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Biomedical subjects

T Ishikawa

Publications and source records attributed to T Ishikawa.

At least 127 records · Page 7Linked to original sources
PubMed2001

Simultaneous discrimination of diastereotopic groups and faces: the first example in intramolecular [3+2] and [2+2+1] cycloaddition reactions.

To explore a novel concept for controlling diastereoselectivity, systematic studies on the sense and degree of diastereotopic groups and face selections in intramolecular [3 + 2] (nitrile oxide and nitrone) and [2 + 2 + 1] (Pauson-Khand) cycloadditions have been conducted. Optically pure methyl (S)-3,4-O-isopropylidene-3,4-dihydroxybutanoate (5) and methyl (S)-2,3-O-isopropylidene-2,3-dihydroxypropanoate (6) were converted to substrate aldehydes (1-4) that bear geminal allyl groups and four types of controllers with the intention of imparting a stereochemical bias to the allylic groups and their faces. The controllers involve 1,2-bis(tert-butyldimethylsiloxy), 1,3-bis(tert-butyldimethylsiloxy), 1,2-acetonide, and 1,3-acetonide groups, which are referred to as 1,2-(TBDMSO)(2), 1,3-(TBDMSO)(2), 1,3-dioxolane, and 1,3-dioxane, respectively. Twelve runs of cycloaddition reactions as combinations between the three types of reactions and the four types of substrates were performed to provide bicyclo[4.3.0] or -[3.3.0] adducts of synthetic importance in which isoxazolidine, isoxazoline, or cyclopentenone segments were fused. For every case, high levels of diastereoselectivity have been achieved: >99% (in eight cases), 82%, and 76% for the discrimination of diastereotopic groups and 68-->99% for the discrimination of diastereotopic faces. On the basis of the absolute structures of the cycloadducts, plausible stereochemical models are proposed.

Journal Article↗
PubMed2001

Cell cycle transition under stress conditions controlled by vertebrate heat shock factors.

The roles of heat shock transcription factors (HSFs) under physiological conditions have recently become the focus of intense study. We generated avian cells lacking two heat-inducible HSFs, HSF1 and HSF3. In addition to complete loss of activation of heat shock genes under stress conditions, these cells exhibited a marked reduction in Hsp90alpha expression under normal growth conditions. Reduction in Hsp90alpha expression caused instability of a cyclin-dependent kinase, Cdc2, and cell cycle progression was blocked mainly at the G2 phase, but also at G1 phase even at mild heat shock temperatures. Restoration of Hsp90alpha expression rescued the temperature sensitivity without induction of HSPS: We demonstrated for the first time a molecular target affected by heat shock in vivo that causes cell cycle arrest in vertebrates and a novel mechanism of stress resistance controlled by vertebrate HSFS:

Animals↗
PubMed2001

Mechanisms underlying presynaptic facilitatory effect of cyclothiazide at the calyx of Held of juvenile rats.

1. Excitatory postsynaptic currents (EPSCs) were recorded using the whole-cell patch-clamp technique at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) in auditory brainstem slices from juvenile rats. 2. Bath application of cyclothiazide (CTZ, 100 microM) significantly increased the amplitude of EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Cyclothiazide increased the magnitude of paired-pulse depression of both AMPA-EPSCs (intervals, 50 and 500 ms) and NMDA-EPSCs (interval, 20 ms). In low Ca(2+), high Mg(2+) solution, CTZ decreased the number of failures and increased the mean amplitude of AMPA-EPSCs more than three-fold. 3. Presynaptic Ca(2+) currents and K(+) currents were directly recorded from the calyceal nerve terminals. These currents were attenuated by CTZ in a reversible manner. The magnitude of inhibition of presynaptic K(+) currents by CTZ (100 microM) was comparable to that by 5 microM 4-aminopyridine (4-AP). Both CTZ and 4-AP slowed the repolarizing phase of presynaptic action potentials. 4. The inhibitory effects of CTZ on presynaptic ion channels were mimicked by a solution having reduced Ca(2+) concentration and 5 microM 4-AP. This solution facilitated EPSCs, but the magnitude of facilitation was significantly less than that caused by CTZ. 5. In the presence of tetrodotoxin (TTX), CTZ increased the mean frequency of miniature EPSCs three-fold. CTZ prolonged their decay time but had no effect on their amplitude. The facilitatory effect of CTZ on the miniature frequency was neither blocked by a protein kinase C inhibitor nor occluded by phorbol ester, suggesting that a distinct mechanism underlies the effect of CTZ. 6. We conclude that CTZ facilitates transmitter release through suppression of presynaptic potassium conductance and stimulation of exocytotic machinery downstream of Ca(2+) influx.

Action Potentials↗
PubMed2001

Chiral helicity induced by hydrogen bonding and chirality of podand histidyl moieties.

[structure: see text] The single-crystal X-ray structure determination of N,N'-bis[(S)-(+)-1-methoxycarbonyl-2-(4-imidazolyl)ethyl]-2,6-pyridinedicarboxamide (L-BHisPA) and the D-isomer (D-BHisPA) derived from the corresponding chiral histidine revealed a left- and right-handed helical conformation, respectively, through intramolecular hydrogen bonding and chirality of the podand histidyl moieties. Furthermore, each helical molecule is connected by continuous intermolecular hydrogen bonds to afford a left- or right-handed helical assembly, respectively, in the crystal packing.

Journal Article↗
PubMed2001

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis.

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography.

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.

Amino Acid Sequence↗
PubMed2001

Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy.

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.

Actins↗
PubMed2001

Spin polarized metastable He*(2(3)S, 1s2s) stimulated desorption of H(+) ions.

The yield of H(+) in the metastable He*(2(3)S, 1s2s) stimulated desorption (MSD) on an H2O/Na/Fe surface has been found to depend on the spin of the incident He*. By combination with the spin-resolved analysis for the surface electronic structure, it is indicated that the neutralization of the hole in the OH 3 sigma molecular orbital, which is created by the incident He*, by Na 3s electrons plays a crucial role in the spin polarization of the H (+) MSD intensity. The dependence of the spin polarization of H(+) MSD on the kinetic energy of the desorbed H(+) has been observed, and it is discussed based on the Menzel-Gomer-Readhead model.

Journal Article↗
PubMed2001

M(R) 77 KDA factor derived from fibroblasts stimulates the invasion ability of breast-cancer cells.

Breast-cancer cells frequently invade into the skin. However, few reports have described the mechanisms responsible for this invasion. In this study, we investigated the effects of skin fibroblasts on the invasion ability of breast-cancer cells, using a modification of Boyden's chamber method, and purified an invasion-stimulating factor from fibroblasts. Conditioned medium of skin fibroblasts, DF-1, significantly stimulated the invasion ability of breast-cancer cells, OCUB-1. The invasion-stimulating factor produced from skin fibroblasts was then partially purified and characterized. The soluble invasion-stimulating factor of fibroblasts was a protein of 77 kDa, as calculated by SDS-PAGE. Although TGF-alpha and bFGF stimulated the invasion of OCUB-1, no inhibition of the 77 kDa factor was achieved with neutralizing antibodies against TGF-alpha and bFGF. These findings suggest that the 77 kDa factor derived from skin fibroblasts might be unique, might play an important role in invasion to the skin and might explain the frequent skin invasion in breast carcinoma.

Biological Factors↗
PubMed2001

Translocation pathway of protein substrates in ClpAP protease.

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPgammaS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through approximately 150 A into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

Adenosine Triphosphatases↗
PubMed2001

Differential sensitivities of the MRP gene family and gamma-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status.

1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075-85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.

ATP-Binding Cassette Transporters↗
PubMed2001

X-ray-excited optical luminescence of impurity atom in semiconductor.

We observed the x-ray-excited optical luminescence (XEOL) of erbium-doped silicon (Si:Er) thin films to make a site-selective x-ray absorption fine structure (XAFS) measurement of an optically active Er atom. The undulator beam was used for the increment of the electron population in the excited state, and following XEOL at an infrared wavelength of 1.54 microm with minimum absorption loss in the host Si was detected. The edge-jump and XAFS oscillation were successfully obtained at the Er L(III)-edge. This spectrum originated from inner-shell excitation and relaxation of only the optically active Er atom, indicating that site-selectivity at an atomic level was achieved.

Journal Article↗
PubMed2001

Multielectron excitations probed by helicity-modulation XMCD at K-edge in 3d transition metal compounds.

Multielectron excitations (MEE) have been investigated using X-ray magnetic circular dichroism (XMCD) at the K-edge in 3d transition metal compounds. In the range of 50-70 eV above the absorption edge, a dichroic signal that is associated with MEE was identified. This phenomenon is thought to occur due to the super Coster-Kronig transition described as the final states (1s)1(3p)5(3d)n+2 resulting from the 3p --> 3d transitions. In order to verify this interpretation, XMCD measurements using the helicity-modulation method and calculations of the dichroic spectrum were performed based on this super Coster-Kronig transition.

Journal Article↗
PubMed2001

Tuning of X-ray phase retarder for magnetic EXAFS spectroscopy in helicity modulation mode.

A technique for polarization tuning over a wide energy range has been developed for magnetic EXAFS spectroscopies using a dia mond x-ray phase retarder in helicity modulation mode. A quick rocking scan using a piezo stage enabled us to tune the angle of retarder crystal with the precision of +/-2 arcsecond within 1 s. Available energy range of circular dichroism measurements has been extended to 6-16 keV by adopting not only undulator gap scan but also the present tuning technique. This technique has been applied to magnetic EXAFS measurements in pure iron at the Fe K-edge. Higher signal-to-noise ratio was obtained in reduced data acquisition time.

Journal Article↗
PubMed2001

Variation of XMCD spectrum with temperature at R L2,3-edges in R3Fe5O12 (R=Gd and Er).

The variation of X-ray magnetic circular dichroism (XMCD) with temperature has been measured at R L2,3-edges in R3Fe5O12 (R=Gd and Er). A drastic change in the spectral profile has been observed at low temperatures. The variation of XMCD peak intensity, corrected by the contribution of Fe with temperature, is in agreement with the magnetization of R sublattice. The results suggest that Fe 3d polarization plays an important role to produce the R L2,3-edges XMCD spectrum.

Journal Article↗
PubMed2001

Undulations in a confined lamellar system with surface anchoring.

We visualize undulations in layered systems using a cholesteric stripe phase with a macroscopic supramicron periodicity. The wave vector of stripe pattern is in the cell's plane. The undulation is induced by an in-plane magnetic field normal to the stripes. The observed displacement of layers is much larger than the value predicted by the Helfrich-Hurault classic theory. We propose a model of undulations that explains the data by finite surface anchoring of layers.

Journal Article↗