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Biomedical subjects

M Ikehara

Publications and source records attributed to M Ikehara.

At least 19 recordsLinked to original sources
PubMed2003

[Liposarcoma of the pleural cavity; report of a case].

A 43-year-old-woman who had sever anterior chest pain visited our hospital on April 3, 2000. A well-defined abnormal shadow was seen in the middle and lower field of the right lung on chest X-ray. Computed tomography showed a large fat density mass in the right pleural cavity with a septum enhanced by contrast medium. Percutaneous needle biopsy revealed lipoma or liposarcoma. Complete resection could be done with combined resection of right lung, lpericardium, parietal pleura and diaphragm. Final histologic diagnosis was well differentiated liposarcoma. There are few reports of liposarcoma arising in the thoracic cavity, we present our case and review the 23 cases reported from the Japanese literatures.

Adult↗
PubMed2000

Prophylactic administration of granulocyte colony-stimulating factor when monocytopenia appears lessens neutropenia caused by chemotherapy for lung cancer.

In a retrospective study, we showed that a monocyte count of <150/microl on days 6 to 8 might be a predictor of grade III or IV neutropenia during cancer chemotherapy given at 3- or 4-week intervals. In the present study, we investigated whether the administration of granulocyte colony-stimulating factor (G-CSF) when monocytopenia appears lessens neutropenia during chemotherapy for lung cancer. Between June 1997 and August 1998, 60 patients who received chemotherapy at 3- or 4-week intervals for unresectable lung cancer were randomized to receive G-CSF (2 microg/kg or 50 microg/m2) when monocytopenia (<150/microl) appeared on days 6 to 8 after chemotherapy (group A) or when neutropenia (<1,000/microl) or leukopenia (<2,000/ microl) appeared after chemotherapy (group B). The administration of G-CSF was stopped when the leukocyte or neutrophil counts reached > 10,000/microl or 5,000/microl, respectively. The blood cells counts were examined three times a week and the degree, duration, and frequency of chemotherapy-induced neutropenia of the two groups were compared. One patient in group A was excluded because whole brain irradiation during chemotherapy was required. Twenty-nine and 30 patients in groups A and B, respectively, received platinum-based chemotherapy and their chemotherapy-induced hematologic toxicities were analyzed. The mean neutrophil count nadir of group A (1,558 +/- 1,771/microl) was significantly higher than that of group B (810 +/- 639/microl, p = 0.032). The duration of grade III neutropenia in group A (1.4 +/- 1.7 days) was significantly shorter than that in group B (2.9 +/- 1.9 days, p = 0.004), and the frequency of grade III neutropenia in group A (48%) was significantly lower than that in group B (83%, p = 0.002). Infectious episodes occurred in five and eight patients in groups A and B, respectively. The durations of G-CSF therapy required by group A and B patients (4.8 +/- 3.1 vs. 4.7 +/- 2.7 days) were not significantly different. Prophylactic administration of G-CSF did not exacerbate anemia or thrombocytopenia induced by chemotherapy. We conclude that the prophylactic administration of G-CSF when monocytopenia appears can lessen neutropenia caused by chemotherapy for lung cancer without increasing the total G-CSF dose.

Adult↗
PubMed1997

Effects of 2'-substituents of the first deoxyguanosine residue in the recognition sequence of EcoRI restriction endonuclease activity.

The effects of 2'-substituents of the first deoxyguanosine on EcoRI activity were examined using synthetic octadeoxynucleotides D(GG*AATTCC) containing 2'-substituted derivatives (G*), i.e., 2'-fluoro-2'-deoxyguanosine (dGfl), 2'-chloro-2'-deoxyguanosine (dGcl), and guanosine (rG). The overall structures of the octamers were very similar, as shown by CD and UV measurements, although their EcoRI reactivities were very different: 100% in 60 min for d(GGAATTCC) and d(GGflAATTCC), 5% in 24 h for d[G(rG)AATTCC], and no cleavage at all in 24 h for d(GGclAATTCC). However, the kinetics showed the octamers exhibit similar binding-affinity to the enzyme (10(-6)-10(-7) M). 31P-NMR analysis suggested the modified octamers change the phosphate backbone conformation in a duplex, since an unusual downfield-shifted signal in the spectra was commonly observed for the modified octamers at low temperature (i.e., a duplex state), which was shifted upfield at high temperature (i.e., a single strand state). The order of the differences was dGcl > rG > dGfl-containing octamers, coinciding with that of the vdW volume of 2'-substituents (Cl > OH > F) and the cleavage reactivities. These findings suggest that steric hindrance by the 2'-substituents causes of conformational change of the phosphate backbone close to the scissile bond, and then interferes with the EcoRI reaction.

Binding Sites↗
PubMed1995

Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67.

Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH.

Adipose Tissue↗
PubMed1994

Kinetic analyses of DNA-linked ribonucleases H with different sizes of DNA.

A series of DNA-linked ribonucleases H with DNA adducts varying in size and sequence, ranging from heptamer to nonamer, were constructed and examined for their ability to cleave the 12-base RNA (5'-CGGAGAUGACGG-3') site-specifically. The DNA-linked RNase H with the 9-base DNA (5'-GTCATCTCC-3') cleaved the 12-base RNA specifically at A6-U7. Kinetic studies revealed that the DNA-linked RNase H with the 8-base DNA (5'-TCATCTCC-3') cleaved it slightly more effectively than that with the 9-base DNA. Factors that may affect the specificity and catalytic efficiency of a DNA-linked RNase H are described.

Base Sequence↗
PubMed1994

Studies of the interactions between Escherichia coli ribonuclease HI and its substrate.

Ribonuclease H (RNase H) recognizes a DNA-RNA hybrid duplex and catalyzes the hydrolysis of the phosphodiester linkages in only the RNA strand. Previously, we developed a method to cleave RNA in a sequence-dependent manner using RNase H and a complementary oligonucleotide containing 2'-O-methylribonucleosides. Since cleavage is restricted to a single site by the modified complementary strand, this system allows kinetic analysis of the RNase H reaction. We describe an investigation of the interactions between RNase HI from Escherichia coli and its substrate, and between the substrate and a metal ion using synthetic oligonucleotide duplexes modified at the cleavage site in combination with the 2'-O-methylribonucleotides. Firstly, the base moiety was changed to interfere with enzyme binding in either the major or minor groove. When 2-N-methylguanine was incorporated into the cleavage site, the Km value for this substrate, containing a methyl group in the minor groove, was 20-fold larger than that for the unmodified substrate, whereas 5-phenyluracil, with a phenyl group residing in the major groove of the duplex, did not affect the affinity. Secondly, the phosphodiester linkage at the cleavage site was changed into a phosphorothioate with a defined configuration. Only the Rp isomer was cleaved at this site in the presence of Mg2+ or Cd2+. These results suggest that the enzyme, but not the metal ion, interacts with the phosphate residue at the cleavage site. Thirdly, the 2'-position of the nucleoside on the 5'-side of the scissile phosphodiester was modified. Alteration of the 2'-hydroxyl function into an amino, fluoro or methoxy group, or removal of this 2'-hydroxyl group, did not affect the affinity for the enzyme, but reduced the reaction rate. An outer sphere interaction of a metal ion with the 2'-hydroxyl group is suggested.

Base Sequence↗
PubMed1994

Nuclear magnetic resonance study of the interaction of T4 endonuclease V with DNA.

T4 endonuclease V catalyzes the DNA strand cleavage in the vicinity of a thymine dimer. In order to obtain insight into the specific recognition mechanism of this enzyme with a thymine photodimer within DNA, the conformations of five different DNA duplexes, [sequence: see text] with which the enzyme can interact, were studied by 1H NMR. DNA I, DNA IV, and DNA V do not contain the TT sequence or a thymine dimer and hence, are expected to bind the enzyme only in a nonspecific manner. DNA II includes a single TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodimer. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure and that the overall structure of B-type DNA is retained. The photodimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The effects of T4 endonuclease V binding on these DNA duplexes were also investigated by 1H NMR. The binding of this enzyme to DNA I, DNA IV, and DNA V causes no alteration in chemical shift values of the imino proton resonances, but the binding to DNA II induces a small downfield shift in the imino proton resonance of GC7.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1994

Role of the Mg2+ ion in the Escherichia coli ribonuclease HI reaction.

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the RP-configuration was cleaved, while the SP-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg(2+)-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.

Bacterial Proteins↗
PubMed1994

Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution.

Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, Tm, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9 degrees C and 2.3 degrees C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.

Base Sequence↗
PubMed1994

Contribution of a hydrogen bond to the thermal stability of the mutant human lysozyme C77/95S.

The structural stability due to a disulfide bridge between Cys77 and Cys95 of the wild-type human lysozyme is partly recovered by a putative hydrogen bond introduced in to the mutant human lysozyme C77/95S, where Cys77 and Cys95 have been replaced by serines (Yamada et al. (1994) Biol. Pharm. Bull., 17, 192 (1994). In order to understand quantitatively the role of the hydrogen bond in the thermal stability of the mutant human lysozyme, we constructed further mutant proteins, C77SC95A in which Cys77 and Cys95 were replaced by serine and alanine, respectively, and C77AC95S, in which Cys77 and Cys95 were replaced by alanine and serine, respectively. From the thermal unfolding studies of these mutant proteins, both C77SC95A and C77AC95S were shown to be destabilized up to -0.81 and -1.32 kcal/mol, respectively, as far as the free energy changes of unfolding were concerned by compared with C77/95A, where both Cys77 and Cys95 were replaced by two alanines. Considering that these decreases in conformational stability are attributable to hydrophobic effects, the hydrogen bond between Ser77 and Ser95, buried in the hydrophobic cavity in C77/95S, was estimated as 3.0 kcal/mol.

Amino Acid Sequence↗
PubMed1993

Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue.

A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification. The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204. The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed. Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult. We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms. Similar superposition with a prokaryotic microbial ribonuclease, RNase St [Nakamura, K. T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y. (1982) Nature 299, 564-566], also indicated Glu58 as a general base. Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072] that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family.

Amino Acid Sequence↗
PubMed1993

Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.

To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.

Base Sequence↗
PubMed1993

pH-dependent thermostabilization of Escherichia coli ribonuclease HI by histidine to alanine substitutions.

Thermal stabilities of mutant ribonuclease HI proteins from Escherichia coli, in which each of five histidine residues was replaced with alanine, were examined at various pHs. Increases in the Tm values were observed at pH 3.0 for four of the mutant proteins, in which each of the four histidine residues exposed to the solvent was mutated, as compared to the Tm of the wild-type protein. The thermostabilization of three of the mutant proteins was dependent on pH, and only observed at low pH. The thermostabilizing effects of the His-->Ala substitutions were cumulative. The temperature of the midpoint of the transition in the thermal unfolding curves, Tm, of the most stable mutant enzyme, in which His 62, His 83, His 124, and His 127 were replaced by Ala, was 5.5 degrees C higher than that of the wild-type enzyme at pH 3.0. The stability of the wild-type protein decreased as the pH was lowered below pH 4, a condition favoring the protonation of carboxyl groups, probably due to unfavorable electrostatic interactions introduced by the increase in positive charges on the protein. Since imidazole groups are positively charged at pH 3.0, it seems likely that thermal stabilization at pH 3.0 by a His-->Ala substitution would be the result of a reduction in such unfavorable electrostatic interactions. These results suggest that amino acid substitutions that cause a decrease in the number of positive charges on the surface of a protein can be used as a general strategy to enhance protein stability at pH values below pH 4.

Alanine↗