Biomedical subjects

T Hirabayashi

Publications and source records attributed to T Hirabayashi.

At least 19 recordsLinked to original sources

PubMed2001

A helical arrangement of beta-substituents of dehydropeptides: synthesis and conformational study of sequential nona- and dodecapeptides possessing (Z)-beta-(1-naphthyl)dehydroalanine residues.

Sequential nona- and dodecapeptides possessing three and four (Z)-beta -(1-naphthyl)dehydroalanine (Delta(Z)Nap) residues, Boc-(L-Ala-Delta(Z)Nap-L-Leu)(n)-OCH(3) (n = 3 and 4; Boc = t-butoxycarbonyl), were synthesized to design a rigid 3(10)-helical backbone for a regular arrangement of functional groups using dehydropeptides. Their solution conformations were investigated by NMR and CD analyses, and theoretical energy calculations. Both peptides were found to adopt a 3(10)-helical conformation in CDCl(3) from their nuclear Overhauser effect spectroscopy (NOESY) spectra, which showed intense cross peaks for N(i)H-N(i+1)H proton pairs, but no cross peaks for C(alpha)(i)H-N(i+4)H pairs. The predominance of a 3(10)-helix was also supported by solvent accessibility of NH resonances. CD spectra of both peptides in tetrahydrofuran showed strong exciton couplets at around 228 nm assignable to naphthyl side chains, which are regularly arranged along a right-handed helical backbone. Chain-length effects on conformational preference in sequential peptide -(Ala-Delta(Z)Nap-Leu)(n)- were discussed based on spectroscopic analysis, energy minimization, and molecular dynamics simulations. Consequently, the repeating number n > or = 3 forms predominantly a right-handed 3(10)-helical conformation. The energy calculation also revealed that the midpoint naphthyl groups of peptide n = 4 are highly restricted to one stable orientation. In conclusion, beta-substituted alpha,beta-dehydroalanine is expected to be a unique tool for designing a rigid molecular frame of 3(10)-helix along which beta-functional groups are regularly arranged in a specific manner.

Alanine↗

PubMed2001

Expression of beta-synuclein in normal human astrocytes.

Although alpha- and beta-synucleins are expressed predominantly in presynaptic nerve terminals, recent studies have demonstrated that alpha-synuclein is also expressed in cultured astrocytes and oligodendrocytes. We determined whether beta-synuclein might be expressed in astrocytes. Beta-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that beta-synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, beta-synuclein immunoreactivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues. Ultrastructurally, beta-synuclein immunoreactivity was found in the cytoplasm of astrocytes, in association with the plasma membrane, ribosomes, rough endoplasmic reticulum and the nuclear outer membrane. The novel expression of beta-synuclein in astrocytes may provide an important insight about the role of this protein.

Astrocytes↗

PubMed2001

Atp-binding cassette transporter ABC2/ABCA2 in the rat brain: a novel mammalian lysosome-associated membrane protein and a specific marker for oligodendrocytes but not for myelin sheaths.

We recently cloned a full-length cDNA of the rat ATP-binding cassette transporter 2 (ABC2, or ABCA2) protein, a member of the ABC1 (or ABCA) subfamily (-ABC1/ABCA1 is a causal gene for Tangier disease) and found it to be strongly expressed in the rat brain. In this study, we identified ABC2 as a lysosome-associated membrane protein that is being localized specifically in oligodendrocytes. The ABC2-immunolabeled cells were detected mainly in the white matter but were also scattered in gray matter throughout the whole brain. In addition, these cells were found to be colocalized with 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) immunoreactivity when the marker antibody for oligodendrocytes was used. However, no such colocalization was observed with markers for other kinds of glial cells. Unlike the CNP antibody, which also intensely stains myelin sheaths in the white matter, ABC2 immunoreactivity was detected only in the cell bodies of oligodendrocytes. At the ultrastructural level, ABC2 immunoreactivity was detected mostly around lysosome and partly in Golgi apparatus by electron microscopy. This was confirmed by immunocolocalization of ABC2 and lysosomal markers in a neuroblastoma cell line. Immunoblotting analysis of ABC2 from the whole brain and the ABC2-transfected cell line revealed bands at approximately 260 kDa. The result of in situ hybridization with a riboprobe for ABC2 matched the results obtained from immunostaining. These findings strongly suggest that ABC2 is a specific marker for oligodendrocytes but not for myelinsheaths and that it is as a novel mammalian lysosome-associated membrane protein involved in myelinization or other kinds of metabolism in the CNS.

ATP-Binding Cassette Transporters↗

PubMed2001

Structural and conformational properties of (Z)-beta-(1-naphthyl)- dehydroalanine residue.

To understand how chemical structure of beta-substituted alpha, beta-dehydroalanine (particularly size and pi conjugation of beta substituent) affects conformational property, x-ray crystallographic analysis was performed on Boc-Ala-Delta(Z) Nap-Val-OMe [Boc: t-butoxycarbonyl; Delta(Z) Nap: (Z)-beta-(1-naphthyl)dehydroalanine; OMe: methoxy] having the naphthyl group as a bulky beta substituent. Single crystals were grown by slow evaporation from an ethanol solution in the triclinic space group P1 with a = 9.528 (3) A, b = 12.410(4) A, c = 5.975(2) A, alpha = 96.77(3) degrees, beta = 102. 81(2) degrees, gamma = 88.74(3) degrees, V = 684.1(4) A3, and Z = 1. Phase determination was carried out by a direct method (SHELEXS), and the final structure was refined to R = 8.1% and R(w) = 9.0% for 1964 observed reflections. The bond lengths and bond angles of the Delta(Z)Nap residue, characterized by a sp(2) hybridized C(alpha) atom, did not differ from those of other dehydroresidues such as Delta(Z) Phe, Delta(Z) Leu, and DeltaVal essentially. The peptide backbone took a type II beta-turn conformation involving an intramolecular hydrogen bond between CO(Boc) and NH(Val), similar to di- or tripeptides containing a Delta(Z) Phe or Delta(Z) Leu residue in the second positions. Here the naphthyl group was found to be nonplanar [chi(2) = 55(1) degrees ] relative to the C(alpha)==C(beta)==C(gamma) plane. The nonplanarity was supported by conformational energy calculation. The molecular packing was stabilized by two kinds of intermolecular hydrogen bonds and van der Waals interactions. Naphthyl groups were arranged in a partially overlapped face-to-face orientation with a center-to-center distance of 5.97 A. For additional information, peptide Boc-(Ala-Delta(Z) Nap-Leu)(2)-OMe was synthesized and its solution conformation was investigated by (1)H-NMR spectroscopy. The hexapeptide showed the tendency to form a 3(10)-helical conformation in solution essentially. Conformational properties of Delta(Z) Nap residue, characterized by a type II beta-turn and 3(10)-helix, were supported by a conformational energy contour map of the Delta(Z)Nap residue.

Alanine↗

PubMed2001

Basic studies on the equivalent cross-relaxation rate imaging (equivalent CRI)--phantom studies.

We have studied saturation transfer in hydrophilic, cross-linked copolymer gels from irradiated polymer protons to observed water protons, using f2 (ppm) profiles of [1 - (I(infinity)/I(0))], [(I(0)/I(infinity)) - 1] or 1/T(IS)(H2O), where I(0) and I(infinity) are the longitudinal magnetization of the observed water protons before and after long-time-f2-irradiation on polymer protons, respectively, and 1/T(IS)(H2O) is the cross-relaxation rate. (A) [1 - (I(infinity)/I(0))] (magnetization transfer ratio, MTR) was used in magnetic resonance imaging (MRI) as the MTR imaging. 1/T(IS)(H2O) (cross-relaxation rate) was used in the imaging of the magnetization transfer rate constant. This method was quite time-consuming compared with MTR imaging. However, f2 (ppm) profiles of [(I(0)/I(infinity)) - 1] correlated well with corresponding profiles of 1/T(IS)(H2O), because [(I(0)/I(infinity)) - 1] is equal to 1/[T(IS)(H2O)/T1(H2O)]. These results lead us to the conclusion that [(I(0)/I(infinity)) - 1] might be applicable to cross-relaxation rate (CR)-like imaging, i.e. equivalent CRI. (B) W (%) (dry weight) profiles of [(I(0)/I(infinity)) - 1] and 1/T(IS)(H2O), obtained by near-resonance f2-irradiation, seem to indicate participation of molecular rigidity and an amount of bound water. However, those values, monitored with off-resonance f2-irradiation, seem to be independent of monomer composition and to indicate mainly participation of rigidity, i.e. W (%) of copolymer gels.

Cross-Linking Reagents↗

PubMed2000

Cloning of an auxin-responsive 1-aminocyclopropane-1-carboxylate synthase gene (CMe-ACS2) from melon and the expression of ACS genes in etiolated melon seedlings and melon fruits.

Two cDNA fragments (pCMe-ACS2 and 3) encoding auxin-responsive 1-aminocyclopropane-1-carboxylate synthase (ACS; EC.4.4.1.14) have been isolated from melon, and the expression patterns of the genes in etiolated melon seedlings and melon fruit have been determined by RT-PCR analysis. The deduced amino acid sequences of pCMe-ACS2 and 3 were homologous to those of AT-ACS6 and 4, which were auxin-responsive ACS genes of Arabidopsis. Both CMe-ACS2 and 3 were auxin-responsive ACS genes and their expressions in roots and hypocotyls were induced by treatment with indole acetic acid (IAA, 100 µM). The mRNA level of CMe-ACS2 in the fruit increased after pollination. Those of both CMe-ACS2 and 3 temporarily increased in the mesocarp tissues at the preclimacteric stage (from day 3 to day 5 after harvest) during ripening, while that of CMe-ACS3 was lower than that of CMe-ACS2. The increase in the mRNA level of CMe-ACS1 (wound- and ripening-induced gene, T. Miki, M. Yamamoto, N. Nakagawa, O. Ogura, H. Mori, H. Imaseki, T. Sato, Nucleotide sequence of a cDNA for 1-aminocyclopropane-1-carboxylate synthase from melon fruits, Plant Physiol. 107 (1995) 297-298.) in the mesocarp tissue was not observed until 5 days after harvest. A genomic DNA encoding CMe-ACS2 was isolated and its nucleotide sequence was determined. Nucleotide sequences resembling the auxin-responsive elements (AuxRE) D1 and D4 (the TGTCTC element) in the GH3 gene from soybean, and the auxin-responsive domain (AuxRD) B in PS-IAA4/5 from pea were found in the 5'-flanking region of the CMe-ACS2 gene.

Journal Article↗

PubMed2000

Localization and regulation of cytosolic phospholipase A(2).

Liberation of arachidonic acid by cytosolic phospholipase A(2) (cPLA(2)) upon cell activation is often the initial and rate-limiting step in leukotriene and prostaglandin biosynthesis. This review discusses the essential features of cPLA(2) isoforms and addresses intriguing insights into the catalytic and regulatory mechanisms. Gene expression, posttranslational modification and subcellular localization can regulate these isoforms. Translocation of cPLA(2)alpha from the cytosol to the perinuclear region in response to calcium transients is critical for the immediate arachidonic acid release. Therefore, particular emphasis is placed on the mechanism of the translocation and the role of the proteins and lipids implicated in this process. The regional distribution and cellular localization of cPLA(2) may help to better understand its function as an arachidonic acid supplier to downstream enzymes and as a regulator of specific cellular processes.

Animals↗

PubMed2000

Cloning, characterization and tissue distribution of the rat ATP-binding cassette (ABC) transporter ABC2/ABCA2.

The ABC1 (ABCA) subfamily of the ATP-binding cassette (ABC) transporter superfamily has a structural feature that distinguishes it from other ABC transporters. Here we report the cloning, molecular characterization and tissue distribution of ABC2/ABCA2, which belongs to the ABC1 subfamily. Rat ABC2 is a protein of 2434 amino acids that has 44.5%, 40.0% and 40.8% identity with mouse ABC1/ABCA1, human ABC3/ABCA3 and human ABCR/ABCA4 respectively. Immunoblot analysis showed that proteins of 260 and 250 kDa were detected in COS-1 cells transfected with ABC2 having a haemagglutinin tag, while no band was detected in mock-transfected cells. After incubation with N-glycosidase F, the mobilities of the two proteins increased and a single band was detected, suggesting that ABC2 is a glycoprotein. Photoaffinity labelling with 8-azido-[alpha-(32)P]ATP confirmed that ATP binds to the ABC2 protein in the presence of Mg(2+). RNA blot analysis showed that ABC2 mRNA is most abundant in rat brain. Examination of brain by in situ hybridization determined that ABC2 is expressed at high levels in the white matter, indicating that it is expressed in the oligodendrocytes. ABC2, therefore, is a glycosylated ABC transporter protein, and may play an especially important role in the brain. In addition, the N-terminal 60-amino-acid sequence of the human ABC1, which was missing from previous reports, has been determined.

ATP-Binding Cassette Transporters↗

PubMed2000

Direct temperature-controlled trapping system and its use for the gas chromatographic determination of organic vapor released from human skin.

For controlling of trap temperature, the relationship between electric resistance of the trap tube and temperature is used. As the electric resistance of the trap tube (20 cm long stainless steel tubing) was very small, such as ca. 0.040 ohm for -70 degrees C and ca. 0.064 ohm for +90 degrees C, it was estimated by using the value of voltage output at both ends of the trap tube when a direct current (5 A) was applied for 6.5 ms at every 100 ms on the trap. By using this temperature measurement, a cycle of trapping is shortened, especially at the process of desorption, because it is possible to set a large increasing rate of temperature, such as 20 degrees C/s. The present trapping system has faster temperature response compared to that with a thermocouple. This system was applied for the study of the releasing of ethanol and water vapors from the human finger, which was treated as follows: dipping in 10% ethanol aqueous solution for 1 min, followed by washing with water and then drying in the air. In this case, a cycle of trapping took 53 s, and the period of total analysis was only 3 min. The present system is an efficient tool for the study of the exhalation of organic vapors from human skin.

Chromatography, Gas↗

PubMed2000

Novel tissue culture method: skeletal muscle implantation under gizzard serous membrane of a chick.

A novel method for a long-term culture of skeletal muscle is described. Skeletal muscle pieces from young chicks were implanted under the gizzard serous membrane of the same chicks. Following muscle degeneration, new well-grouped muscle fibers were formed by the fusion of myocytes that differentiated from surviving satellite cells, and the regenerated muscle tissues were maintained in position for longer than 60 days. The implants were in the vital circulatory system, receiving trophic and oxygen supplies, and are completely free from motor nerve innervation and cell contamination with exogenous muscle cells, not as in intra-muscular implantation. Therefore, this tissue culture method should be useful for studying skeletal muscle regeneration and maturation over a long period. Furthermore, osteogenesis and feather development were also found in the implants of embryonic limbs by using the same method. These observations showed that not only skeletal muscle tissues but also other tissues could be cultured under the gizzard serous membrane.

Actins↗

PubMed2000

Heterogeneity of chicken slow skeletal muscle troponin T mRNA.

The troponin T (TnT) transcripts in chicken slow skeletal muscle were characterized by S1 nuclease mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5'-RACE. We found two kinds of transcripts in the 5'-region, one having the codon for alanine (position 135-137), C (258), and A (262) and the other lacking the codon and having T (258) and G (262) instead of C and A. In the 3'-region, we found four single base substitutions at 703 (T or C), 774 or T), 797 or T), and 827 (G or A). Four of the six substitutions lead to amino acid changes in chicken sTnT isoforms. We determined the genomic structure of the 3'-region of the chicken sTnT gene. The region includes 7 exons corresponding to position 249-891 of the chicken sTnT cDNA and no alternative exon, showing that the 3'-heterogeneity in sTnT transcripts was due to allelic variation. J. Exp. Zool. 286:149-156, 2000.

Amino Acid Sequence↗

PubMed2000

Cellular distribution of the splice variants of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC(1)-R) in the rat brain by in situ RT-PCR.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide and its specific receptor (the PAC(1) receptor) is widely distributed in the rat brain. It has been reported that alternative splicing of the region encoding the third intracellular loop of the PAC(1) receptor generates six isoforms which are differentially coupled to signal transduction pathways, but the precise distribution and localization of these splice isoforms in the brain remain to be determined. Using the initial specific primer pairs which correspond to the 'hip' or 'hop' types of receptors for the solution-phase reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated that the major splice variants of the PAC(1) receptor in various regions of the rat brain are the short splice isoform 'PAC(1)-R-s' which does not contain either the 'hip' or 'hop' cassette and the another splice isoform, 'PAC(1)-R-hop', which contains the 'hop' cassette. With an innovative molecular histochemical technique, in situ RT-PCR, we determined that these two splice isoforms are both intensely expressed in the mitral cells of the olfactory bulb, the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and neocortex, and many neurons in the nuclei of hypothalamus and thalamus as well as other regions. The initial mapping of the cell type-specific expression of these two splice variants of the PAC(1) receptor provides the basis for a better understanding of the functional significance of the PAC(1)-R and its ligand PACAP in various brain regions.

Alternative Splicing↗

PubMed2000

Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution.

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.

Peptides↗

PubMed2000

Agarose isoelectric focusing for the detection of many isoforms and high molecules in muscle protein analysis.

Two-dimensional gel electrophoresis with agarose in the first dimension was established. The technique was reported previously and was now much improved without changing the basic procedure, presenting "a protein map" of chicken skeletal muscle, including almost all components such as myosin heavy chains (200 kDa) and dystrofin (400 kDa). Application of this technique for the analysis of all components was also successful with the liver and lens. Since large molecules can get into the agarose in the first dimension, the study on protein interaction was also reported with troponin components as a model system.

Animals↗

PubMed2000

Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber.

Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B-type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber.

Alternative Splicing↗

PubMed2000

Is endogenous isoprene the only coeluting compound in the measurement of breath pentane?

Pentane is a widely used index of lipid peroxidation. Although isopentane, an isomer of pentane, is a major component of ambient air in urban areas, many studies have disregarded the possibility that this compound is coeluted in the measurement of breath pentane. In the present study, a gas chromatograph equipped with a cold trap apparatus and a large-bore glass capillary column was used for determination of pentane, isopentane and isoprene in breath and ambient air. Isoprene was detected in all subjects at a concentration higher than that in the ambient air. However, the concentrations of breath pentane and isopentane were similar to, or less than, those of the ambient air. We suggest that great care is required in the measurement of breath pentane so that endogenous isoprene and ambient isopentane are not coeluted.

Adult↗

PubMed2000

Splice variants of PAC(1) receptor during early neural development of rats.

The specific pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, PAC(1)-R, consists of at least seven isoforms, and they are differentially coupled to signal transduction pathways by alternative splicing. We have found that the major splice variants of the PAC(1) receptor seen during development are the short splice isoform, PAC(1)-R-s (which does not contain either the "hip" or "hop" cassette), and another form, PAC(1)-R-hop (which contains the "hop" cassette). We also have applied an innovative molecular histochemical technique, in situ reverse transcription-polymerase chain reaction (RT-PCR), and determined that these two splice isoforms are colocalized in the neuroepithelia from the primitive streak stage.

Alternative Splicing↗

PubMed2000

M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and lambda-independent pathways in PC12D cells.

We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif 268 [Ebihara, T. & Saffen, D. (1997) J. Neurochem. 68, 1001-1010]. Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G-protein Rho. Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho-dependent and Rho-independent pathways. Moreover, the Rho-independent activation of zif 268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif 268 SREs is mediated by Ras. To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction. Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE-dependent induction of the zif 268 gene within a Ras-independent pathway.

Animals↗