.

The removal of heavy metals from plating factory wastewater with economical materials was investigated by the column method. Montmorillonite, kaolin, tobermorite, magnetite, silica gel and alumina were used as the economical adsorbents to wastewater containing Cd(II), Cr(VI), Cu(II) and Pb(II). This removal method of heavy metals proved highly effective as removal efficiency tended to increase with increasing pH and decrease with increasing metal concentration. The removal percentages by adsorption onto montmorillonite, tobermorite, magnetite, and silica gel showed high values for all metals. From the results for the heat of adsorption, the adsorption process in the present study might be chemisorption. The proposed method was successfully applied to the removal of Cd(II), Cr(VI) and Cu(II) in rinsing wastewater from plating factory in Nagoya City, Aichi Prefecture, Japan. Since the economical adsorbents used can be obtained commercially because they are easily synthesized, the wastewater treatment system developed is rapid, simple and cheap for the removal of heavy metals.

Adsorption↗

PubMed2003

Molecular cloning and cDNA sequence analysis of carboxypeptidases A1, A2 and B from the Japanese flounder Paralichthys olivaceus.

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus. The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306-309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn(2+) ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.

Amino Acid Sequence↗

PubMed2003

Unequal distribution of otx1 mRnas among cleavage stage blastomeres in the teleost, Leucopsarion petersii (shiro-uo).

The otx genes belong to the orthodenticle gene family and play important roles in anterior brain development in vertebrates. We isolated two cDNA sequences, one homologous to human and zebrafish otxl and another homologous to zebrafish otx3, from the teleost Leucopsarion petersii (shiro-uo), which belongs to the family of gobies in the Perciformes. During embryogenesis of shiro-uo, otx1 and otx3 were expressed in the fore- and mid-brain throughout development in a manner similar to that observed in other vertebrates so far studied. However, otx-1 mRNA was also present at earlier stages and we obtained unique results using in situ hybridization and RT-PCR analysis demonstrating that otx-1 signals showed a distinct increase in the upper blastomeres, but not in the lower blastomeres, at the 8-cell stage. These stronger signals were maintained in the animal pole blastomeres during the 16-cell to 64-cell stages, followed by a gradual decrease during blastula stages. Such unexpected unequal distribution of otx1 mRNA revealed that blastomeres at early cleavage stages already showed non-equivalence in the embryogenesis of shiro-uo.

Amino Acid Sequence↗

PubMed2002

cDNA sequence and tissue expression of Fugu rubripes prion protein-like: a candidate for the teleost orthologue of tetrapod PrPs.

We report the isolation and characterization of a cDNA coding for Fugu rubripes prion protein (PrP)-like of 180 amino acids which includes the PrP-conserved hydrophobic region homologous to that of Xenopus PrP. In addition to the hydrophobic region, Fugu PrP-like has several features common to PrPs, such as a signal sequence, a basic nature (pI 9.7) and a single intron in the 5' untranslated region. A possible glycosyl phosphatidylinositol (GPI) anchor site also exists in PrP-like. In expression analysis, PrP-like mRNA was detected in retina, skin, and brain, all of which express PrP mRNA in mammals. In a genome fragment clone (T002589, 31945 bp) sequenced by the Fugu Genomics Project, PrP-like located between KIAA0168 and SLC231A homologues. In human chromosome 20p13, PrP, Doppel, KIAA0168, and SLC231A align in this order. The close gene arrangement between the Fugu and human genomes suggests that Fugu PrP-like is a real orthologue of human PrP. However, Fugu PrP-like does not possess tandem repeats or a region with two glycosylation sites and a disulphide bridge. We do not declare that the cloned Fugu PrP-like represents fish PrP due to structural inconsistency, but believe that it will offer new insights into the evolution of PrPs from fish to tetrapods.

Amino Acid Sequence↗

PubMed2002

Determination of manganese in herbal medicine samples by slurry-sampling electrothermal atomic absorption spectrometry with a metal tube atomizer.

An ultrasonic slurry-sampling electrothermal atomic absorption spectrometry with a molybdenum tube atomizer has been developed for the determination of manganese in herbal medicine samples. Ten percent glycerol solution was used as the slurry medium. The optimum pyrolysis temperature was 400 degrees C. The detection limit was 69 fg (3xS/N). Matrix element interference was checked and it was found that glycerol as a chemical modifier eliminated the interference. The amounts of manganese in herbal medicines determined by the proposed method are in good accordance with those measured in dissolved acid-digested samples. The method enables rapid calibration, and simple and rapid analysis of manganese in herbal medicine samples at low cost.

Herbal Medicine↗

PubMed2002

Development of neuropeptide Y-related peptides in the digestive organs during the larval stage of Japanese flounder, Paralichthys olivaceus.

cDNAs encoding three neuropeptide Y (NPY)-related peptides were identified from the Japanese flounder. One peptide corresponds to NPY, and its mRNA was expressed in the brain and retina. The second peptide corresponds to peptide YY (PYY), and its mRNA was expressed in the brain but surprisingly not in the digestive organs. This suggests that PYY lacks function as a digestive hormone in flounder. The third peptide corresponds to peptide Y (PY), and its mRNA occupied the brain and the endocrine cells of the intestine and the pancreatic accessory islets, but not of the principal islet. This suggests that PY has a dual role as a neuropeptide and a digestive hormone. In flounder larvae, PY cells appear in the intestinal epithelia at 3 days posthatching (dph), when larvae start feeding, in contrast to the endocrine pancreas at 30 dph. Therefore, the endocrine system of PY, which probably regulates the pancreatic enzyme secretion, develops synchronously with the start of feeding in the intestine but about 1 month later in the endocrine pancreas in flounder.

Amino Acid Sequence↗

PubMed2002

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.

Amino Acid Sequence↗

PubMed2002

mRNA expression of pancreatic enzyme precursors and estimation of protein digestibility in first feeding larvae of the Japanese flounder, Paralichthys olivaceus.

An understanding of digestibility in marine fish larvae is required to formulate a diet to replace zooplankton. Using flounder, this study was aimed at determining which digestive enzymes are synthesized in the larval pancreas, and how the proteins are cleaved in the digestive canal. Whole mount in situ hybridization indicated that the mRNA of all digestive enzyme precursors examined, including trypsin, chymotrypsin, elastase, carboxypeptidase A and B, and lipase, was expressed in the pancreas of first feeding larvae at 3 days post-fertilization. In the larvae before differentiation of the stomach, protein digestion in the digestive canal mainly depends on pancreatic proteases. So, to evaluate protein digestibility in the larval digestive canal, the digestion of proteins by pancreatic extract was monitored by gel electrophoresis. It was indicated that thyroglobulin, albumin and lactate dehydrogenase were rapidly cleaved to polypeptide fragments, but ferritin and catalase exerted resistance to proteolysis, suggesting that digestibility in the larval digestive canal differs depending on protein species.

Animals↗

PubMed2002

cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese flounder, Paralichthys olivaceus.

To better understand the digestive physiology and phylogeny of the pancreatic serine proteases of teleosts, we cloned trypsin, chymotrypsin and elastase from flounder (Paralichthys olivaceus). Fifty phage plaques randomly chosen from a flounder pancreatic cDNA library were found to contain three species of trypsin, two species of chymotrypsin and four species of elastase. cDNAs of two species of carboxypeptidase A, one carboxypeptidase B and lipase were also obtained. In total, 23 out of 24 digestive enzyme cDNAs were those of proteolytic enzymes. Such a high ratio of proteolytic enzyme cDNA in the pancreas may reflect the carnivorous feeding habits of flounder. A phylogenetic comparison of the peptide sequences of flounder enzymes with those of other teleosts and mammals suggested that duplication of trypsin, chymotrypsin and elastase occurred before the divergence of the ray finned fish. It is also hypothesized that functional descendants of both duplicated genes of elastase exist in the teleosts and mammals, whereas only one of the genes of trypsin and chymotrypsin gave rise to the functional descendants in the teleosts but not in the mammals.

Amino Acid Sequence↗

PubMed2002

A homologue of human placental protein, PP11, and mouse T cell-specific protein, Tcl-30, in exocrine pancreas of a teleost (Paralichthys olivaceus).

We cloned a 1401-bp cDNA encoding a novel pancreatic protein (PPSB) with two cysteine-rich somatomedin B-like domains from Japanese flounder (Paralichthys olivaceus). PPSB is predicted to be composed of 385 amino acids, including a signal sequence. The peptide sequence shares high homology with human placental protein 11 (PP11) and mouse T-cell specific protein (Tcl-30), which both contain a single somatomedin B-like domain. PPSB shares 47% and 44% identity with PP11 and Tcl-30, respectively. The unique point of PPSB is that an additional, somatomedin B-like domain is tandemly inserted. Unlike PP11 and Tcl-30, PPSB mRNA is specifically expressed by the exocrine pancreatic acinar cells, together with trypsinogen. Since PP11 has serine protease activity, it is predicted that its teleost homologue, PPSB, may function as a pancreatic digestive enzyme.

Amino Acid Sequence↗

PubMed2002

Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B. rapa.

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.

Amino Acid Sequence↗