Biomedical subjects

Takashi Konno

Publications and source records attributed to Takashi Konno.

14 recordsLinked to original sources

PubMed2006

Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking.

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.

Bacterial Proteins↗

PubMed2005

Effect of chymase on intraocular pressure in rabbits.

Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1-31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), attenuated the ocular hypertension induced by chymase. Endothelin-1 (1-31) also caused ocular hypertension, which was inhibited by a selective endothelin ET(A) receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1-31) and subsequent activation of endothelin ET(A) receptors are involved in the development of ocular hypertension induced by chymase.

Animals↗

PubMed2005

Photoreactions between [60]fullerene and various aromatic tertiary amines.

The photoreactions of [60]fullerene with aromatic tertiary amines such as N,N-dimethylaniline and N,N-dimethyl-1-naphthylamine gave two or three types of [60]fullerene adducts. The reaction efficiency in the series of p-substituted N,N-dimethylanilines remarkably increased with increasing electron-donating properties of aromatic tertiary amines employed.

Journal Article↗

PubMed2005

2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production.

Adenosine↗

PubMed2005

Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma.

Adenosine↗

PubMed2005

Paradoxical inhibition of protein aggregation and precipitation by transglutaminase-catalyzed intermolecular cross-linking.

Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced beta-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked beta-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular cross-linking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions.

Animals↗

PubMed2005

Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking.

Two different types of physical bonding have been proposed to involve in the formation of neuronal inclusions of patients with neurodegenerative diseases such as Alzheimer's, Parkinson's, and polyglutamine diseases. One is the noncovalent bonding that stabilizes the amyloid-type fibrous aggregates, and the other is the covalent cross-linking catalyzed by tissue transglutaminase. The cross-linking is subdivided into the inter- and intramolecular cross-linking. Little attention has been paid to the pathological roles of the intramolecular cross-linking. To elucidate the possible interplay between the intramolecular cross-linking and the amyloid-type fibril formation, we performed an in vitro aggregation analysis of three intracellular amyloidgenic proteins (a domain of tau protein, alpha-synuclein, and truncated yeast prion Sup35) in the presence of tissue transglutaminase. The analysis was performed in low concentrations of the proteins using techniques including thioflavin T binding and mass spectrometry. The results demonstrated that the amyloid-type fibril formation was strongly inhibited by the transglutaminase-catalyzed intramolecular cross-linking, which blocked both the nucleation and the fiber extension steps of the amyloid formation. Far-UV CD spectroscopy indicated that the cross-linking slightly altered the backbone conformation of the proteins. It is likely that conformational restriction imposed by the intramolecular cross-links has impaired the ordered assembly of the amyloidgenic proteins. Nonamyloid type aggregation was also suppressed by the intramolecular cross-links. On the basis of the results, we proposed that tissue transglutaminase is a modulator for the protein aggregation and can act defensively against the fibril deposition in neurons.

Adult↗

PubMed2005

Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin.

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 micros after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.

Circular Dichroism↗

PubMed2004

Anionic contribution for fibrous maturation of protofibrillar assemblies of the human tau repeat domain in a fluoroalcohol solution.

Tau protein forms fibrous aggregates in the brain of patients with Alzheimer's disease. This type of aggregation in vitro is promoted efficiently by polyanions and anionic micelles. Here, we report another cosolvent system that induces the fibrous aggregation of human tau four-repeat domain (tau4RD). The protein aggregation was primarily achieved by a nonanionic agent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), while the ionic condition was modified by inorganic salts. The aggregation analysis by three spectroscopic methods revealed a two-phase kinetics of the aggregation of tau4RD in the presence of HFIP at approximately 4-6%. Large increases in the light-scattering, the thioflavin-binding, and the secondary structure content of tau4RD have progressed within a few minutes at 37 degrees C, which was followed by another slower aggregation phase. Electron microscopic analysis demonstrated that the amorphous granules are formed in the faster step, which acquired a fibrous shape in the slower step in the solution containing NaCl. In the absence of the salt, however, the fibrous maturation was inhibited. Examination of various salt species in place of NaCl demonstrated that binding of anions to the precursor aggregates was essential for the fibrous maturation. On the basis of the results, we proposed an aggregation scheme of tau in which the formation of a thioflavin-binding intermediate occurred ahead of its fibrous maturation. The anionic environment was suggested to play a crucial role in the fibrous maturation and, therefore, could be an in vivo determinant of the morphology of the aggregates of tau.

Adult↗

PubMed2004

Pressure-dissociable reversible assembly of intrinsically denatured lysozyme is a precursor for amyloid fibrils.

Although a diversity of proteins is known to form amyloid fibers, their common mechanisms are not clear. Here, we show that an intrinsically unfolded protein (U), represented by a disulfide-deficient variant of hen lysozyme with no tertiary structure, forms an amyloid-like fibril after prolonged incubation. Using variable pressure NMR along with sedimentation velocity, circular dichroism, and fluorescence measurements, we show that, before the fibril formation, the protein forms a pressure-dissociable, soluble assemblage (U'(n)) with a sedimentation coefficient of 17 S and a rich intermolecular beta-sheet structure. The reversible assemblage is characterized with a Gibbs energy for association of -23.3 +/- 0.8 kJ.mol(-1) and a volume increase of 52.7 +/- 11.3 ml.mol(-1) per monomer unit, and involves preferential interaction of hydrophobic residues in the initial association step. These results indicate that amyloid fibril formation can proceed from an intrinsically denatured protein and suggest a scheme N <==>U <==>U'(n)-->fibril as a common mechanism of fibril formation in amyloidogenic proteins, where two-way arrows represent reversible processes, one-way arrow represents an irreversible process, and N, U, and U'(n)represent, respectively, the native conformer, the unfolded monomeric conformer, and the soluble assemblage of unfolded conformers.

Amyloid↗

PubMed2004

Effects of 2-alkynyladenosine derivatives on intraocular pressure in rabbits.

We evaluated the activities of 2-alkynyladenosine derivatives, relatively selective adenosine A2 receptor agonists, in the intraocular pressure regulation in rabbits. An adenosine A2 receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) decreased intraocular pressure, while another A2 receptor agonist 2-(phenylamino)adenosine transiently increased it. The first group of 2-alkynyladenosine derivatives (1-hexyn-1-yl derivatives) caused a transient increase followed by decrease in intraocular pressure, while the second group (1-octyn-1-yl and 6-cyano-1-hexyn-1-yl derivatives) only decreased it. The second group is also effective in the ocular hypertensive models induced by water-loading and alpha-chymotrypsin. The outflow facility was increased by a 1-octyn-1-yl derivative. Both increase and decrease in intraocular pressure induced by 2-alkynyladenosine derivatives were inhibited by an adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropyl xanthine. These findings suggest that 2-alkynyladenosine derivatives may affect intraocular pressure via adenosine A2 receptor, and 2-alkynyladenosine derivative-induced ocular hypotension is due to the increase of outflow facility.

Adenosine↗

PubMed2004

[Role of adenosine in intraocular pressure].

Adenosine is thought to participate in the regulation of intraocular pressure since adenosine and several adenosine derivatives increase and/or decrease intraocular pressure. This article reviews the involvement of adenosine receptors in the regulation of intraocular pressure and the possible application of relatively selective adenosine A(2)-receptor agonists, 2-alkynyladenosine derivatives (2-AAs), as novel drugs for treatment of glaucoma. We found that some 2-AAs decreased intraocular pressure in normotensive rabbits. Moreover, these 2-AAs are also effective in the ocular hypertensive models induced by water-loading and alpha-chymotrypsin. In addition, the ocular hypotension induced by 2-(1-octyn-1-yl) derivative was inhibited by an adenosine A(2)-receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not by an adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropyl xanthine. Moreover, the outflow facility was increased by the 2-(1-octyn-1-yl) derivative. These findings suggest that 2-AAs may affect intraocular pressure via adenosine A(2)-receptor, and 2-AAs-induced ocular hypotension is due to the increase in outflow facility. Some 2-AAs may be novel drugs against ocular hypertension and/or glaucoma, although additional studies are required to characterize the effects of 2-AAs on regulation of intraocular pressure in detail.

Adenosine↗

PubMed2003

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe.

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.

Amino Acid Motifs↗

PubMed2003

Structural dissection of alkaline-denatured pepsin.

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.

Amino Acid Sequence↗