Yasuo Konishi

The Road to Advanced Glycation End Products: A Mechanistic Perspective

Protein glycation is a slow natural process involving the chemical modification of the reactive amino and guanidine functions in amino acids by sugars and carbohydrates-derived reactive carbonyls. Its deleterious consequences are obvious in the case of long-lived proteins in aged people and are exacerbated by the high blood concentration of sugars in diabetic patients. The non-enzymatic glycation of proteins occurs through a wide range of concurrent processes comprising condensation, rearrangement, fragmentation, and oxidation reactions. Using a few well established intermediates such as Schiff base, Amadori product and reactive a-dicarbonyls as milestones and the results of in vitro glycation investigations, an overall detailed mechanistic analysis of protein glycation is presented for the first time. The pathways leading to several advanced glycation end products (AGEs) such as (carboxymethyl)lysine, pentosidine, and glucosepane are outlined, whereas other AGEs useful as potential biomarkers of glycation are only briefly mentioned. The current stage of the development of glycation inhibitors has been reviewed with an emphasis on their mechanism of action.

Factors affecting molecular characteristics of whey protein gelation

Abstract The effects of pH, protein concentration, NaCl, heating temperature and time on the gelation of a whey protein concentrate (WPC) and the associated changes in the molecular conformation of the individual whey proteins were studied using polyacrylamide gel electrophoresis, high performance liquid chromatography and Fourier transform infrared spectroscopy. Heat denaturation was studied using differential scanning calorimetry. The results obtained showed that varying WPC concentration affected textural properties of gels without any observed differences in the molecular behavior of α-lactalbumin and β-lactoglobulin, while heating temperature, pH and NaCl affected both molecular and textural characteristics. WPC formed firm gels at temperatures above 70 °C, at alkaline pH range and in the absence of NaCl. When the whey proteins were heated, α-lactalbumin did not aggregate above pH 7 but denatured readily to form aggregates at acid pH while β-lactoglobulin aggregated at both acid and alkaline pH regions. Denaturation of α-lactalbumin at neutral pH resulted in an increase in viscosity while denaturation of β-lactoglobulin resulted in gel formation. The aggregation of WPC, particularly at alkaline pH, was attributed mainly to β-lactoglobulin and only minimally to α-lactalbumin.

The dihydro-β-agarofuran sesquiterpenoids

Covering: January 1990 to June 2006 Dihydro-β-agarofuran sesquiterpenoids are a structurally diverse class of natural products based on tricyclic 5,11-epoxy-5β,10α-eudesman-4-(14)-ene skeleton. Between January 1990 and June 2006, 462 new dihydro-β-agarofuran sesquiterpenoids of 74 structural types have been isolated from about 64 species of Celastraceae, 3 species of Hippocrateaceae and one species of Lamiaceae. The present review covers the chemical and biological activity research of dihydro-β-agarofuran sesquiterpenoids in the past 16 years. The chemical research includes structural classification into sesquiterpene polyesters and macrolide sesquiterpene pyridine alkaloids, synthesis of dihydro-β-agarofuran as well as extraction, isolation and purification methods. The biological activity research includes activities such as multidrug resistance (MDR) reversal activity, HIV inhibition, cytotoxicity, antitumor activity, antifeedant activity and insecticidal activity with some insights to their modes of actions.

High Accuracy Molecular Weight Determination and Variation Characterization of Proteins Up To 80 ku by Ionspray Mass Spectrometry

A quadrupole mass spectrometer with an ionspray interface was used to measure the molecular weight (MW) of proteins up to 80,000 u . With the improvements in instrument cahbration by a statistical averaging method and in data analysis by a gaussian curve-fitting method, precision of MW determination as high as 12 ppm was achieved with equine myoglobin (MW 16,950.4 ± 0.2 u). Exact MW determination of three components in cerato-ulmin revealed that the two minor ones had lost amino acid residues Ser and Ser-Asp, respectively, from the major component (MW 7618.4 ± 0.2 u). MW classifIcation of eight components in the Fab fragment of a monoclonal antibody revealed that one set of four had MW − 47,540 u and the other − 47,640 u. The MW difference of 100.2 ± 0.6 u between fragment 1 and 2, attributed to inhomogeneous cleavage at the Fab C-terminus, was probably due to one additional Thr in 1. The MW of bovine serum albumin (BSA) was found to be 66,431.5 ± 1.3 u, − 164 u higher than the calculated sequence MW, most probably because of the incorrectness in the previously reported BSA amino acid sequence. The MW of human serum transferrin (79,556.8 ± 1.7 u) was shown to be 4414 u higher than the sequence MW, pointing to a glycosylation of 22.7 sugar units in this protein. The greater complexity in bovine serum transferrin (MW 78,030.5 ± 1.8 and 78,326 ± 3.3 u for the two major components) was correlated with the heterogeneity in the glycosylation.

Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones.

ABSTRACT Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 μM versus Km = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km = 5.8 μM) and the highest catalytic efficiency (kcat/Km ratio of 7.2 × 105 M−1 s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

Stepwise refolding of Acid-denatured myoglobin: Evidence from electrospray mass spectrometry

Application of electrospray mass spectrometry (ES/MS) to a protein refolding study was demonstrated. Acid denaturation of equine myoglobin was reversed by adding various amounts of ammonium hydroxide to the protein that was unfolded in 10% acetic acid. The protein refolding process was followed by ES/MS, in which both the changes in the protein charge-state distribution and mass were monitored. The ES/MS results show that the pH-dependent renaturation of the acid-denatured myoglobin is stepwise, consisting of two major steps. The unfolded polypeptide chain first refolds to establish a compact nativelike structure, without the assistance of the heme prosthetic group. The newly formed binding cavity then retains the heme group by noncovalent interactions. It is also shown that inclusion of a stabilizing buffer, such as ammonium acetate, in the protein solution is greatly beneficial to the ES/MS detection of intact noncovalent globin/heme complex.

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N′‐acetyl [D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow‐tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α‐thrombin (K i=3.7±0.3 × 10−10 M) and bovine α‐thrombin (1.8±0.7 × 10−9 M). P79 showed saturable inhibition of plasma APTT. The P1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site‐directed tripeptide [D‐Phe‐Pro‐ArgΨ(COCH2)CH3COOCH3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α‐thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α‐thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α‐thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???‐acetyl[D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH2‐ and COOH‐terminal recognition sites.

Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-d-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

Antifungal activity of alkaloids from the seeds of Chimonanthus praecox.

Two alkaloids, D-calycanthine (1) and L-folicanthine (2), were isolated from the active MeOH extract of the seeds of Chimonanthus praecox LINK. The structures of the two compounds were established by (1)H- and (13)C-NMR, and MS (FAB, ESI) analyses. In the in vitro tests, compounds 1 and 2 showed significant inhibitory activities against five plant pathogenic fungi Exserohilum turcicum, Bipolaris maydis, Alternaria solani, Sclerotinia sderotiorum, and Fusarium oxysportium, among which B. maydis was found to be the most susceptible to 1 with an EC(50) value of 29.3 microg/ml, followed by S. sderotiorum to 2 with an EC(50) value of 61.2 microg/ml. To our knowledge, this is the first report of isolation and LC/MS/MS identification as well as of antifungal properties of these alkaloids from the seeds of this plant.