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Biomedical subjects

A Ueno

Publications and source records attributed to A Ueno.

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

Photoinduced hydrogen evolution with peptide dendrimer-multi-Zn(II)-porphyrin, viologen, and hydrogenase.

To construct an artificial photosynthetic system, multi-Zn(II)-mesoporphyrins in peptide dendrimers were equipped as a photosensitizer of photoinduced hydrogen evolution in a four-component system (electron donor, photosensitizer, electron carrier, and catalyst), so that hydrogen was evolved effectively by the dendrimer architecture, for the first time. The hydrogen evolution activity was correlated to the photoreduction ability of viologen by the Zn-porphyrin-peptide dendrimers. Additionally, using positively charged methyl-viologen as an electron carrier, the photoinduced hydrogen evolution function with the positively charged peptide dendrimer was superior to that with the negatively charged peptide dendrimer, despite that the positive dendrimer did not strongly bind the positively charged methyl-viologen with the electrostatic interaction. By contrast, when zwitterionic propylviologen sulfonate was used, photoreduction and hydrogen evolution properties were identical between the positively and the negatively charged dendrimers. These results demonstrated that the dynamic interaction between the positive dendrimer and methyl-viologen was preferable for the photoreduction and hydrogen evolution, and that the three-dimensional assembly of Zn(II)-mesoporphyrins using the peptide dendrimers was effective as a photosensitizer in the artificial photosynthesis.

Amino Acid Sequence↗
PubMed2001

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand.

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.

Amino Acid Sequence↗
PubMed2001

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells.

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Alkaline Phosphatase↗
PubMed2001

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA.

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.

Amino Acids↗
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

Inhibition of nitric oxide synthase induces intestinal mucosal damage and increases mortality in rats treated by FK506.

INTRODUCTION: Despite the beneficial immunosuppressive effects of FK506 during small intestine transplantation, FK506 appears to have direct toxic effects on the intestine. The mechanisms of FK506-induced intestinal damage is unclear, and whether nitric oxide (NO) is involved in the mechanism has not been well defined. This study was designed to evaluate the effects of NG-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, on small intestinal damage in rats treated with FK506. MATERIALS AND METHODS: Wistar rats weighing 240-260 g, aged 11 weeks, were administered FK506 (5 mg/kg/day i.m) and/or L-NAME (5 mg/dl in drinking water) for 10 days. Body weight gain, diarrhoea and mortality were observed during experiment. At the end of experiment, the intestinal specimens were excised for histological evaluation. In addition, the effects of L-aginine treatment (1 g/dl in drinking water) were evaluated in this study. RESULTS: L-NAME administration time-dependently induced diarrhoea and high mortality in the rats treated with FK506. At the end of 10 days treatment, 7 of 12 rats (58.3%) suffered from diarrhoea and 5 of 12 rats (41.7%) died in the FK506 + L-NAME group (vs. FK506 group, p = 0.05). A significant loss of body weight was also found in the rats treated with FK506 + L-NAME (-52.2 +/- 28.8 g, in FK506 + L-NAME group vs. -14.3 +/- 8.7 g in FK506 group, p = 0.001). In parallel with the severe diarrhoea and high mortality, the loss of villi, hemorrhage and necrosis (grade 5 of pathological damage) was seen in the small intestinal mucosa of rats treated with FK506 + L-NAME. L-arginine treatment in part prevented diarrhoea, mortality and pathological damage of small intestinal mucosa induced by L-NAME. CONCLUSIONS: Inhibition of NOS induces intestinal mucosal damage and increases mortality in rats treated with FK506. L-arginine treatment can in part prevent the injury induced L-NAME. The present study suggests that NO, as an important protective factor, may be involved in the FK506-induced intestinal damage.

Administration, Oral↗
PubMed2001

Effects of nitric oxide synthesis inhibition on FK506-induced nephrotoxicity in rats.

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, N omega-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-beta-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.

Animals↗
PubMed2001

[Inflammation-allergy and prostanoids. (1). Prostanoids in experimental inflammatory reaction].

It is known that prostaglandins (PGs) modify the inflammatory reaction in concert with other biologically active mediators. However, characteristics of these interactions or modulating actions have not yet been clarified well. Recently, the production of mice with specific receptor deficiencies by using the gene targeting procedure for PG receptors has accelerated elucidation of the roles of PGs through correlation of their phenotypes and experimental features. Here I discuss roles of PGs in experimental paw edema, the writhing reaction of a pain model, and regulation of cytokine production, as determined using some PG-receptor-deficient mice. From the experiment of carrageenin-induced paw edema in IP receptor-deficient mice, with an indomethacin or bradykinin antagonist, we conclude that bradykinin initially induces paw swelling and then stimulates the release PGI2, which in turn enhances the swelling with bradykinin. By comparing the writhing responses in IP-deficient and wild-type mice, we found that PGI2 is a main mediator for this pain reaction. However, in the LPS-pretreated mice, not only PGI2 but also other PGs produced by COX-2 may be involved in pain induction. Production of TNF alpha and IL-10 was modified with PGI2 or PGE2; the production of TNF alpha was down-regulated by the stimulation via IP-, EP2- or EP4 receptor, but that of IL-10 was up-regulated by these receptors, resulting in an anti-inflammatory effect.

Animals↗
PubMed2001

Intravesicular Fas localization in epithelial cells of castrated rat prostate glands.

Androgenic steroids regulate the development and size of mammalian prostate epithelial cells. To evaluate the relationship between Fas-Fas ligand system and apoptosis in prostate epithelial cells of the castrated rats, we have examined immunocytochemical localization of Fas antigen in the castrated rat prostate glands at a series of different times. We used a rabbit polyclonal anti-Fas antibody with a streptavidin-biotin method and confocal laser scanning method or an immunogold method. Fas immunolocalization was examined in ventral lobes of prostate glands taken from intact or castrated adult male Wistar rats on day 1, 2, 3, 4 and 5 by light or electron microscopy. At a light microscopic level, the castrated prostate epithelial cells showed mostly Fas immunolocalization in their apical parts of cytoplasm on day 2 after the castration. In addition, their extent of the Fas expression was expanded throughout the cytoplasm in proportion to the androgen ablation periods, and later the Fas expression was detected at luminar or basolateral sides of the epithelial cells. Both immunogold labeling with ultrathin sections and immunoperoxidase technique with cryostat sections demonstrated that Fas was localized mainly in secretory granules of the castrated prostate epithelial cells and some parts of their cell membranes at later stages. Our immunocytochemical findings showed that Fas expression was time-dependently induced in most of the prostatic epithelial cells after castration of rats. The rate of Fas-expressing epithelial cells was too high and inconsistent with the previously reported rate of TUNEL-positive ones. The membrane-associated Fas may have little effect on the apoptosis in the present case, bacause a lot of soluble Fas was secreted from the prostatic epithelial cells. A further study is needed to clarify some significance of the secretory Fas in the prostatic epithelium after the rat castration.

Animals↗
PubMed2001

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene.

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001). We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

Animals↗
PubMed2001

Advantages of the digital X-ray system in dental identification of persons with reference to two murder cases.

Direct digital X-ray technology was applied to dental identification of victims in two murder cases using the Compuray system. In both cases the digital radiography proved to be simple to use, quick and effective, allowing superimposition, enlargement and transportability to a mortuary. These are the first reported uses of the technology in Japan and further development promises the transmitability of data and images electronically to remote locations, further enhancing its usefulness. Comparing the skull with the dental ante-mortem X-ray films and records of a specific person who was reported "missing", we found many identical points between the two, especially in regard to the X-ray findings with the Compuray. In both cases we obtained a large number of X-ray images in a remarkably short time and this was very useful for identification by means of the teeth.

Adolescent↗
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

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2.

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE(2) biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE(2) generation. Arg(110), a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A(2) allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE(2) biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

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↗