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Biomedical subjects

H Mihara

Publications and source records attributed to H Mihara.

At least 37 records · Page 2Linked to original sources
PubMed2001

Construction of peptide conjugates with peptide nucleic acids containing an anthracene probe and their interactions with DNA.

We designed and synthesized the peptide nucleic acid (PNA)-peptide conjugates having anthracene chromophores and investigated their interactions with calf thymus DNA, [d(AT)(10)](2), [d(GC)(10)](2), and [d(AT)(10)dA(6)](2). Considering the synthesis compatibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of alpha-helix peptides, we attempted to attach thymine PNA oligomers at the C-terminus of a 14 amino acid alpha-helix peptide that contained a pair of artificial intercalators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, fluorescence and circular dichroism properties. The results observed in this study showed that the designed peptide folded in an alpha-helix structure in the presence of calf thymus DNA, [d(AT)(10)](2), and [d(AT)(10)dA(6)](2) with the chromophores at the side-chain being fixed with a left-handed chiral-sense orientation. The alpha-helix and the anthracene signals were not observed for [d(GC)(10)](2). Incorporation of thymine PNA oligomers into the designed alpha-helix peptide increased the DNA binding ability to [d(AT)(10)dA(6)](2) with increasing the length of the PNA without changing the conformations of the peptide backbone and the anthracene side-chains.

Anthracenes↗
PubMed2001

Follow-up study of conservative treatment for atlantoaxial rotatory displacement.

Rotatory displacement of the atlantoaxial joint is one of the causes of torticollis in children. Some of these cases show persistent symptoms and may lead to rotatory fixation; however, only a few studies have been directed to the prognosis of atlantoaxial rotatory displacement after conservative treatment. Clinical and radiographic reviews were performed in 35 patients (16 male and 19 female) with rotatory displacement of the atlantoaxial joint. The mean age at admission was 6.5 (range: 2-12) years old, and the mean follow-up period was 3.4 (1.4-5.8) years. All patients successfully achieved reduction after 2 to 3 weeks of continuous halter traction; however, 9 patients (25.7%) experienced recurrence, and 7 patients (20.0%) had a slight torticollis at follow-up. The duration of symptoms before treatment affected the recurrence rate, and the torticollis was apt to remain in cases with severe displacement at admission.

Atlanto-Axial Joint↗
PubMed2001

Malignant lymphoma of the bone associated with systemic sarcoidosis.

A 57-year-old woman was hospitalized with malignant lymphoma of the right talus. After treatment, complete remission was obtained. Gallium-67 scintigraphy to confirm the remission demonstrated increased uptake in the whole body skeletal muscle, especially in her thighs. Biopsy of right gastrocnemius muscle showed epithelioid granuloma. Serum angiotensin-converting enzyme activity (ACE) and lysozyme had increased to several times the normal range. We diagnosed her disease as bone-associated sarcoidosis-lymphoma syndrome. Human herpes virus 8 (HHV-8) genome was examined in the bone marrow specimen, and the relationship between sarcoidosis-lymphoma syndrome and HHV-8 was discussed.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Design, synthesis and peroxidase-like activity of 3alpha-helix proteins covalently bound to heme.

As a model of artificial peroxidase, de novo designed three-alpha-helix proteins, 3alpha-H9 and 3alpha-H12, covalently bound to Fe(III)-mesoporphyrin IX were synthesized and examined for a peroxidase-like activity. The activity was regulated according to the positions of His residues in the proteins, and the His residues played a role in an acid-base catalytic function.

Amino Acid Sequence↗
PubMed2000

Construction of two-stranded alpha-helix peptides based on influenza virus M1 protein selectively bound to RNA.

Various 2alpha-helix peptides were designed and synthesized based on the RNA-binding region of matrix protein M1 in influenza virus. The binding properties of the peptides to model ssRNA, ssDNA, dsDNA, and virus RNA were examined by the fluorescence studies of a dansyl group incorporated into the peptides. The peptide containing the hydrophilic residues of M1 RNA-binding region bound RNAs selectively.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of designed peptides that undergo structural transition from alpha helix to beta sheet and amyloid fibril formation.

BACKGROUND: Conformational alteration and fibril formation of proteins have a key role in a variety of amyloid diseases. A simplified model peptide would lead to a better understanding of underlying mechanisms whereby protein misfolding and aggregation occur. Recently, we reported the design of peptides that undergo a self-initiated structural transition from an alpha helix to a beta sheet and form amyloid fibrils. In this study, we focus on two glutamine residues in the peptide, and report a mutational analysis of these residues. RESULTS: A coiled-coil alpha-helix structure bearing a hydrophobic adamantanecarbonyl (Ad) group at the N terminus was designed (parent peptide Ad-QQ). In neutral aqueous solution, the double Gln-->Ala mutant (Ad-AA) underwent the alpha-->beta structural transition within four hours, which was similar to the case of Ad-QQ. In contrast, two kinds of single Gln-->Ala mutant (Ad-QA and Ad-AQ) required three days for the transition. Furthermore, Ad-QQ and Ad-AA formed amyloid fibrils, whereas Ad-QA and Ad-AQ did not. Interestingly, however, Ad-QA and Ad-AQ complementarily assembled into the fibrils when they were mixed. CONCLUSIONS: The Gln-->Ala substitution in the peptide significantly alters the alpha-->beta transitional properties and the ability to form amyloid fibrils. A heterogeneous assembly of two peptide species into the fibrils is also presented. These results suggest that the secondary structural transition and self-assembly into the well-organized fibril may depend strictly on the primary structure, which determines the beta-sheet packing. The results might provide insights into misfolding and fibril formation of disease-associated mutant proteins.

Amino Acid Sequence↗
PubMed2000

Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method.

We have utilized sequence information from an antiheme monoclonal antibody to develop novel porphyrin-binding peptides. Several peptides which have an intramolecular disulfide bond in different positions and different chain lengths were prepared. The affinities of peptides for meso-tetrakis(4-carboxyphenyl)porphyrin were increased by an appropriate conformational restraint using a disulfide bond. Detailed studies with a representative 12-peptide, 12C4, whose length was reduced from 20 residues of the complementarity-determining region (CDR), indicated that both the hydrophobic and electrostatic interactions were essential factors in the peptide-porphyrin binding. Moreover, two-dimensional 1H NMR spectroscopy revealed the conformation of the peptide and the critical residues for the porphyrin-binding. According to the obtained results, a further minimized 9-peptide, 9L, was successfully redesigned with a sequence capable of forming a beta-turn instead of a disulfide bond. Furthermore, affinity maturation studies of 9L were performed by using a combinatorial approach such as the spot-synthesis method. Peptides with an improved affinity for porphyrins were prepared by systematic amino acid replacement. Thus, the design of peptides targeted to porphyrins was demonstrated by the combination of antibody information and the rationally designed combinatorial method.

Amino Acid Sequence↗
PubMed2000

A high-throughput screening utilizing intramolecular fluorescence resonance energy transfer for the discovery of the molecules that bind HIV-1 TAR RNA specifically.

A 16-residue peptide, including the Tat(49-57) sequence was labeled with a fluorescein and a tetramethylrhodamine at its N- and C-terminus, respectively. This double dye-labeled peptide was prepared as a tracer for high-throughput screening utilizing intramolecular fluorescence resonance energy transfer (FRET). The binding of the competitor molecules for HIV-1 TAR RNA were monitored and dissociation constants of those molecule were determined by using this tracer. This novel screening system might be useful to discover the drug for HIV-1 TAR RNA.

Drug Evaluation, Preclinical↗
PubMed2000

Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.

Carbon-Sulfur Lyases↗
PubMed2000

Degradation of human Aurora2 protein kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway.

Human Aurora2 was originally identified by its close homology to yeast IPL1 and fly aurora, which are key regulators of chromosome segregation and a family of serine/threonine kinases. Here we demonstrate that the Aurora2 protein is degraded rapidly after G2/M phase release in mammalian cells. Aurora2 protein has a rapid turnover rate with a half-life of approximately 2 h. In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of unstable proteins. The treatment of mammalian cells with proteasome inhibitors blocks Aurora2 degradation. Furthermore, Aurora2 is polyubiquitinated in vivo and in vitro using anaphase-promoting complex (APC). These results demonstrate that Aurora2 protein is turned over through the APC-ubiquitin-proteasome pathway. Oncogene (2000) 19, 2812 - 2819

Anaphase-Promoting Complex-Cyclosome↗
PubMed2000

Construction of alpha-helix peptides with beta-cyclodextrin and dansyl units and their conformational and molecular sensing properties.

In order to apply de novo peptide design to molecular sensing, we designed and synthesized a-helical peptides with beta-cyclodextrin (beta-CDx) as a binding site and a dansyl unit (Dns) as a fluorescence sensing site. The conformational and molecular sensing properties of the peptides with beta-CDx and Dns in various positions were investigated. Circular dichroism and fluorescence measurements revealed that beta-CDx and Dns form intramolecular complexes which depend on their positions in the peptides. In the 17 residual peptides named EK3 and EK3R, in which beta-CDx and Dns were introduced at the fourth and the eighth positions (EK3) or at the eighth and the fourth positions (EK3R), Dns was deeply included in the CDx cavity and formed a more stable self-inclusion complex with CDx than in the peptides EK6 and EK6R, in which these moieties were at the eighth and the fifteenth positions or at the fifteenth and the eighth positions, respectively. The stability of the self-inclusion complex between beta-CDx and Dns controlled the a-helix structure as well as the binding and sensing abilities for the exogenous guests. These results demonstrate the usefulness of peptide tertiary structure for arranging CDx and other functional units, and suggest that this approach is important in the development of a new type of CDx-based sensory system.

Amino Acid Sequence↗
PubMed2000

Cervical myelopathy caused by C3-C4 spondylosis in elderly patients: a radiographic analysis of pathogenesis.

STUDY DESIGN: A radiographic analysis of elderly patients with cervical spondylotic myelopathy, particularly those with involvement of the C3-C4 level. OBJECTIVES: To elucidate the potential contributors to the higher incidence of pathology at C3-C4 in the elderly. SUMMARY OF BACKGROUND DATA: In this study, the elder patients showed a greater predilection for involvement of the C3-C4 lesion compared with their younger counterparts. No previous study has addressed C3-C4 pathology in elderly patients. METHODS: This study included 18 patients, 10 men and 8 women, with cervical spondylotic myelopathy caused by C3-C4 disorders (group I). For the purpose of comparison, 18 younger patients (less than 50 years of age) with myelopathy (group II) and 30 volunteers over the age of 65 (group III) were also investigated. Mean age at admission was 73.5 years for group I, 42.4 years for group II, and 73.4 years for group III. Radiographic analysis, using static and dynamic radiographs, was performed to evaluate the morphologic features. RESULTS: The mean spinal canal diameter for groups I and II was significantly smaller than that for group III. Group I exhibited greater C2-C7 lordosis. The aged population, group I and group III, showed greater C3-C4 angulation associated with C4 forward inclination in neutral standing position as compared with younger patients. Regarding dynamic factors, group I showed the largest segmental motion at C3-C4, and, conversely, the smallest mobility at the lower segments, with significant differences. CONCLUSIONS: Using radiographic analysis, morphologic features that predispose patients to disorders of the C3-C4 motion segment were evaluated. These features included 1) greater C3-C4 angulation associated with age-related postural change and 2) hypermobility at the C3-C4 segment compensating for decreased mobility at the lower segments.

Adult↗
PubMed2000

cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis.

Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.

Amino Acid Sequence↗
PubMed2000

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA.

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.

Amino Acid Sequence↗
PubMed2000

Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase.

Escherichia coli CsdB, a NifS homologue with a high specificity for L-selenocysteine, is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that belongs to aminotransferases class V in fold-type I of PLP enzymes and catalyzes the decomposition of L-selenocysteine into selenium and L-alanine. The crystal structure of the enzyme has been determined by the X-ray crystallographic method of multiple isomorphous replacement and refined to an R-factor of 18.7% at 2.8 A resolution. The subunit structure consists of three parts: a large domain of an alpha/beta-fold containing a seven-stranded beta-sheet flanked by seven helices, a small domain containing a four-stranded antiparallel beta-sheet flanked by three alpha-helices, and an N-terminal segment containing two alpha-helices. The overall fold of the subunit is similar to those of the enzymes belonging to the fold-type I family represented by aspartate aminotransferase. However, CsdB has several structural features that are not observed in other families of the enzymes. A remarkable feature is that an alpha-helix in the lobe extending from the small domain to the large domain in one subunit of the dimer interacts with a beta-hairpin loop protruding from the large domain of the other subunit. The extended lobe and the protruded beta-hairpin loop form one side of a limb of each active site in the enzyme. The most striking structural feature of CsdB lies in the location of a putative catalytic residue; the side chain of Cys364 on the extended lobe of one subunit is close enough to interact with the gamma-atom of a modeled substrate in the active site of the subunit. Moreover, His55 from the other subunit is positioned so that it interacts with the gamma- or beta-atom of the substrate and may be involved in the catalytic reaction. This is the first report on three-dimensional structures of NifS homologues.

Amino Acid Sequence↗
PubMed2000

High incidence of silent myocardial ischemia in elderly patients with non insulin-dependent diabetes mellitus.

The present study was designed to reveal the incidence of silent myocardial ischemia in asymptomatic elderly non-insulin-dependent diabetic (NIDDM) patients (aged over 60 years). As a first step screening, maximal treadmill exercise test was performed. Of 140 patients studied, 54 (38.6%) were unable or not expected to achieve diagnostic levels of exercise during treadmill testing. A positive exercise test was noted in 39 of 86 (45.3%) subjects. As a second step examination, dipyridamole thallium scintigraphy was performed for 93 subjects who exhibited a positive exercise test and could not perform a maximal exercise test. Abnormal perfusion pattern was found in 39 of 93 (41.9%), who were finally considered to have a silent myocardial ischemia. Coronary angiography was performed in 18 subjects with diagnosis of silent myocardial ischemia, who gave their consent. Significant coronary artery stenosis was in fact found in 17 of 18 (94.4%) subjects studied, confirming a very high positive predictive value of this diagnostic procedure. In conclusion, elderly NIDDM patients (aged over 60 years) had an extremely high prevalence (estimated 26.3%) of silent myocardial ischemia. This evidence suggests that early and intensive detection may be needed as a part of routine care for this group.

Aged↗
PubMed2000

Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions.

We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.

Bacterial Proteins↗