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Increasing evidence suggests that the metal homeostasis is involved in the pathogenesis of various neurodegenerative diseases including senile type of dementia such as Alzheimer’s disease, dementia with Lewy bodies, and vascular dementia. In particular, synaptic Zn2+ is known to play critical roles in the pathogenesis of vascular dementia. In this article, we review the molecular pathways of Zn2+-induced neurotoxicity based on our and numerous other findings, and demonstrated the implications of the energy production pathway, the disruption of calcium homeostasis, the production of reactive oxygen species (ROS), the endoplasmic reticulum (ER)-stress pathway, and the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) pathway. Furthermore, we have searched for substances that protect neurons from Zn2+-induced neurotoxicity among various agricultural products and determined carnosine (β-alanyl histidine) as a possible therapeutic agent for vascular dementia.

Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l -isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10−3 /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10−3 /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10−3 /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.

Increasing evidence suggests that amyloid formation, i.e., self-assembly of proteins and the resulting conformational changes, is linked with the pathogenesis of various neurodegenerative disorders such as Alzheimer’s disease, prion diseases, and Lewy body diseases. Among the factors that accelerate or inhibit oligomerization, we focus here on two non-genetic and common characteristics of many amyloidogenic proteins: metal binding and asparagine deamidation. Both reflect the aging process and occur in most amyloidogenic proteins. All of the amyloidogenic proteins, such as Alzheimer’s β-amyloid protein, prion protein, and α-synuclein, are metal-binding proteins and are involved in the regulation of metal homeostasis. It is widely accepted that these proteins are susceptible to non-enzymatic posttranslational modifications, and many asparagine residues of these proteins are deamidated. Moreover, these two factors can combine because asparagine residues can bind metals. We review the current understanding of these two common properties and their implications in the pathogenesis of these neurodegenerative diseases.

Carnosine (β-alanyl-L-histidine) is a small dipeptide with numerous beneficial effects, including the maintenance of the acid-base balance, antioxidant properties, chelating agent, anti-crosslinking, and anti-glycation activities. High levels of carnosine and its analogue anserine (1-methyl carnosine) are found in skeletal muscle and the brain. Zinc (Zn)-induced neurotoxicity plays a crucial role in the pathogenesis of vascular dementia (VD), and carnosine inhibits Zn-induced neuronal death. Here, the protective activity of carnosine against Zn-induced neurotoxicity and its molecular mechanisms such as cellular Zn influx and Zn-induced gene expression were investigated using immortalised hypothalamic neurons (GT1-7 cells). Carnosine and anserine protected against Zn-induced neurotoxicity not by preventing increases in intracellular Zn(2+) but by participating in the regulation of the endoplasmic reticulum (ER) stress pathway and the activity-regulated cytoskeletal protein (Arc). Accordingly, carnosine and anserine protected against neurotoxicity induced by ER-stress inducers thapsigargin and tunicamycin. Hence, carnosine and anserine are expected to have future therapeutic potential for VD and other neurodegenerative diseases.

Photoaffinity labeling enables analysis of weak biological interactions through formation of a covalent bond between ligands and receptor proteins upon photolysis of photoreactive groups. The photoreactive group, 3-trifluoromethyl-3-phenyldiazirine, has many advantages over other photoreactive groups like phenylazide and benzophenone, but its use is limited due to the difficulty to incorporate this photophore within macromolecules. The chemoselective ligation of versatile photoreactive units to macromolecules is a reliable solution for this difficulty; this approach easily attaches the useful 3-trifluoromethyl-3-phenyldiazirine photophore to biomacromolecules. Photoreactive bromide unit bearing 3-trifluoromethyl-3-phenyldiazirine is chemoselectively attached to phosphorothioate linkages of modified oligonucleotides to prepare photoreactive DNA, which is then used in a photoaffinity electrophoretic mobility shift assay for analyzing the proteins bound to a specific DNA motif. This review focuses on the preparation of photoreactive biomacromolecules such as peptides, proteins, oligosaccharides, and DNA based on our photoreactive unit technique and describes their application as novel approaches for the photoaffinity labeling of biomacromolecules.

Prion diseases are progressive neurodegenerative diseases that are associated with conformational changes that convert normal cellular prion protein (PrP(C)) into an abnormal pathogenic prion protein (PrP(Sc)). It is widely recognized that prion diseases are forms of transmissible amyloidosis and are considered to be protein-misfolding diseases (conformational diseases), a category that also includes Alzheimer's disease. Trace elements play crucial roles in the conformational change affecting PrP(C), and increasing evidence suggests that PrP(C) is a metal-binding protein that is involved in the homeostasis of Cu, Zn, and Fe. In this article, we review the current understanding of links between trace elements and the conformational change to PrP(Sc), based on our studies using synthetic prion peptides, as well as other new findings. We also focus on PrP(Sc)-induced disruption of Ca homeostasis as a molecular mechanism for neurodegeneration in prion diseases. Possible roles of carnosine (ß-alanyl histidine) as a candidate neuroprotective substance use in prion diseases are also discussed.

糖質に分類されるシアル酸は、様々な生体機能を有していることが知られている。従って、シアル酸を含む糖鎖を化学合成することは、生命現象の解明に寄与するものと考えられる。しかし、シアル酸を糖供与体とする糖鎖合成は困難とされており、近年多くの研究者らによって、新たな処方が開発されてきている。我々は、シアル酸が持つ生体機能の興味を持ち、これを有する糖鎖誘導体 (5) の合成を試みた。, The sialic acids are now defined as a family of carbohydrates, have very important biological functions. Thus, it considers that oligosaccharides containing them employ elucidation of biological phenomena. However, for synthesis of them glycosylations using donor of sialic acid derivative have been very hard to achieve both high yields and stereoselectivity of them. But recently, Synthesis methods of oligosaccharides containing sialic acids were developed by many researchers. Highly regioand α-stereoselective sialyl glycoside syntheses have been achieved by using, as glycosyl donors, the 2-thioglycoside of Neu5Ac. We were interested in biological functions of oligosaccharide containing sialic acid, and tried to synthesize sialyl lewis X and synthesized trisaccharide derivative which was precursor of sialyl lewis X, α-L-Neu5Ac(2 → 3)β-D-Gal(1 → 4)β-D-GlcNAc-MP.

光アフィニティーラベル法は、糖鎖- レクチンの相互関係を明らかにする最も効果的な手法である。従来法では、プローブ合成時に還元末端糖が開環してしまっていたが、我々は、完全な糖鎖構造 (D- ラクトース) からなるリガンドとフェニルジアジリン基を有する分子プローブを開発した。次の問題点として、糖鎖は複雑な構造を有していることから、本分子プローブの合成法の確立が重要であると考えた。我々は、オルソゴナルグリコシル化法により糖鎖部分の合成を目指したが、反応時に糖受容体と糖供与体の脱離基が置換されてしまい、目的化合物が得られなかった。そこでトリクロロアセトイミデート法を用い、糖鎖合成に着手したところ、D- ガラクトース-D- グルコサミンから成る二糖誘導体が得られ分子プローブ(1、2、3) へと導かれた。, Photoaffinity labeling technology is the most efficient method for elucidation of carbohydrate - lectin interactions. However, when carbohydrate probes are synthesized according to conventional this method, the reducing terminus of the sugar is opened to provide an acyclic structure. We were undertaken to solve this problem, and developed new molecular probe containing complete oligosaccharide structure (D-lactose) and phenyldiazirine group for elucidation of carbohydrate - lectin interactions. Next problem, establishment of synthesis method of molecular probe is important subject because carbohydrates are composed of various complex structures. We tried synthesis of oligosaccharides using orthogonal glycosylation strategy. But when glycosyl donor and glycosyl acceptor were coupled by promoter, leaveing group of glycosyl donor (-F group) was substituted to leaving group of glycosyl acceptor (-SPh group). We synthesized Galp-GlcpNAc disaccharide derivatives using glycosyl trichloroacetimidate strategy, and then were obtained 3 types of molecular probes containing ligand and photoreactive group (1, 2, 3).

アカパンカビ(Neurospora crassa)は培養液中のカルシウム濃度の変化により大きくその成長率が変わる.そのため培養中のカルシウム濃度が異なると,アカパンカビの細胞内の組成も変化すると考えられる.今回,培養中のカルシウム濃度がアカパンカビの細胞タンパク質の組成にどのような変化を与えるか調べた.その結果,野生株で細胞外カルシウム濃度に依存して量が変化する4種類のタンパク質の存在が明らかになった.カルシウムホメオスタシスに欠陥があり,高濃度カルシウム存在下では成長が抑制される突然変異株ca-1(19)を用い同様に調べた結果,低濃度カルシウムで培養したときにも2種類のタンパク質の量が増加していることが明らかになった.これら分子量91 と32 kDaの2つのタンパク質は,アカパンカビのカルシウムストレスを示すマーカータンパク質になる可能性がある., Because mycelial growth of filamentous fungus, Neurospora crassa, is dependent on the extracellular calcium concentration, calcium condition during the culture stage may affect the structural onstitution of Neurospora cell. In this study, we examined the effect of extracellular calcium concentration on the protein constitution of Neurospora cells, and found four proteins in wild-type strain which varied in quantity depending on extracellular calcium concentration. Two of four candidate proteins were highly expressed even at low extracellular calcium concentration in the case of defective mutant strain in calcium homeostasis, ca-1(19), which could not grow in high extracellular calcium concentration. These results suggest that two proteins of which molecular mass are 91 and 32 kDa are candidates for marker proteins of calcium stress in Neurospora cell.

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

A method for analyzing the structural alterations in Asn or Asp residues was developed by using the peptides related to neuronal conformational diseases, i.e., the prion protein (PrP)(106-126) and the Alzheimer's amyloid beta (A beta) protein(6-28). The alterations were analyzed by reversed-phase (RP) HPLC, because the peptides containing the structurally altered residues were diastereoisomers of each other, and they were separated with the mobile phase containing an MeCN/sodium phosphate solution and NaCl. The amount of L-Asp, L-isoAsp, D-Asp, or D-isoAsp residues in each PrP peptide isomer was simultaneously quantified by carrying out single HPLC analysis; these residues were generated by the deamidation of the Asn residue. Only 0.3% of the newly generated peptide containing the D-Asp residue was detected. Furthermore, the investigation of the partial fragment of the A beta protein revealed that the present method possessed the ability of simultaneous analysis of the isomerizations of two Asp residues. These results implied that the present method was highly sensitive and reduced the time required for the analysis. This method may accelerate the elucidation of the PrP and A beta protein functions, because the structural alterations of Asn and Asp have been reported to influence these functions.

紫外線は様々な生体分子に影響を与えることが知られている.今回,我々はUV-B紫外線に対するアミノ酸およびペプチドの安定性を調べた.15種類のペプチドにUV-Bを照射した実験結果から,Trp, His, Met, Phe, Tyrの5種類のアミノ酸を含むペプチドの安定性が低いことがわかった.アミノ酸およびペプチドの安定性は共存するアミノ酸の有無で大きく変化することも明らかになった.Metは共存するアミノ酸があるときのみUV-B照射で減少し,αAクリスタリンを構成するペプチド断片は,タンパク質に紫外線を照射したときに容易に分解した.これらの結果は紫外線を吸収しやすいアミノ酸が他のアミノ酸やペプチド断片の安定性に影響を与えていることを示唆している., Ultraviolet (UV) light has been reported to influence on various biological components. In this study, we examined the stability of amino acids and peptides against the irradiation of UV-B, the light with 320-400 nm wavelength range. The experiments using 15 types of peptides suggested that the peptides containing five amino acids, Trp, His, Met, Phe, and Tyr were easily reduced by UV-B irradiation. Both stabilities of amino acid and peptide against UV-B irradiation were differed by the presence of coexistent of other amino acids. The amino acid, Met was drastically reduced by UV-B irradiation only in the case of coexistent of other amino acids, and the peptides located in the ��Acrystallin protein also were easily degraded by the irradiation. These results may suggest that amino acids, which easily absorb UV light, had an effect on the stabilities of other amino acid and some peptide fragments.

軟骨細胞に局在するシステインプロテアーゼであるカテプシンKは,骨の差異九州に関わる酵素である。カテプシンKの阻害剤は骨粗鬆症の治療薬の候補になり得る。スクリーニングや酵素の機能を調べるためには組み換えカテプシンKを大量に作成することが必要である。我々はカイコ幼虫を用いた発現系が分泌型酵素であるカテプシンKの発現に適していると考えた。C末端にmycタグを付けたカテプシンK遺伝子をカイコ用の発現ベクターに挿入し,限界希釈法で組み換えウイルスを得た。このウイルスをカイコ培養細胞に感染させてカテプシンKの発現場所を調べたところ,細胞内で発現していた。細胞外への分泌が観察されなかったのでカイコ幼虫での発現をあきらめ,培養細胞からの精製を試みた。DEAE-セファロースカラム,Mono-Sカラム,抗myc抗体アフィニティーカラムで順次精製した結果,非活性を細胞可溶性画分の約300倍に高めることに成功した。, Cathepsin K, which is localized in osteoclasts, plays an important role of bone resorption. Selective inhibitors of cathepsin K therefore can be promising therapeutic candidates for the treatment of diseases such as osteoporosis. Recombinant cathepsin K protein is necessary for screening the therapeutic candidates and elucidating the enzymatic functions because the amount of native cathepsin K is very low. Silkworm is one of the most attractive hosts for large-scale production of eukaryotic secretory proteins. The gene for fusion cathepsin K protein with myc-tag at the C-terminus was inserted into expression vector for silkworm and recombinant viruses were isolated by end-point dilution assay. Cathepsin K was expressed in the cell lysate, but not in the medium, and consequently we gave up the idea of cathepsin K expression in the silkworm. Thus, we tried to isolate cathepsin K protein from the cultured cells, and purified it by sequential operations with DEAE-Sepharose column, Mono-S column, and anti-myc antibody column. The purification fold of cathepsin K activity increased approximately 300 times in comparison with that in soluble fraction of cell lysate.

Formation of a covalent bond using photoreactive groups allows one to maintain the complex between the ligand and its binding protein even under denaturing conditions. Thus, the photoreactive groups functioned as the powerful hook in the fishing of specific binding proteins. This is a very useful feature for developing the efficient and sensitive methods in molecular biology. Among the photoreactive groups, carbene-generating phenyldiazirine is a first choice for the application to the methods because the use of phenyldiazirine seems to be promising for achieving efficient crosslinking. This review describes improvements of phenyldiazirine usability and an application of phenyldiazirine to displayed phage screening. We synthesized the photoreactive diazirine units for a solution to preparing photoreactive ligands. Since the photoreactive units can be easily integrated into physiological ligands such as peptides, proteins, DNAs, and sugars by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. We applied the phenyldiazirine to screening of displayed phages, and investigated the screening efficiency. The phages displayed specific-binding protein were extremely concentrated by using photoreactive peptide bearing diazirine. High efficiency in the screening is due to carry out intensive washing which removes almost all the unspecific binding phages. Moreover, such application overcomes the unavoidable drawback of photoreactive groups, the low efficiency of crosslinking because the isolated genes can be amplified.

オピオイド結合細胞接着分子(OBCAM)タンパク質は、イムノグロブリンのスーパーファミリーに属し、エンケファリンやβ-エンドルフィンなどのオピオイドリガンドを認識する。我々は、大腸菌で組換えOBCAMタンパク質と、β-エンドルフィンの部分配列をもとにジアジリンとビオチンをもつ光アフィニティーリガンドとを製作し、光アフィニティーラベル実験を行った。光照射により両者が共有結合で結ばれた複合体を形成した。しかしながら、[Leu]-エンケファリン,[D-Ala^2, D-Leu^5]-エンケファリンおよびβ-エンドルフィンを過剰に加えても、この複合体形成を阻害できなかった。光アフィニティーリガンドと同一の構造をもつ(ジアジリンをもつ)ペプチドアナログのみが複合体形成を阻害した。このペプチドアナログ中のジアジリンを光分解したものは阻害活性を失った。これらの結果は、オピオイドペプチド中のジアジリン構造がOBCAMタンパク質との親和性を増強させていることを示している。, An opioid-binding cell-adhesion molecule (OBCAM) protein, which is an immunoglobulin superfamily protein, recognizes various opioid ligands including enkephalin and β-endorphin. The molecular mechanisms of ligand-binding interface of the proteins are not yet clear. We prepared a bacterial expressed OBCAM protein using pGEX4T expression system, and photoaffinity ligand bearing diazirine and biotin, which was constructed using the partial sequence of β-endorphin, Tyr-Gly-Gly-Phe-Leu-Thr-Ser-Glu-Lys-Ser. Crosslinking formation between OBCAM protein and photoaffinity ligand was confirmed by SDS-PAGE analysis. Neither excess amount of [Leu]-enkephalin, [D-Ala^2, D-Leu^5]-enkephalin, nor β-endorphin inhibited the crosslinking formation. The diazirine-bearing peptide that had an identical amino acid sequence to that of the photoaffinity ligand only inhibited the crosslinking formation, and the removal of diazirine on the peptide by photolysis resulted in loss of the inhibitory activity. These results show that diazirine group on the opioid ligand enhanced its binding affinity to the OBCAM protein.

Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20-60 S at 37 degrees C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

Asp58 and Asp151 in human lens alphaA-crystallin invert and isomerize to d-beta-aspartyl residues with age. Here, we report that the racemization rate constants (k) of Asp58 and Asp151 residues in human recombinant alphaA-crystallin at 37 degrees C are 3.72 +/- 0.8 x 10(-4) and 10.7 +/- 0.7 x 10(-4)/day, respectively. The activation energy of racemization of Asp58 and Asp151 in the protein was 27.0 +/- 0.5 kcal/mol and 21.0 +/- 0.5 kcal/mol, respectively. The time required for the D/L ratio of Asp58 and Asp151 to approximate to 1.0 (D/L ratio of Asp = 0.99) at 37 degrees C was estimated as 20.9 +/- 3.7 and 6.80 +/- 0.4 years, respectively. Thus, Asp151 is more susceptible to racemization than Asp58, consistent with data from short model peptides. However, the racemization rates of both Asp58 and Asp151 residues in the protein were twice as rapid as in model peptides. These results indicate that the racemization of Asp residues in alphaA-crystallin may be influenced not only by the primary structure but also by the higher order structure around Asp residues in the protein.

Zinc is an extremely most abundant trace element in the brain. Substantial amounts of zinc exist in the presynaptic vesicles, and are released with glutamate during the neuronal excitation. Synaptically-released zinc is believed to play crucial roles in normal brain functions. Therefore, zinc deficiency impairs brain development and capabilities of learning and memory. Notwithstanding, recent studies have indicated that excess zinc is linked with several neurodegenerative diseases and has a causative role in delayed neuronal death after transient global ischemia. We have developed the sensitive assay system for zinc neurotoxicity in vitro using GT1-7 cells (immortalized hypothalamic neurons) to elucidate the functions of zinc in neurodegenerative diseases. Pharmacological experiments have exhibited the involvement of energy failure, metal-metal interaction, and disruption of calcium homeostasis in zinc-induced neurotoxicity. It is inferred that zinc might play its part in brain functions as Janus, an ancient Roman god with two faces, and that zinc homeostasis is essential for the neuronal survival. Our assay system provides a good method for screening the protective substances of zinc neurotoxicity as a therapeutic target of the global ischemia.

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.

胎盤組織に局在するカテプシン(システインプロテアーゼ)は、ここ数年で8種類も同定されたが、いずれも遺伝子レベルの発現が確認されているだけである。本研究では、妊娠後11.5日から15.5日までのマウス胎盤でのみ発現するカテプシン-P/J (Cat-P/J)および-6 (Cat-6) に注目し、組換えタンパク質の製作による機能解明を目指した。両組換えタンパク質は大腸菌pET発現系で製作した。蛍光基質での解析により、Cat-Pはカテプシンの典型的基質であるZ-Phe-Arg-MCAとαスロンビンの基質であるBoc-Val-Pro-Arg-MCAの両者を、Cat-6は後者のみを切断することが明らかになった。これらの活性は非常に弱いものであったが、両者の基質特異性の差が初めて明らかになった。次に高活性の組換え体を得る目的で、昆虫細胞発現系での製作を試みた。組換えCat-P/Jの発現は、感染開始から4日目の培地中で最大となることを確認したが、精製するには至らなかった。, More than twenty cathepsins, a major component of trhe lysosomal proteolytic system, are identified. In the last decade, the cathepsins which show tissue-restricted expression pattern have been disconered. In placenta, eight novel genes related to cathepsin L have been recently identified. To characterize the cathepsins highly expressed in placenta, we prepare two recombinant cathepsins, cathepsins-P/J(Cat- P/J) and -6(Cat-6) by E. coli expression system with pTE-3 vector. Cat-P/J hydrolyzed both A-Phe-Arg-MCA, a typical substrate for cathepsins, and Boc-Val-Pro-Arg-MCA, a substrate for a-thronbin, while Cat-6 hydrolyzed only Boc-Val-Pro-Arg-MCA. Though these activities are very weak, this is the frist report describing the difference of substrate specificity between Cat-P/J and -6. Next we tried preparing the recombinant Cat-P/J by a baculovirus expression system becouse it had several advantage to produce functional soluble proteins. The soluble recombinant Cat-P/J wa secreted into the culture medium, and its maximum amount was observed at 4days post-infection. However, the purification of Cat-P/J was not achieved because of small amount of the recombinant proteins.

Recently, we reported the amino acid sequence of chicken alpha1-acid glycoprotein (chicken alpha1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken alpha1-AGP using tryptic digests of carbamidomethylated chicken alpha1-AGP, carbamidomethylated completely deglycosylated chicken alpha1-AGP (cd-alpha1-AGP), and nonreduced denatured cd-alpha1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides mlz 3037.4 (amino acid sequences 69-76 plus 161-183) and 3453.3 (amino acid sequences 69-80 plus 161-183), the two disulfide bonds of chicken alpha1-AGP were determined to be located at Cys 6-Cys 146 and Cys 73-Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated.

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp 151 in recombinant human αA-crystallin. First, four types of dodecapeptide fragment ( 146 IQTGLD 151 ATHAER 157 ) in which the Asp residue was either l -Asp, d -Asp, l -isoAsp or d -isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp 151 -containing peptide fragments isolated from the tryptic hydrolysate of recombinant αA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing d -Asp, l -isoAsp and d -isoAsp, were generated when αA-crystallin was incubated for 140 days at 37 °C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing l -isoAsp and d -isoAsp represented dodecapeptide fragments containing l -isoAsp 151 and d -isoAsp 151 , respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing d -Asp represented dodecapeptide fragments containing d -Asp 151 . The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp 151 residue in human αA-crystallin, the degree of isomerization from l -Asp to d -Asp can be determined to a level as low as 0.3%.

We describe a rapid HPLC method for measuring the isomerization levels of Asp-residues within peptides. This method can detect the isomerization of Asp-residue at a level of less than 1% without performance of acid hydrolysis. Using this method, we further studied the isomerization of Asp-residues in the αA-crystallin f(55–65) fragment of sequence TVLDSGISEVR (T6). Four isomers of T6 peptides, which contain L-Asp, L-isoAsp, D-Asp, and D-isoAsp, respectively, were synthesized. Following incubation of any one of these peptides, four possible isomers of T6 peptides were found in an aqueous buffer. The isoAsp/ Asp ratios of peptides in these solutions were found to reach 3.6–3.8 after incubation at 90°C for 72 hr.

application/pdf, An efficient synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based phenylalanine derivatives is described. Alkylation of a chiral glycine equivalent with a spacer containing (3-trifluoromethyl)phenyldiazirinyl bromides enables us to create photoreactive L-phenylalanine derivatives. After introduction of biotin at the spacer, the biotinylated and photoreactive amino acid was applied for L-amino acid oxidase and incorporated into a substrate binding site. These compounds will be powerful tools not only for photoaffinity labeling to elucidate properties of bioactive peptides but also as trifunctional photophors to introduce a ligand skeleton. (C) 2002 Elsevier Science Ltd. All rights reserved.

Two novel peptides that inhibit cathepsin B were isolated from a tryptic and chymotryptic hydrolysate of human serum albumin, and designated as Cabin-A1 and -A2. Cabin-A1 and -A2 were purified by reversed-phase HPLC and identified as Ser-Leu-His-Thr-Leu-Phe and Phe-Gln-Asn-Ala-Leu, respectively. These peptides correspond to f(65-70) and f(403-407) of human serum albumin. Human albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg), which corresponds to f(210-218), was also isolated as a potent cathepsin B inhibitor. Synthetic Cabin-A1, -A2, and human albutensin A showed dose-dependent inhibition of cathepsin B, with K(i) values of 2.4, 290, and 3.8 microM, respectively.

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.

One of the major events occurring at biological interfaces is the specific recognition of bioactive ligands by their receptor proteins. The elucidation of interacting partners is an immediate entrance into the discovery of medicinal leads. The method of photoaffinity labeling enables the direct probing of target protein through a covalent bond introduced between a ligand and its specific receptor. Thus, the photoaffinity labeling is applied in two stages of drug discovery and development processes. First, the method is useful for the screening of early leads. If the binding site analysis of target protein is important for defining a particular pharmacophore, the photoaffinity labeling will give the structural information at the contact point of drugs with receptors. Second, emerging new technologies, combinatorial chemistry, recombinant DNA techniques, and high-throughput analysis, are extending the potential of photoaffinity labeling to become a rapid and more sensitive means for the identification of drug-receptor pairs as well as the elucidation of molecular recognition mechanism at drug-receptor interfaces. This review focuses on several recent impacts of photoaffinity labeling as a useful tool for drug discovery and developments.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

We previously described a novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated Acein-1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein-2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed-phase gradient and isocratic high performance liquid chromatography. Acein-2 was found to be a tripeptide, Leu-Ile-Tyr, which is thought to correspond to f(518–520) of human α2-macroglobulin. The synthetic tripeptide showed a potent dose-dependent inhibition of ACE, with an IC 50 value of 0.82 μmol/l. Lineweaver–Burk plots suggested that Acein-2 as well as the previously described Acein-1 are non-competitive inhibitors.

We optimized continuous elution electrophoresis (CEE) for rapid purification of PCR products. After PCR amplification, the reaction mixture is applied directly to CEE, and then the PCR products in the size range from 200 to 1500 bp are purified within nearly 10 min. CEE is able to separate two DNA fragments differing in length by 50 bp. As judged by ligation efficiency, the quality of PCR products separated by CEE is equal to that purified by extraction from the melting gels. CEE reduces operational time because purification of the PCR products is a repetitional procedure in recombinant DNA techniques.

Zinc (Zn) is an essential trace element that is abundantly present in the brain. Although Zn plays crucial roles in learning and memory, numerous studies have indicated that the disruption of Zn homeostasis, namely both depletion and excess Zn, causes severe damage to neurons and is linked with various neurodegenerative diseases including Alzheimer's disease and vascular dementia. Here, we review the current understanding of the role of Zn in the pathogenesis of these neurodegenerative diseases. Based on our findings and other numerous studies, Zn acts as a contributor to Alzheimer's disease in the oligomerization, and as a protector in the neurotoxicity of Alzheimer's β-amyloid protein. Furthermore, Zn plays a central role in ischemia-induced neuronal death and the pathogenesis of vascular dementia. Involvement of Ca(2+) dyshomeostasis and endoplasmic reticulum (ER) stress in the mechanism of Zn-induced neurotoxicity are suggested. We also discuss the possible role of carnosine (β-alanyl histidine), a dipeptide that is present in the brain, as a protective substance for neuronal injury.

The effects of calmodulin antagonists and inhibitors of protein kinases and phosphatases on light-induced phase shifting were investigated in Neurospora crassa. Calmodulin antagonists, namely, trifluoperazine, chlorpromazine, and W-7, almost completely inhibited the light-induced phase shifting without having any effect on the circadian clock itself. Chlorpromazine was less effective in inhibiting the light-induced phase shifting than trifluoperazine. W-5, a dechlorinated analogue of W-7, failed to inhibit the light-induced phase shifting at the same concentration as that at which W-7 was effective. These results suggest that calmodulin is required during signal transduction from the light-perceiving system to the circadian clock in N. crassa. Inhibitors of protein phosphorylation did not inhibit the light-induced phase shifting, although these inhibitors completely inhibited mycelial growth. Trifluoperazine partially inhibited the phosphorylation of three proteins when phosphorylation was assayed in vitro.

We have developed two novel 3-phenyl-3-trifluoromethyldiazirinyl bis-glucose derivatives to investigate the properties of the adipocyte glucose transporter GLUT4. These compounds were substituted by electron-withdrawing (iodo and nitro) groups on the aromatic ring of 3-phenyl-3-trifluoromethyldiazirine photophore and were found to be more photosensitive than compounds without such substituents. The compounds were used as inhibitors of insulin-stimulated glucose transport activity in order to assess half-maximal inhibition or relative affinity values for GLUT4. The affinities were found to be 60-130 times higher than the parent compound D-glucose. Because of the increased photo-reactivity and high affinity these compounds will be useful in studies directed at further elucidation of GLUT4 function.

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.

Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer'sβ-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasisviathe amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases orα-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Zinc (Zn) is an essential trace element that is abundantly present in the brain. Despite its importance in normal brain functions, excess Zn is neurotoxic and causes neurodegeneration following transient global ischemia and plays a crucial role in the pathogenesis of vascular-type dementia. We found that GT1-7 cells (immortalized hypothalamic neurons) are more vulnerable to zinc-induced neurotoxicity than other cultured neuronal cells are. Further, we investigated the molecular mechanisms of Zn-induced neurotoxicity in vitro using GT1-7 cells. Pharmacological evidence based on our own results and those of numerous other studies has indicated the significance of calcium dyshomeostasis in the mechanisms of Zn-induced neuronal injury. We developed a screening system for substances, which prevent Zn-induced neurotoxicity, with the aim of identifying a treatment for vascular-type dementia. Here, we review in detail the characteristics and mechanisms of Zn-induced neuronal death in relation to calcium homeostasis. The potential role of carnosine (s-alanyl histidine), a dipeptide present in the brain as an endogenous protective substance, in neuronal injury is also discussed.

Full-grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G2-like stage. Injection of the contents of germinal vesicles (GV-content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus-like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation-promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full-grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G2-like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G2-specific cytostatic factor (G2-CSF). G2-CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G2/M transition in the cell cycle of early embryonic cells.

Alpha-Crystallin, which forms a huge multimeric complex that is essential for maintaining eye lens transparency, is one of the major proteins in the lens. The protein, which exists as isoforms alphaA and alphaB, functions as a molecular chaperone to restore the original conformations of distorted constituent proteins in the lens. This function is important to maintain the transparency of the lens, because there is no protein turnover in the lens. Abnormal aggregation of constituent proteins in the lens has been reported in cataract patients, and deamidation of Asn as well as racemization and isomerization of Asp have been found in the alpha-Crystallin of these patients. While the establishment of a quick and facile analytical method is eagerly anticipated to investigate the relevance of the isomerization to pathological states such as cataracts, differentiating the isomerization states is still not performed routinely. Here, we report the differentiation and semiquantitative analysis of an isoaspartic acid (betaAsp) in human alpha-Crystallin using postsource decay on a MALDI-TOF mass spectrometer incorporating a curved field reflectron. Our reproducible results of analyzing synthetic and tryptic peptides containing betaAsp corroborated results obtained using a previously reported diagnostic ion, y(l-n+1) - 46, for the differentiation of betaAsp. The relative content of betaAsp in the peptide was successfully estimated from a unique ratio, y(l-n):y(l-n+1), corresponding to cleavages at the C- and N-termini, respectively, of the isomeric residues. The betaAsp content was consistent with measurements obtained independently by reversed phase HPLC analysis. Experiments in which neighboring amino acids adjacent to betaAsp/Asp were substituted revealed that the ratio between y(l-n) and y(l-n+1) reflected the isomerization status, while the diagnostic ion was observed only in the peptides that included an arginine residue at their C-terminus. Postsource decay experiments utilizing both the diagnostic ion and the characteristic fragment pattern could be applied to various kinds of peptides containing betaAsp.

We have developed two novel 3-phenyl-3-trifluoromethyldiazirinyl bis-glucose derivatives to investigate the properties of the adipocyte glucose transporter GLUT4. These compounds were substituted by electron-withdrawing (iodo and nitro) groups on the aromatic ring of 3-phenyl-3-trifluoromethyldiazirine photophore and were found to be more photosensitive than compounds without such substituents. The compounds were used as inhibitors of insulin-stimulated glucose transport activity in order to assess half-maximal inhibition or relative affinity values for GLUT4. The affinities were found to be 60-130 times higher than the parent compound D-glucose. Because of the increased photo-reactivity and high affinity these compounds will be useful in studies directed at further elucidation of GLUT4 function.

Formation of a covalent bond using photoreactive groups allows one to maintain the complex between the ligand and its binding protein even under denaturing conditions. Thus, the photoreactive groups functioned as the powerful hook in the fishing of specific binding proteins. This is a very useful feature for developing the efficient and sensitive methods in molecular biology. Among the photoreactive groups, carbene-generating phenyldiazirine is a first choice for the application to the methods because the use of phenyldiazirine seems to be promising for achieving efficient crosslinking. This review describes improvements of phenyldiazirine usability and an application of phenyldiazirine to displayed phage screening. We synthesized the photoreactive diazirine units for a solution to preparing photoreactive ligands. Since the photoreactive units can be easily integrated into physiological ligands such as peptides, proteins, DNAs, and sugars by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. We applied the phenyldiazirine to screening of displayed phages, and investigated the screening efficiency. The phages displayed specific-binding protein were extremely concentrated by using photoreactive peptide bearing diazirine. High efficiency in the screening is due to carry out intensive washing which removes almost all the unspecific binding phages. Moreover, such application overcomes the unavoidable drawback of photoreactive groups, the low efficiency of crosslinking because the isolated genes can be amplified.

Neurotoxicity of Alzheimer's beta-amyloid protein (AbetaP) is central to the pathogenesis of Alzheimer's disease (AD). Recent approaches have emphasized the importance of AbetaP oligomerization, which causes synaptic degeneration and neuronal loss, finally leading to the pathogenesis of AD. Although the precise molecular mechanism of AbetaP neurotoxicity remains elusive, our and other numerous findings have demonstrated that AbetaP directly incorporated into neuronal membranes formed calcium-permeable ion channels (amyloid channels) and resulted in an abnormal elevation of the intracellular calcium levels. The formation of amyloid channels and the abnormal increase of intracellular Ca(2+) have also been commonly observed in other neurodegenerative diseases, including conformational diseases such as prion disease or dementia with Lewy bodies. This article reviews the current understanding of the pathology of AD based on the hypothesis that the disruption of calcium homeostasis through amyloid channels may be the molecular basis of AbetaP neurotoxicity. The potential development of preventive agents is also discussed.