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Biomedical subjects

Y Yamagata

Publications and source records attributed to Y Yamagata.

At least 73 records · Page 4Linked to original sources
PubMed1996

Three-dimensional structure of a DNA repair enzyme, 3-methyladenine DNA glycosylase II, from Escherichia coli.

The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

Amino Acid Sequence↗
PubMed1996

Ca2+/calmodulin-dependent protein kinase II in septally kindled rat brains: changes in protein level, activity and subcellular distribution in hippocampus and cerebral cortex.

The protein level and the activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in homogenate from septally kindled rat brains were quantitatively compared with those from paired controls 2 weeks after the final stimulation. The major alpha subunit level was decreased, while the activity was increased in crude homogenate from hippocampus and parietal cortex of kindled animals, indicating an apparent increase in the specific activity of CaM kinase II in these regions of the kindled brains. No such changes were observed in cerebellum. After the separation of crude homogenate into the soluble and particulate fractions, the ratio of CaM kinase II activity recovered in the soluble fraction was increased in hippocampus and parietal cortex, indicating a change in subcellular distribution of CaM kinase II in the kindled state.

Animals↗
PubMed1996

The structure, stability, and folding process of amyloidogenic mutant human lysozyme.

The physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.

Amyloidosis↗
PubMed1996

Syntheses and anti-inflammatory activities of O-acyloximes. II.

Novel O-acyloximes having an acetyl group or N-protected amino acid as an O-acyl group were synthesized by reaction with acetyl chloride or by a mixed anhydride method. 4'-Morpholinoacetophenone oxime (oxime-2) was determined to be the (E) isomer by X-ray crystal structure analysis. The anti-inflammatory activities of the test compounds were assessed in terms of the inhibitory effect on increased vascular permeability induced by histamine, and several compounds were assessed together by means of the carrageenan-induced paw edema assay. In general, acetyl oximes and tert-butyloxycarbonylphenylalanyl oximes showed inhibitory action on increased vascular permeability. Particularly important for the appearance of anti-inflammatory activity was direct attachment of the acetyl group to the oxime. Of the two isoforms of cyclooxygenase (COX-1 and COX-2), COX-1 activity was inhibited by oxime-2 and 4'-piperidinoacetophenone oxime (oxime-3) with IC50 values of 50 and 130 microM, respectively, while COX-2 activity was not inhibited. The in vitro inhibitory effect of oxime-2 and oxime-3 on COX-1 activity decreased with O-acylation of the oximes.

Animals↗
PubMed1996

A long-term follow-up study of asymptomatic hematuria and/or proteinuria in adults.

Between January 1, 1983 and December 31, 1992, 805 patients with asymptomatic proteinuria and/or hematuria were selected in the mass screening of 56,269 adults. We conducted prospective long-term follow-up studies of these patients and evaluated their clinical findings and renal histology. They were divided into three groups according to the first dipstick urinalysis findings: 478 patients with pure hematuria (H), 150 patients with concomitant hematuria and proteinuria (H & P), and 177 patients with proteinuria (P). The mean follow-up period was 5.80 +/- 4.42 years. Of the 478 patients with H, a specific cause of hematuria was found in 46 (9.6%), the remaining 432 (90.4%) patients were diagnosed as having asymptomatic hematuria (ASH). During the follow-up period, in the ASH patients, hematuria disappeared in 44.2%, 43.7% had persistent microhematuria without proteinuria, and 10.6% manifested proteinuria, none of the patients showed renal insufficiency. Of the 150 patients with H & P, 134 were diagnosed as having asymptomatic H & P. During the follow-up period, the hematuria and proteinuria disappeared in 16.4% of these patients, the proteinuria disappeared in 8.2%, and 14.9% of the patients showed renal insufficiency. Of the 177 patients with P, 151 were diagnosed has having asymptomatic P. During the follow-up period, proteinuria disappeared in 23.2%, and 10.6% showed renal insufficiency. Renal biopsy was performed in 151 patients in the study population who had a moderate degree of proteinuria; 68.2% of these patients had IgA nephropathy, 12.6% had non-IgA mesangial proliferative GN, 6.0% had membranous nephropathy, 5.3% had minimal change, and 2.6% had focal and segmental glomerular sclerosis. This study of the mass screening of urinalysis in asymptomatic adults showed that although the patients with pure hematuria did not exhibit renal insufficiency, 10.6% of these patients were proteinuric during the follow-up period. Therefore, careful observation and management are needed in these patients.

Adolescent↗
PubMed1995

Contribution of hydrophobic residues to the stability of human lysozyme: calorimetric studies and X-ray structural analysis of the five isoleucine to valine mutants.

In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynamic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional structure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at pH 2.7 revealed the following. (1) The stabilities of all the mutant proteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) The destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were identical to that of the wild-type protein, and only small structural rearrangements were observed locally around some of the mutation sites. The most striking change among the mutant proteins was found in the mutant protein, 159V, which contains a new water molecule in the cavity created by the mutation. The thermodynamic stabilities of the mutant proteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined.

Calorimetry, Differential Scanning↗
PubMed1995

The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21.

The gene for an alkaline serine protease from alkalophilic Bacillus sp. NKS-21 (subtilisin ALP I) was cloned, and its nucleotide sequence was determined. The gene (aprQ) contained an open reading frame of 1125 bp, encoding a primary product of 374 amino acids. The mature protease, composed of 272 amino acids, was preceded by a putative signal sequence of 37 amino acids and a pro-sequence of 65 amino acids. The mature protease conserved the catalytic triad, Asp, His, and Ser, as subtilisin BPN' or other subtilisins, and the subtilisin ALP I might belong to the subtilisin super family. The primary structure of subtilisin ALP I was compared and discussed with those of 13 subtilisins, 5 subtilisins from alkalophilic Bacillus, and 8 from neutrophiles. Low homology was shown between subtilisin ALP I and subtilisins from alkalophiles or subtilisins from neutrophiles. Forty-five amino acid residues of the mature protein of subtilisin ALP I were entirely independent of other subtilisins. According to the homology of ALP I with other subtilisins, subtilisin ALP I might be in the middle point between alkaline subtilisins and neutral ones.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis (natto) No. 16.

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.

Amino Acid Sequence↗
PubMed1995

A new alkaline serine protease from alkalophilic Bacillus sp.: cloning, sequencing, and characterization of an intracellular protease.

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.

Amino Acid Sequence↗
PubMed1995

Specific effect of magnesium ion on 2',3'-cyclic AMP synthesis from adenosine and trimeta phosphate in aqueous solution.

Phosphorylation of adenosine by trimetaphosphate was investigated using various catalysts in aqueous solution under mild conditions at pH approximately 7.0 and at 41 degrees C. The product was primarily 2',3'-cyclic AMP together with smaller amounts of ATP. Magnesium ion was found to have a remarkable catalytic effect of approximately one hundred times greater than the other chemicals tested. The mechanism for the specific effect of magnesium ion is discussed.

Adenosine↗
PubMed1995

Molecular and enzymatic properties of an aspartic proteinase from Rhizopus hangchow.

An aspartic proteinase, rhizopuspepsin (EC 3.4.23.21), from Rhizopus hangchow was purified. The M(r) and isoelectric point were determined as ca 37,000 and 4.5, respectively. The first 19 amino acids in the N-terminal region were SGSGVVPMTDYEYDIEYYG. The contents of the alpha-helix, beta-structure and random coil were calculated to be ca 7.5, 88.9 and 2.7%, respectively. The enzyme can activate trypsinogen at pH 3.0. The activity was completely inactivated by pepstatin A. The specificity and mode of action of the enzyme were investigated with oxidized insulin B-chain at pH 3. The enzyme hydrolysed primarily two peptide bonds, the Leu15-Tyr16 bond and the Tyr16-Leu17 bond, while additional cleavage of the bonds, Ala14-Leu15 and Phe24-Phe25 was also noted.

Amino Acid Sequence↗
PubMed1995

Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS.

We purified a new extracellular serine proteinase (designated subtilisin Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6, and its properties were characterized. Its optimum pH was at 10.0, when succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) was used as a substrate. The substrate specificity of subtilisin Sendai was determined with oxidized insulin B-chain and fluorogenic peptidyl-MCA substrates. The isoelectric point of subtilisin Sendai was over 11.0. The molecular mass of the enzyme was estimated as 28,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The circular dichroism spectrum of the enzyme was measured, and we discuss the relationship between the secondary structure of the enzyme and alkaline stability at pH 12 in comparison with that of subtilisin NAT. The structural gene (aprS) was cloned and sequenced. The deduced amino acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-sequence of 86 amino acids. The homology of the primary structure for 13 subtilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences near these amino acid residues were well conserved. As a special feature, it was observed that there was an extensive number of negatively charged amino acids in the pro-region of subtilisin Sendai and alkaline subtilisins. This was different from those of subtilisin from neutrophiles.

Amino Acid Sequence↗
PubMed1995

Increase in synapsin I phosphorylation implicates a presynaptic component in septal kindling.

Synaptic plasticity in the CNS is thought to be an important component of learning and memory. Kindling is an animal model of synaptic plasticity in which repetitive local electrical stimulation eventually leads to a generalized motor seizure. Once established, the sensitivity of kindled animals to this epileptic condition is long-lasting. An increase in synaptic efficacy appears to underlie the plastic changes observed in kindling but the molecular mechanisms involved remain unknown. Here we demonstrate that the phosphorylation state of synapsin I, a synaptic vesicle-associated protein which has been implicated in the regulation of neurotransmitter release, is significantly increased in hippocampus and parietal cortex of rats two weeks after the establishment of septal kindling. Furthermore, K(+)-evoked release of L-glutamate is significantly increased in synaptosomes prepared from cerebral cortex of kindled animals. Thus, changes within the presynaptic nerve terminal may contribute, at least in part, to the long-lasting modification in neuronal function induced by kindling.

Animals↗
PubMed1995

Model building of DNA repair enzyme 3-methyladenine DNA glycosylase-DNA complex.

We report the model structure of enzyme-DNA complex based on X-ray structure of 3-methyladenine-DNA glycosylase II and mutagenic studies. The enzyme structure does not have any recognizable DNA binding motifs. The DNA was located in the large cleft between the two all alpha domains with the damaged adenine base moiety (3-methyladenine) of DNA duplex positioned in the entrance region of the cleft including the tryptophan and aspartic acid residues. For the target 3-methyladenine to interact with these residues, the 3-methyladenine has to flipped out from the DNA helix. A number of the positively charged amino acid residues adopt at the negatively charged phosphate moieties. It is necessary to kink the DNA helix slightly to form the good interactions between both molecules. The damaged base must be held in place by a number of interactions including the weak van der Waals and electrostatic attractions.

Binding Sites↗
PubMed1994

Functional changes of dextran-modified alkaline proteinase from alkalophilic Bacillus sp.

A serine alkaline proteinase (EC 3.4.21.62) from Bacillus sp. (ALPase I) was modified with the 2,4-dialdehyde derivative of clinical dextran (dialdehyde dextran). The modified preparation was purified using an ion-exchange column and gel filtration. The modified enzyme contained 75% carbohydrate by weight. The isoelectric point (pI) of ALPase I was converted from 8.2 to approximately 5.0 by this modification. The specific activity of the dextran-modified ALPase I was 56% of that of the native enzyme when milk casein was used as a substrate. It also had some superior characteristics: the thermostability of the modified enzyme at pH 10.0 was about 10-15 degrees C higher than that of control. In organic solvents such as n-hexane, benzene, and toluene, the hydrolysis reaction of the modified ALPase I for the fluorogenic substrate, succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-methylcoumaryl-7-am ide (Suc-Ala-Ala-Pro-Phe-MCA), was several times higher than that of the native. This modification greatly improved the stability of ALPase I against nonionic and anionic surfactants. After exposure to lauryl benzene sulfonate and sodium lauryl sulfonate the modified enzyme retained over 95 and 90% of its activity, respectively, but the native enzyme lost its activity. We conclude that modification of serine proteinases with dialdehyde-dextran might be a useful method for improving enzyme character for enzyme technology.

Amino Acid Sequence↗
PubMed1994

Demethylation of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase: synthesis and characterization of O6,9-dimethylguanine coordinated with cis-platin at the N7-position.

In order to investigate the chemical characteristics of O6-methylguanines, the substrate of O6-methylguanine-DNA methyltransferase, cis[PtCl(NH3)2(O6,9-dimethylguanine-7)]NO3, a 1:1 complex of cis-platin with O6,9-dimethylguanine, was synthesized and its demethylation rate, pKa, X-ray structure and cytotoxicity were determined.

Animals↗