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Biomedical subjects

H Takata

Publications and source records attributed to H Takata.

At least 19 recordsLinked to original sources
PubMed1998

Cyclic glucans produced by the intramolecular transglycosylation activity of potato D-enzyme on amylopectin.

Potato D-enzyme catalyses an intramolecular transglycosylation reaction on amylose to produce cycloamylose, a novel cyclic alpha-1, 4 glucan. To determine if a similar activity could be observed with a high molecular weight branched substrate, recombinant potato D-enzyme was incubated with amylopectin. The substrate was converted into two fractions of lower molecular mass. Fraction I comprised 15% cyclic molecules of which the majority contained both alpha-1,4 and alpha-1,6 links. These were shown to be branched molecules with branches shorter than those in amylopectin. Fraction II comprised 80% cyclic molecules of which the majority contained only alpha-1,4 links (cycloamylose). Since fraction II appeared before fraction I, we propose that D-enzyme first catalysed the cyclisation of the outer side chains of amylopectin and then the cyclisation of inner chains to produce branched clusters. These results demonstrate that D-enzyme can catalyse the transfer of branched glucans, and suggest novel ways in which it may participate in starch metabolism in plants.

Amylopectin↗
PubMed1998

L* protein of the DA strain of Theiler's murine encephalomyelitis virus is important for virus growth in a murine macrophage-like cell line.

Strain GDVII and other members of the GDVII subgroup of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and cause acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. On the other hand, strain DA and other members of the TO subgroup of TMEV are less virulent and establish a persistent infection in the spinal cord, which results in a demyelinating disease. We previously reported that GDVII does not actively replicate in a murine macrophage-like cell line, J774-1, whereas DA strain productively infects these cells (M. Obuchi, Y. Ohara, T. Takegami, T. Murayama, H. Takada, and H. Iizuka, J. Virol. 71:729-733, 1997). In the present study, we used recombinant viruses between these strains of the two subgroups to demonstrate that the DA L coding region of DA strain is important for virus growth in J774-1 cells. Additional experiments with a mutant virus indicate that L* protein, which is synthesized out of frame with the polyprotein from an additional alternative initiation codon in the L coding region of TO subgroup strains, is a key determinant responsible for the cell-type-specific restriction of virus growth. L* protein may play a critical role in the DA-induced restricted demyelinating infection by allowing growth in macrophages, a major site for virus persistence.

Animals↗
PubMed1998

[Experimental study of percutaneous hot ethanol injection therapy (PHEIT) by continuous heating device for hepatocellular carcinoma].

Percutaneous ethanol injection therapy (PEIT) is widely used as a local treatment for hepatocellular carcinoma (HCC). However, because only a small amount of ethanol can be used in one PEIT session and because the antitumor effect is limited, this modality is indicated only when there are three or fewer tumors and when the tumor diameter is < or = 3 cm. To obtain a more potent and certain antitumor effect, we have devised a new treatment called percutaneous hot ethanol injection therapy (PHEIT), and developed a Continuous Heating Device with which ethanol can be heated and locally injected at a specified temperature. The continuous Heating Device is composed of three major components: a syringe heater, a needle thermocontroller, and a needle tip thermosensor. A disposable syringe filled with liquid is inserted into the syringe heater, which heats the liquid to a desired temperature by adjusting the voltage. The needle thermocontroller is a puncture guide needle to which a heating device has been attached. The needle-tip thermosensor constantly measures, displays and records the temperature of the liquid at the needle tip during injection. Also, because the Continuous Heating Device is a closed-circuit system, there is no risk of accidental a fire, which ensures procedural safety. It is also possible to use this device to safely heat and inject a variety of other liquids, such as physiological saline and anticancer agents and thus contribute to the widespread development of ultrasound-guided injection therapy.

Animals↗
PubMed1997

Cyclodextrins are not the major cyclic alpha-1,4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose.

The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha-, beta-, and gamma-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic alpha-1, 4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha-1,4-glucans and into the final major product, beta-CD. CGTase from Bacillus macerans also produced large cyclic alpha-1, 4-glucans except that the final major product was alpha-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.

Amylose↗
PubMed1997

The linear plasmid pDHL1 from Debaryomyces hansenii encodes a protein highly homologous to the pGKL1-plasmid DNA polymerase.

Both the linear plasmids, pDHL1 (8.4 kb) and pDHL2 (9.2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15.0 kb) in the same host cell for their replication. A 3.5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3.5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4.2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the alpha subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5' non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids.

Amino Acid Sequence↗
PubMed1997

Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Amino Acid Sequence↗
PubMed1997

Influence of distal nerve segment volume on nerve regeneration in silicone tubes.

The influence of a volume of a distal nerve segment upon nerve regeneration in an 8-mm gap created within a silicone tube was examined. The rats were randomly divided into four groups. Each group had 5 mm, 1 mm, or a half volume of 1-mm nerve segment (a nerve piece of 1 mm transected longitudinally) inserted into the distal end of a silicone tube of 11 mm. The empty group without a nerve segment was used as control. Diameters of regenerated cylindrical structure between the nerve ends in the silicone tube were measured under an operation microscope and myelinated axon diameter, myelinated axon density, myelin sheath width, and ratio of myelinated axon area to total cross sectional area were measured using the transverse sections at the midpoint of the silicone tube at 6 weeks after surgery. Although there was a significant difference in all of those parameters between the control group and any of the remaining three groups, no significant difference was found between any pair of these three groups. The results of this study indicated that the degree of nerve regeneration does not correlate with the volume of a distal nerve segment and even a very small piece can play an important role in supporting regenerating nerve beyond a definitive gap.

Animals↗
PubMed1997

Histomorphometric analysis of the response of rat tibiae to shape memory alloy (nitinol).

The bone reaction to nitinol (Ni-Ti), a metal with shape memory, and other materials inserted transcortically and extending into the medullary canal of rat tibiae was quantitatively assessed using an image processing system. The materials examined were implants, all of the same shape and size, composed of nitinol, pure titanium (Ti), anodic oxidized Ti (AO-Ti), a titanium alloy (Ti-6Al-4V) and pure nickel (Ni). While the other four implant materials were progressively encapsulated with bone tissues, Ni was encapsulated with connective tissues through the 168-day experimental period, and the Ni implants showed no bone contact at any time during the experimental period. Histometric analysis revealed no significant difference among the tissue reactions to Ti, AO-Ti and Ti-6Al-4V, but Ni-Ti implants showed significantly (P < 0.01) lower percentage bone contact and bone contact area than any of the other titanium or titanium alloy materials.

Alloys↗
PubMed1997

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1997

Effect of tPA on regional lung perfusion in unilobar canine pulmonary thromboembolism.

We investigated effects of tissue-type plasminogen activator (tPA) on regional pulmonary arterial hemodynamics in pulmonary thromboembolism (PTE) in a canine model of unilobar PTE. Ten beagle dogs were divided into two groups-tPA (n = 5) and control group (n = 5). In each dog an artificial blood circuit (ABC) consisting of a silicone tube and a cannulation-type electromagnetic blood flowmeter probe was placed at the left lower pulmonary artery. A unilobar PTE was induced by placing autologous clots into a metallic coil inside the ABC. The CO2 sampling tubes were positioned at the orifice of the left lower bronchus and the trachea, and the end-expiratory CO2 partial pressure (PET(CO2)) was measured. In the tPA group, blood flow at the left lower pulmonary artery (LL-flow) was improved to near baseline within approximately 30 min of receiving tPA, and PET(CO2) at the left lower bronchus (LL-PET(CO2)) increased in direct correlation with LL-flow. The hemodynamic improvement after tPA therapy correlated with the partial pressure of the regional pulmonary expiratory CO2. Moreover, it was suggested that changes in physiologic conditions in PTE were not determined by clot quantity alone.

Animals↗
PubMed1996

Structure of the cyclic glucan produced from amylopectin by Bacillus stearothermophilus branching enzyme.

The thermostable branching enzyme (BE, EC 2.4.1.18) from Bacillus stearothermophilus TRBE14 produces large cyclic glucans from waxy rice amylopectin similar to those obtained from amylose as described elsewhere [H. Takata, T. Takaha, S. Okada. M. Takagi, and T. Imanaka, J. Bacteriol., 178 (1996) 1600-1606]. The structure of the product (P-1) from the late-stage reaction was analyzed in detail. The weight-average degree of polymerization (dpw) of P-1 was 900. Its chain-length distribution was not significantly changed compared with that of amylopectin, although the amount of long chains (dp > 38) was slightly decreased. The cyclic component of P-1, which was isolated by the extensive action of glucoamylase, had dpw of 49. Three point five alpha-1,6 linkages were directly involved in the formation of the ring structure with several non-cyclic side chains linked to the ring. Based on these results, the action and new roles of BE are discussed.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center.

The substrate specificity and the transglycosylation activity of neopullulanase was altered by site-directed mutagenesis on the basis of information from a three-dimensional structure predicted by computer-aided molecular modeling. According to the predicted three-dimensional structure of the enzyme-substrate complex, it was most likely that Ile-358 affected the substrate preference of the enzyme. Replacing Ile-358 with Trp, which has a bulky side chain, reduced the acceptability of alpha-(1-->6)-branched oligo- and polysaccharides as substrates. The characteristics of the I358W-mutated enzyme were quite different from those of wild-type neopullulanase and rather similar to those of typical starch-saccharifying alpha-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for alpha-(1-->6)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlled by manipulating the hydrophobicity around the attacking water molecule, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, and Ser-422, which were located on the entrance path of the water molecule might be involved. The transglycosylation activity of neopullulanase was increased by replacing one of the three residues with more hydrophobic amino acid residues; Y377F, M375L, and S422V. In contrast, the transglycosylation activity of the enzyme was decreased by replacing Tyr-377 with hydrophilic amino acid residues, Asp or Ser.

Amino Acid Sequence↗
PubMed1996

Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan.

Potato D-enzyme was purified from recombinant Escherichia coli, and its action on synthetic amylose (average Mr of 320,000) was analyzed. D-enzyme treatment resulted in a decrease in the ability of the amylose to form a blue complex with iodine. Analysis of the products indicated that the enzyme catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic alpha-1,4-glucan (cycloamylose). Confirmation of the cyclic structure was achieved by demonstrating the absence of reducing and nonreducing ends, resistance to hydrolysis by glucoamylase (an exoamylase), and by "time of flight" mass spectrometry. The degree of polymerization of cycloamylose products was determined by time of flight mass spectrometry analysis and by high-performance anion-exchange chromatography following partial acid hydrolysis of purified cycloamylose molecules and was found to range from 17 to several hundred. The yield of cycloamylose increased with time and reached >95%. D-enzyme did not act upon purified cycloamylose, but if glucose was added as an acceptor molecule, smaller cyclic and linear molecules were produced. The mechanism of the cyclization reaction, the possible role of the enzyme in starch metabolism, and the potential applications for cycloamylose are discussed.

Chromatography, Gel↗
PubMed1996

Cyclization reaction catalyzed by branching enzyme.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1995

[The relationship between blood sugar control and vascular complications in elderly diabetic patients--investigative study in 17 hospitals of Kochi Prefecture. Kochi Working Group on Diabetes Mellitus].

The aim of this study was to clarify whether the control state of fasting blood sugar can influence the occurrence of diabetic microangiopathy and macroangiopathy even in elderly patients with diabetes mellitus. In Kochi Prefecture 18 internal physicians participated in evaluating clinical courses of 898 patients, consisting of 466 males and 432 females, for an average of 69 months. Elderly cases aged 65 years or more old (group 1) consisted of patients who were aged 71.8 +/- 5.2 years old (M +/- SD). The average age of 481 adult cases under 65 years of age (group II) was 54.4 +/- 8.4 years old. Between the good and poor control groups, there was no difference in terms of blood pressure, body mass index (BMI) and serum lipids. Arteriosclerotic diseases such as myocardial infarction, cerebral infarction and arteriosclerosis obliterans appeared about as frequently in both the good and the poor control groups, while microangiopathies such as retinopathy, nephropathy and neuropathy were significantly more frequent in the poor control group compared to the good control group. The same tendency concerning these complications was seen in group II. Concerning treatment, diet treatment without drug treatment was significantly more frequent in the good control group compared to the poor control group, while hypoglycemic agents and subcutaneous insulin injection were used more often in the poor control group, the more severe state of diabetes mellitus in the latter group. Concerning the main reasons for good control, successful diet treatment was cited most often, followed by regular intake of medications. On the other hand disturbed diet treatment was the most frequent reason for poor control, indicating the strategic importance of diet treatment. Arteriosclerotic diseases were found more often in group I than group II, while the frequency of microangiopathies was similar. Concerning sexes, male patients tended to suffer more often from arteriosclerotic diseases than female patients, but the frequency of microangiopathy was similar. From the above findings it was concluded that poor control of fasting blood sugar level was related to microangiopathies in both non-elderly adult and elderly patients.

Aged↗
PubMed1994

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

1,4-alpha-Glucan Branching Enzyme↗