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Biomedical subjects

H Matsuzawa

Publications and source records attributed to H Matsuzawa.

At least 19 recordsLinked to original sources
PubMed1995

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47.

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Amino Acid Sequence↗
PubMed1995

Analysis of target organs for the latency of murine cytomegalovirus DNA using specific pathogen free and germfree mice.

Cytomegalovirus (CMV) establishes a latent infection in its host; however, the organ sites of viral latency and its mechanism still remain to be fully clarified. To elucidate this issue, a latent infection with murine (M) CMV was attempted to induce in mice and the organ sites of the latent viral genome were examined for more than one year by a polymerase chain reaction (PCR). As a result, latent MCMV DNA was detectable in both the lung and the spleen as late as 59 weeks after infection. The heart was also observed to be a target organ of latent MCMV DNA, though the amount of viral DNA was much less than that seen in the lung and spleen. In germfree (GF) mice, on the other hand, no such latent viral DNA was observed in the spleens, while it was seen, but to a significantly smaller degree, in the lungs and the hearts than in the same organs of specific pathogen-free (SPF) mice. The amount of infectious virions generated in the host appeared to be almost equal between the GF and SPF mice. The above findings therefore suggest that the spleen, lung and heart are target organs for MCMV latency and the indigenous bacterial flora, which are not colonizing in GF mice, play an important role in the establishment of such viral latency in SPF mice.

Amino Acid Sequence↗
PubMed1995

Three-dimensional anisotropy contrast magnetic resonance imaging of the rat nervous system: MR axonography.

Anisotropy of apparent diffusivities (Dapp) of water molecules is known to be an excellent means for elucidating detailed information regarding neuronal fiber direction and density in live subjects (axonography). Nevertheless, determination of eigenvalues and eigenvectors of the apparent diffusion tensor, D xi app, the necessary process for the treatment of D xi app in axonography, is cumbersome and the quality of the reconstructed images based on estimated parameter values of D xi app has often been disappointing. We present here a simple algorithm which allows for the extraction of the anisotropic term vector, DAapp, from D xi app in pictorial form, which in turn provides a novel color-coded contrast for magnetic resonance imaging (MRI). MRI images constructed using this technique, three-dimensional anisotropy contrast (3DAC), provided impressive anatomic resolution of the CNS, comparable to in vitro histology. Directional information of DAapp expressed by hue of color clearly correlated to fiber direction, while magnitude of DAapp expressed by intensity of color correlated to fiber diameter. Magnetic resonance axonography (MRX) utilizing 3DAC technique possesses significant potential for various biological and clinical applications.

Algorithms↗
PubMed1995

Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter.

The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.

Bacteriophage T7↗
PubMed1995

Molecular cloning of the genes for pyruvate kinase of two bacilli, Bacillus psychrophilus and Bacillus licheniformis, and comparison of the properties of the enzymes produced in Escherichia coli.

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.

Amino Acid Sequence↗
PubMed1995

Magnetic resonance axonography of the rat spinal cord: postmortem effects.

The recent development of magnetic resonance (MR) axonography, which uses three-dimensional anisotropy contrast (3DAC), a new algorithm for the treatment of an apparent diffusion tensor, has provided an unprecedented opportunity for visualizing the anatomical details of the spinal cord in live animals. In this study, the authors investigated the sensitivity of the 3DAC method in detecting pathological conditions by obtaining chronological MR axonography of the rat spinal cord immediately after induction of cardiac arrest. The results clearly demonstrated that 3DAC is highly sensitive to any perturbation of physiological conditions. Trichromatic coefficient analyses indicated postmortem changes observed pictorially are indeed due to loss of anisotropy. The study further indicated the presence of at least two independent factors responsible for observed physiological anisotropy. Considering its rather simple implementation process and high anatomical resolution as well as its sensitivity to pathological alteration, MR axonography based on the 3DAC method appears to be the ideal noninvasive imaging technique for assessment of the spinal cord in biomedicine.

Animals↗
PubMed1994

Fermentation conditions for efficient production of thermophilic protease in Escherichia coli harboring a plasmid.

Escherichia coli TG1, transformed with an expression plasmid pAQN carrying the aqualysin I (AQI) gene derived from Thermus aquaticus YT-1 under the control of the tac promoter, was cultivated under various conditions in order to find fermentation conditions for the efficient production of the thermophilic protease, AQI. The amount of AQI produced was closely related to the growth phase at the time of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, and the highest production was obtained when it was added during the exponential growth phase. The addition of yeast extract had a greater effect on AQI production than did Polypeptone or casamino acids, and AQI productivity increased from 1.1 x 10(3) kU/g to 2.7 x 10(3) kU/g cells when 2 g/l yeast extract was supplied. Furthermore, the specific growth rate improved from 0.35 h-1 to 0.89 h-1 when 5 g/l yeast extract was supplied. The culture temperature also affected AQI gene expression. When the temperature was shifted from 37 degrees C to 34 degrees C at the time of IPTG induction, 19 kU/ml enzymatically active AQI was obtained, corresponding to a 28% increase over the amount produced in a batch culture without a shift. This is about a 44-fold higher yield than was obtained from the original strain, T. aquaticus YT-1.

Biotechnology↗
PubMed1994

Carboxypeptidase Taq, a thermostable zinc enzyme, from Thermus aquaticus YT-1: molecular cloning, sequencing, and expression of the encoding gene in Escherichia coli.

The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Taq prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases. However, a His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Taq is a novel type of zinc-dependent metallocarboxypeptidase.

Amino Acid Sequence↗
PubMed1992

Conformational equilibrium of an enzyme catalytic site in the allosteric transition.

The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.

Allosteric Regulation↗
PubMed1992

A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli.

The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.

Base Sequence↗
PubMed1992

Glutamyl-tRNA synthetase from Thermus thermophilus HB8. Molecular cloning of the gltX gene and crystallization of the overproduced protein.

The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.

Adenosine Triphosphate↗
PubMed1992

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.

Amino Acid Sequence↗
PubMed1992

[Clinical application of phase contrast angiography].

"Phase Contrast Angiography" is a new technique of Magnetic Resonance Angiography as reported by Dumoulin CL et al. Using this technique, we can obtain images of vessels (angiograms) without injection of contrast medium. We present the results of phase contrast angiography on cerebral and spinal vascular disease. We utilized the General Electric SIGNA, 1.5 tesla NMR. One hundred and ninety-one cases of cerebral and spinal vascular diseases were scanned using phase contrast angiography. Included were 90 cases of occlusive vascular disease, 16 cases of moyamoya disease, 39 cases of arteriovenous malformation, and 28 aneurysms. The phase contrast angiography uses flow encoding gradient pulses, which impart a velocity-dependent phase shift to the transverse magnetization of moving spins. The resulting image contains only information from the moving spins; while information from stationary tissue remains suppressed. In cases using 3-D angiogram, we made 32 images 6 degrees apart in their projection direction and displayed them on a video terminal. We were able to visualize occlusions of vessels, aneurysms, bypassed vessels, and abnormal vessels of arteriovenous malformations. Retrospective evaluation comparing phase contrast angiography with conventional angiography of the stenotic findings on the horizontal portion of middle cerebral arteries (64 vessels of 32 patients), resulted in a false positive ratio of 39.1%. We obtained clinically valuable results regarding the hydrodynamics of patients using "phase contrast angiography" non-invasively. These results reveal not only "anatomical" images of vessels, but also "functional" images, which are sensitive to the pattern of the blood flow. This study would strongly suggest that phase contrast angiography presents a valuable tool for the clinical diagnosis of cerebral and spinal vascular diseases.

Cerebral Angiography↗
PubMed1992

[Quantitative analysis of cerebrospinal fluid dynamics in syringomyelia using cine MRI with pre-saturation].

Some reports show the qualitative analysis of cerebrospinal fluid (CSF) pulsation in the subarachnoid space and the syrinx using cine magnetic resonance imaging (MRI). However, few reports studied the quantitative analysis of CSF pulsation. We report here the results of quantitative analysis of CSF pulsation using the cine MRI with pre-saturation pulse. Using flow phantom, we calibrated the correlation between the true velocity and calculated velocity acquired from the movement of pre-saturated low signal. Three cases of syringomyelia with Chiari malformation, two cases of traumatic syringomyelia, and three normal volunteers were examined using this technique, and we could obtain time-velocity curves of CSF in both subarachnoid spaces and syrinx. Although obvious pulsation of CSF was observed in the syrinx of all Chiari malformations, no pulsation of CSF was observed in the syrinx of traumatic syringomyelia. CSF in the syrinx moves upward in the early systolic cardiac cycle, downward in the systolic cycle, and upward again in the diastolic cycle. Patterns of CSF pulsation in syrinx were similar to those in the subarachnoid space, but "phase" of these pulsations differed from case to case. These results suggest the close relationship between CSF pulsation in the syrinx and CSF obstruction at the cranio-vertebral junction. Dissociation of the "phase" of CSF pulsation in the syrinx and subarachnoid space may be significant in the analysis of the pathogenesis of syringomyelia.

Adult↗
PubMed1991

Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin.

Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme.

Acylation↗
PubMed1991

Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8. Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis.

The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. By expression of the T. thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography. The amino acid sequence of T. thermophilus MetRS showed low identities (approximately 25%) with those of MetRSs from E. coli, and cytoplasm and mitochondria of Saccharomyces cerevisiae. However, the amino acid residues in the binding sites for ATP and the anticodon and the 3' terminus of tRNA(Met) are highly conserved among the four MetRSs. T. thermophilus MetRS has a zinc finger-like sequence with all the three cysteine residues and a histidine residue. By site-directed mutagenesis of one of the cysteine residues (Cys127) of T. thermophilus MetRS, the SH group was found to be important for methionyl-tRNA synthesis. Just upstream of the structural gene for T. thermophilus MetRS there is a short open reading frame which codes for a methionine-rich peptide and is partly overlapped with an alternative terminator/antiterminator structure, suggesting that transcription of this gene is regulated by attenuation. Further upstream a region contains a nucleotide sequence homologous to that of the 5' half of T. thermophilus initiator tRNA(Met).

Amino Acid Sequence↗