Biomedical subjects

K Ohgi

Publications and source records attributed to K Ohgi.

At least 37 records · Page 2Linked to original sources

PubMed1998

A protease sensitive region of plant and animal ribonucleases belonging to the RNase T2 family.

Recombinant RNase LE from tomato and squid liver RNase Tp, typical plant/animal type RNases belonging to the RNase T2 family, were subjected to limited digestion with several proteases, and the cleavage sites were analyzed by Edman degradation. Recombinant RNase LE was cleaved specifically at the 24th Lys by lysylendopeptidase and trypsin, and RNase Tp was cleaved at the 21st Glu by V8 protease. These cleavage sites are located very close to those where the cleavage during preparation of several animal RNase T2 family enzymes was observed. From this finding, it was concluded that the short segment around the 20th amino acid residue in plant/animal RNases is located on the surface of the molecules and forms loops, and is thus very sensitive to proteases.

Amino Acid Sequence↗

PubMed1998

Primary structure of a squid acid and base non-specific ribonuclease.

Squid (Todarodes pacificus) liver RNase (RNase Tp) was purified. RNase Tp was a base non-specific and acid RNase. Upon hydrolysis of RNA, RNase Tp released four mononucleotides in the order of G > A > U > C. RNase Tp consisted of two peptides with 198 and 23 amino acid residues. The amino acid sequences of these peptides were analyzed. The large peptide had two unique segments containing most of the active site amino acid residues of RNase T2 family enzymes. From the comparison of the sequence of short peptide with the sequences of the other RNase belonging to RNase T2 family RNases, it was found that the amino acid sequence of the short peptide was very similar to that of the C-terminal portion of RNases of the RNase T2 family. Thus, we concluded that the short peptide was a C-terminal part of RNase Tp. The molecular mass of the protein moiety of RNase Tp was 25,582 daltons. The amino acid sequence of RNase Tp most resembles that of oyster RNase (91 amino acid residues identical) in the RNase T2 family RNases. However, the N-terminal portion of RNase Tp was unusually similar to those of plant RNases, rather than the other animal RNases.

Acids↗

PubMed1998

[Basic principles and historical consideration of MR cholangiopancreatography].

Basic principle of MR cholangiopancreatography (MRCP) is heavily T2-weighted imaging (hydrography) to use bile and pancreatic juice as "natural contrast medium". Firstly developed sequence for MRCP was a CE-FAST sequence, which employed time-reversed FID signal. The current most popular sequences for MRCP are single-shot fast spin-echo sequences, which are divided into three types (2D single slice, 2D mutiple slice and 3D methods). The advantage of 2D single slice method is conveniently obtained projection imaging within a few seconds of examination time. Both 2D multiple slice and 3D methods consists of a MIP image and a series of source images. The MIP image creates global images of pancreatico-biliary system. The source images provide detailed evaluation of various anatomical structures and abnormalities. By using these sequences properly, MRCP can yield valuable informations of pancreatico-biliary diseases non-invasively.

Biliary Tract↗

PubMed1998

[Optimization of pulse sequences for MR cholangiopancreatography with FASE].

Various types of pulse sequences for MR cholangiopancreatography (MRCP) have been developed in the past ten years. FASE (fast advanced spin echo) is one of the single-shot fast spin-echo sequences developed by Toshiba. By using this sequence, 2D single slice, 2D multiple slice and 3D methods can be provided. We routinely employ 2D single slice and 3D methods. The merit of 2D single slice method is conveniently obtained projection imaging within short data acquisition time. On the other hand, 3D method can yield detailed evaluation of various anatomical structures and abnormalities by using thin-slice source images. O2-inhalation study is especially recommended in case of 3D method. With the proper knowledge of sequence characteristics, MRCP using FASE can provide valuable informations of pancreatico-biliary diseases.

Biliary Tract↗

PubMed1998

[Elimination of artifacts in MRCP: technical consideration].

Artifacts of MR cholangiopancreatography (MRCP) include fluid-filled gastrointestinal tracts, respiratory motion artifacts, spasm of Oddi's sphincter, vascular compression of bile ducts, overlapping of various anatomical structures and bright signal of surrounding fatty tissue. In this article, various technical considerations to eliminate these artifacts were described for the better imaging analysis of MRCP. The use of high-concentration ferric ammonium citrate (Ferriseltz) is recommended to reduce bright signal of fluid-filled gastrointestinal tracts. In case of long breathhold sequences, O2-inhalation study is useful to eliminate respiratory motion artifacts. Careful attention should be paid to the spasm of Oddi's sphincter and the vascular compression of bile ducts to avoid erroneous interpretation of MRCP findings.

Arteries↗

PubMed1997

Role of histidine 46 in the hydrolysis and the reverse transphosphorylation reaction of RNase Rh from Rhizopus niveus.

In order to study the reaction mechanism of RNase Rh from Rhizopus niveus, the rates of cleavage of four 2',3'-cyclic nucleotides by mutant enzymes of RNase Rh, H46F, H109F, E105Q, and K108L were measured. H46F is virtually inactive towards cyclic nucleotides, but H109F hydrolyzed these substrates at 0.7-4.5% of the rates of the native RNase Rh. The other mutants hydrolyzed 2',3'-cyclic nucleotides at 15-20% of the rates of the native enzyme. Relative enzymatic activities towards four cyclic nucleotides of H109F in the hydrolysis reaction (2nd step) were much higher than in the transphosphorylation reaction (the 1st step). In the presence of a 13-fold excess of uridine, H109F catalyzed the transphosphorylation reaction of 2',3'-cyclic AMP (A>p) to ApU. However, this reaction was not catalyzed by H46F mutant or native RNase Rh. These results showed that His46 is crucial to the hydrolysis reaction, and to the reversed reaction of the transphosphorylation reaction. We suggest that His46 in RNase Rh plays a major role in these reactions by acting as a base catalyst to activate water and the 5'-hydroxyl group of nucleosides, respectively.

Binding Sites↗

PubMed1997

Primary structure of base non-specific and acid ribonuclease from bullfrog (Rana catesbeiana).

A base non-specific acid ribonuclease (RNase RCL2) was purified from bullfrog liver [H. Yagi et al. Biol. Pharm. Bull., 18, 219-222 (1992)]. The sequence study and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster, drosophila and chicken liver suggested that the RNase RCL2 consisted of two components, large protein fraction (182 amino acid residues) and peptide 2 (20 amino acid residues) or peptide 1 (18 amino acid residues), and that both components bind with disulfide bridge. The RNase preparation was probably formed from a single polypeptide protein by processing with some proteases. The amino acid sequence of RNase RCL2 showed that the RNase belongs to the RNase of RNase T2 family and its sequence most resembles chicken liver acid RNase. In RNase RCL2, the amino acid residues which consist of the active site and major base recognition site of RNase Rh, a typical RNase of RNase T2 family, are very well conserved except for Tyr57 (RNase Rh numbering), and part of the amino acid residues of the minor base recognition site (Phe101 and Pro92) are also conserved.

Amino Acid Sequence↗

PubMed1997

Enzymatic properties of double mutant enzymes at Asp51 and Trp49 and Asp51 and Tyr57 of RNase Rh from Rhizopus niveus.

Mutation of Asp51 of a base-nonspecific RNase, RNase Rh, to Ser, Thr, or Gln makes the enzyme more preferential for the dinucleoside phosphate (XpY) having G and C at the 5'-side (X). On the other hand the mutation of one of the B1 site components, Tyr57 to Trp, and Trp49 to Phe makes the enzyme more preferential for purine bases and pyrimidine bases, respectively. In this study, to obtain more specific RNases and RNases with different base specificity, we prepared double-mutant enzymes that have Ser, Thr, and Asn at the 51st position and Trp at the 57th position or Phe at the 49th position, and their enzymatic specificities were studied with XpYs as substrates. The double-mutant enzymes D51SY57W and D51TY57W are more guanylic acid preferential than the mother single-mutant enzymes, D51S and D51T, respectively. They are extremely guanylic preferential RNases. D51NY57W is more a guanylic acid preferential enzyme than D51N, but cytidylic acid preference is of a similar order to that of D51N. The double mutant enzymes D51NW49F and D51TW49F showed an increased cytidylic acid preference as well as guanylic acid preference as compared to the mother single-mutant enzymes, D51T and D51N. The results of analysis of base specificity by the release of mononucleotides from RNA and the rates of hydrolysis of homopolynucleotides led to the same conclusion as in the case of the hydrolysis of XpY.

Dinucleoside Phosphates↗

PubMed1997

The base specificities of tomato ribonuclease (RNase LE) and its Asp44 mutant enzyme expressed from yeast cells.

RNase LE from cultured tomato cells is a member of the RNase T2 family. It is, however, distinguishable from RNase Rh from Rhizopus niveus, a typical RNase of the RNase T2 family, by its CD spectrum in the 200-250 nm region. In order to reinvestigate the base specificity of RNase LE and to study the role of Asn44 in RNase LE, which is considered to correspond to the base recognition site Asp51 of RNase Rh, RNase LE, and its Asp mutant at the 44th position were expressed from yeast cells with the same expression system as RNase Rh [K. Ohgi, et al., J. Biochem., 109, 776-785 (1991)]. RNase LE with four extra amino acid residues at the 2nd amino acid residue of mature RNase LE and its Asp44 mutant were secreted from yeast cells to give a yield of 10 mg/liter and 0.5 mg/liter culture broth, respectively. The expressed RNase LE (RNase RNAP LE) had the same characteristics as native RNase LE in the CD spectrum and specific activity. This is the first example of the expression of plant RNase from microbes and in sufficient amount to perform further enzymological research. The base specificity of RNase LE was guanylic acid preferential and that of N44D was changed to a more adenylic acid preference as compared to that of RNase LE. These experiments showed that Asn44 of RNase LE is crucial for base recognition as the case of Asp51 in RNASE Rh, and also suggested that the base recognition mechanism of RNase LE is very similar to that of RNase Rh.

Amino Acid Sequence↗

PubMed1997

[A case of spinal hemorrhage associated with abdominal aortic coarctation].

We report a 59-year-old male with abdominal aortic coarctation presented as paraplegia due to spinal hemorrhage caused by the rupture of abnormally dilated spinal artery. In coarctation of aorta, coarctation is usually located in the aortic isthmus which could be the cause of cervical and upper thoracic myelopathy. However, there has been no report of abdominal aortic coarctation with hemorrhagic transverse myelopathy. In this case hemorrhage occurred after surgical treatment and prescribed warfarin may have exaggerated the outcome.

Aorta, Abdominal↗

PubMed1997

Primary structure of porcine spleen ribonuclease: sequence homology.

The primary structure of porcine spleen RNase (RNase Psp1) was investigated as a mean of assessing the structure-function relationship of base non-specific ribonucleases of animal origin. N-terminal analysis of RNase Psp1 yielded three N-terminal sequences. These peptides were separated by gel-filtration on Superdex 75HR, after reduction and S-carboxymethylation of RNase Psp1. Determination of the amino-acid sequence of these peptides indicated that the RNase Psp1 preparation consisted of three peptides having 20 (RCM RNase Psp1 pep1), 15 (RCM RNase Psp1 pep2), and 164 (RCM RNase Psp1 pro) amino-acid residues, respectively. It possessed two unique segments containing most of the active site amino-acid residues of the RNases of the RNase T2 family. The alignment of these three peptides in RNase Psp1 was determined by comparison with the other enzymes in the RNase T2 family. The overall results showed that RCM RNase Psp1 pep1 and RCM RNase Psp1 pep2 are derived from the N-terminal and C-terminal regions of RNase Psp1, respectively, probably by processing by some protease. The molecular mass of the protein moiety of RNase Psp1 was 23235 Da.

Amino Acid Sequence↗

PubMed1996

The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution.

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.

Amino Acid Sequence↗

PubMed1996

Preoperative simultaneous cisplatin- or carboplatin-based chemotherapy and radiotherapy for squamous cell carcinoma of the oral cavity.

BACKGROUND: Encouraging results have been reported with cisplatin- or carboplatin-based chemotherapy regimens and simultaneous irradiation treatment in advanced and unresectable head and neck head and neck cancer. We have therefore examined the effectiveness of such therapy on tumor control, survival, and toxicity in patients with advanced oral squamous cell carcinoma. METHODS: Forty-one patients with squamous cell carcinoma of the oral cavity (including soft palate) were treated preoperatively with cisplatin or carboplatin, and 5-fluorouracil or peplomycin in combination with simultaneous irradiation to a target volume of 40Gy, and 2-6 weeks later, curative surgery was performed. RESULTS: Thirty-eight patients (91.7%) had Stage III or IV disease, and three patients had Stage II lesions. The preoperative clinical responses of the primary tumor were: 25 patients (61.0%) achieved a complete response (CR), 15 (36.6%) a partial response (PR), only 1 patient (2.4%) had stable disease or no change (NC). The overall response rate was 97.6%. Histological effects according to the grading system of Shimosato and coworkers [Jpn J Clin Oncol 1:19-35, 1971] were seen in 38/41 (92.7%). Of clinical CR patients, 73.9% were also histologic negative for tumor. Side effects of this therapy were relatively low and reversible. With a median follow-up of 52.8 months (range 17-92 months), 5-year cumulative survival rates were 81.5% for all patients, 100% for Stage II, 88.6% for Stage III, and 76.4% for Stage IV patients, respectively. There was no significant postoperative morbidity. CONCLUSIONS: This preoperative chemoradiotherapy regimen was highly active, well tolerated, and appeared to have a survival benefit even for advanced carcinomas of the oral cavity.

Adult↗

PubMed1996

Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus.

In order to establish the role of Tyr57 and Trp49 in the enzymatic reaction of RNase Rh, several mutant enzymes at Tyr57 and Trp49 were prepared by protein engineering and their enzymatic properties were investigated. Among the four mutant enzymes at Trp49 (W49F, W49Y, W49A, and W49I), W49F showed 16% of the activity of the native enzyme, but the others (W49Y, W49A, and W49I) showed greatly decreased activity. The data showed that Trp49 is very important for the enzyme activity. Among 8 mutant enzymes at the 57th position, Y57F and Y57W showed similar enzymatic activity toward RNA to that of the wild-type enzyme, but the others (Y57G, Y57A, Y57V, Y57M, and Y57K) are more active toward RNA and less active toward XpGs. The reason for the apparent increase for RNA activity is discussed from the view point of substrate inhibition. It is noteworthy that W49F and Y57W became more pyrimidine base- and purine base-preferential, respectively.

Amino Acid Sequence↗

PubMed1996

Enzymatic properties of mutant forms of RNase Rh from Rhizopus niveus as to Asp51.

In order to determine the role of Asp51 of RNase Rh from Rhizopus niveus, enzymes with mutations at the 51st position, D51N, D51E, D51Q, D51S, D51T, D51A, and D51K, were prepared, and their enzymatic properties were investigated as to specific activity and base specificity. All the mutant enzymes showed relatively high activity toward poly I and poly C, and markedly reduced activity toward poly A and poly U. In particular, the enzymatic activities toward poly I of D51T and D51S were higher than that of RNase RNAP Rh. Among the mutant enzymes, D51N, D51S, and D51T showed more than ca. 30% of the activity of RNase Rh, when RNA, poly I and poly C were used as substrates, respectively. The substitution of Ala, Glu, or Lys at Asp51 is unfavorable for enzymatic activity. Among XpGs (X = A, G, U, or C), D51N, D51S, and D51T showed higher activity toward GpG then CpG. Therefore, Asp51 in RNase Rh plays a critical role in the adenylic acid preference of RNase T2 family enzymes. Our results obtained with a protein engineering technique provide basic insights into the control of the base specificity of RNase Rh.

Amino Acid Sequence↗

PubMed1996

Enzymatic activities of several K108 mutants of ribonuclease (RNase) Rh isolated from Rhizopus niveus.

We previously investigated the role of the Lys108 residue of ribonuclease (RNase) Rh from Rhizopus niveus, and suggested that Lys108 probably acts to stabilize the pentacovalent intermediate, and that an Arg residue could replace the role of Lys108. In RNase Le2 from Lentinus edodes, a homologous enzyme of RNase Rh, Lys108 is replaced by Thr. In this paper, the enzymatic properties of a K108T mutant and its analogous enzyme, K108S, were investigated to determine the effect of Thr and its analog, Ser at the 108th position on enzyme activity. The enzymatic properties of these mutant enzymes were compared with those of other mutant enzymes at this position (K108M, K108A, K108L). The results showed that Thr and Ser could replace Lys108 but resulted in only 2-20% of the activity of the native enzyme depending on the substrates used.

Amino Acid Sequence↗

PubMed1996

Base specificity and primary structure of poly U-preferential ribonuclease from chicken liver.

The primary structure and base specificity of chicken liver RNase CL1 which has been reported by Miura et al. [Chem. Pharm. Bull., 32, 4053-4060 (1984)] as poly U-preferential RNase, were extensively studied. The sequence study of this enzyme and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster and Drosophila melanogaster suggested that RNase CL1 consists of three peptides with 17, 19, and 163 amino acid residues. The amino acid sequence of these three peptides were identified. The two small peptides are joined to the large peptide by disulfide bridges. The amino acid sequence of RNase CL1 had 62 (31.2%) and 63 residues (31.6%) identical with oyster RNase and D. melanogaster RNase, respectively, and belongs to the RNase T2 family RNase. Reassessment of the base specificity of RNase CL1 found that it is guanylic acid, then uridylic acid-preferential, and not poly U preferential.

Amino Acid Sequence↗

PubMed1995

Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus.

In order to elucidate on the mechanism of action of RNase Rh from Rhizopus niveus, we investigated the role of Lys108, which is conserved among the RNase T2 family RNases except for two cases. The RNase activities of Lys108 mutant RNases, RNase RNAP K108R and K108L, are about 33.5 and 3.1% of that of the wild type enzyme, respectively. The relative rates of cleavage of dinucleoside phosphates by these two mutant enzymes were comparable to those with RNA as a substrate. The kinetic parameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, G, U, and C) were measured. The data indicated that the Km values of the two mutant enzymes are similar to those of the wild-type enzyme. The rates of release of the four nucleotides from RNA by digestion with the mutant enzymes were in the order A > G > U > C, which is qualitatively the same as that of the wild-type enzyme. From these data, we concluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispensable for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition state as proposed in the case of Lys41 in RNase A, or may polarize the phosphate moiety of the substrate.

Amino Acid Sequence↗