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Biomedical subjects

Takashi Nakazawa

Publications and source records attributed to Takashi Nakazawa.

At least 19 recordsLinked to original sources
PubMed2007

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Binding Sites↗
PubMed2006

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone.

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.

Acetic Anhydrides↗
PubMed2006

Strain differences in the responsiveness between Sprague-Dawley and Fischer rats to nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor.

To determine a rat strain appropriate for carcinogenicity testing of FYX-051, a xanthine oxidoreductase inhibitor, we performed a 4-week oral toxicity study by administering 0.3, 1 and 3 mg/kg, and 1, 3 and 10 mg/kg of FYX-051 to male Sprague-Dawley (SD) and Fischer (F344) rats, respectively. Histopathology revealed that the degree of FYX-051-induced nephropathy was 3-fold stronger in SD rats than in F344 rats. Our previous study demonstrated that the key factor of species differences in FYX-051-induced nephropathy is purine metabolism. This observation led us to examine the involvement of purine metabolism in differences among two strains of rats. However, purine metabolism was proven not to be implicated as an important factor. Subsequently, other factors responsible for the strain differences were examined. FYX-051-induced increases in plasma xanthine concentrations were higher in SD rats than in F344 rats, suggesting more remarkable effects on pharmacodynamics in the former than the latter. Urinary volume was greater in F344 rats administered 10 mg/kg of FYX-051 (6.8 ml/h/kg) than in SD rats administered 3 mg/kg of FYX-051 (5.0 ml/h/kg), implying easier xanthine excretion in the former. Urinary xanthine solubility was 55 mg/dl in F344 rats aged 6 weeks, in contrast to 38 mg/dl in SD rats of the same age. Also, there were no significant differences in exposure levels at the same dose between SD and F344 rats. The outcomes of exposure levels and renal histopathology in both rats suggest the possibility that F344 rats could be exposed to a 3-fold higher amount of drug than SD rats in a carcinogenicity bioassay. The present study, therefore, suggested that strain differences of nephrotoxicity were caused by the combined effects of pharmacodynamics, xanthine excretion capacity, and urinary xanthine solubility. Furthermore, these results indicate that F344 rats would be a suitable strain for the carcinogenicity study of FYX-051.

Animals↗
PubMed2006

Metabolic profile of FYX-051 (4-(5-pyridin-4-yl-1h-[1,2,4]triazol-3-yl)pyridine-2-carbonitrile) in the rat, dog, monkey, and human: identification of N-glucuronides and N-glucosides.

FYX-051, 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl)pyridine-2-carbonitrile, is a novel xanthine oxidoreductase inhibitor that can be used for the treatment of gout and hyperuricemia. We examined the metabolism of FYX-051 in rats, dogs, monkeys, and human volunteers after the p.o. administration of this inhibitor. The main metabolites in urine were pyridine N-oxide in rats, triazole N-glucoside in dogs, and triazole N-glucuronide in monkeys and humans, respectively. Furthermore, N-glucuronidation and N-glucosidation were characterized by two types of conjugation: triazole N(1)- and N(2)-glucuronidation and N(1)- and N(2)-glucosidation, respectively. N(1)- and N(2)-glucuronidation was observed in each species, whereas N(1)- and N(2)-glucosidation was mainly observed in dogs. With regard to the position of conjugation, N(1)-conjugation was predominant; this resulted in a considerably higher amount of N(1)-conjugate in each species than N(2)-conjugate. The present results indicate that the conjugation reaction observed in FYX-051 metabolism is unique, i.e., N-glucuronidation and N-glucosidation occur at the same position of the triazole ring, resulting in the generation of four different conjugates in mammals. In addition, a urinary profile of FYX-051 metabolites in monkeys and humans was relatively similar; triazole N-glucuronides were mainly excreted in urine.

Animals↗
PubMed2006

Study on the mechanism of photosensitive dermatitis caused by ketoprofen in the guinea pig.

To investigate the mechanism on photosensitive dermatitis caused by ketoprofen (KP) in humans, the following experiments were performed by topical application on guinea pigs. The phototoxicity study involving treatment with 10% solution of KP, its enantiomers (R-KP and S-KP), loxoprofen, and flurbiprofen revealed no phototoxic reactions. In the photoallergenicity study, KP and its enantiomers (0.5-2% solution) induced skin reaction at all dosages; however, loxoprofen and flurbiprofen (1-5% solution) did not induce such a photoallergenic reaction. These results suggest that the chemical structure of the benzophenone chromophore in KP would be one of the important factors for induction of the photoallergy since both loxoprofen and flurbiprofen do not possess this structure and hence lack photoallergenic potential. Furthermore, to assess time profiles of KP concentration in the skin and plasma, guinea pigs received a repeated topical application of R-KP and S-KP at a dosage of 40 mg/kg over a period of 3 days. Plasma KP concentrations were extremely low as compared to skin KP concentrations and were not detected at 72 h after the final dosing. At 24 h after the final dosing, KP concentrations in the skin with R-KP and S-KP treatment were 187.4 and 254.7 microg/g, respectively, and their half-lives were 80.5 and 84.4 h, respectively. KP concentrations at 336 h after final dosing were 11.3 microg/g for R-KP and 15.7 microg/g for S-KP treatment. The acylglycerol-combined KP concentrations at 336 h were 2% or less as compared to KP concentrations with R-KP and S-KP treatment. There were no differences in KP concentrations in the skin between R-KP and S-KP and in combined KP concentrations between the enantiomers. The present study indicates that photosensitive dermatitis after topical application of KP in humans, caused by photoallergenicity and not phototoxicity, can be reproduced in the animal testing, and suggests that the skin reaction may be caused by the long period of retention of KP in the skin.

Administration, Topical↗
PubMed2006

A pediatric patient with neuro-Behçet's disease.

Behçet's disease is rare in childhood. We describe a 10-year-old boy with neuro-Behçet's disease (NB) who presented with fever, headache, vertigo, and hearing loss. An examination of the cerebrospinal fluid (CSF) revealed pleocytosis as well as elevated protein and interleukin (IL)-6 levels. Brain magnetic resonance imaging (MRI) showed hyperintensity of the right thalamus and midbrain on T2-WI, and gadolinium (Gd) enhancement of left acoustic nerve origin. HLA-B51 was positive. Prednisolone combined with methotrexate resulted in a complete remission. Brain MRI and the CSF IL-6 level were useful for the diagnosis and monitoring of this pediatric patient with NB.

Behcet Syndrome↗
PubMed2005

Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10.

X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.

Circular Dichroism↗
PubMed2005

Simultaneous treatment with citrate prevents nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor, in rats.

The possible mechanism of the underlying nephropathy found in the rat toxicity study of FYX-051, a xanthine oxidoreductase inhibitor, was investigated. Rats received oral treatment of either 1 or 3 mg/kg of FYX-051, with and without citrate for four weeks to elucidate whether nephropathy could be caused by materials deposited in the kidney. Furthermore, analysis of the renal deposits in rats was also performed. Consequently, interstitial nephritis comprising interstitial inflammatory cell infiltration, dilatation, basophilia and epithelial necrosis of renal tubules and collecting ducts, deposits in renal tubules and collecting ducts, and so forth was seen in six of the eight rats and in all eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, with the intensity in the 3 mg/kg group being moderate to severe. In the simultaneous treatment with citrate group, however, no alterations were observed in the kidney, except for minimal interstitial nephritis in one instance in the 3 mg/kg FYX-051 + citrate group along with an increased urinary pH, leading to an increase in xanthine solubility. Analysis of intrarenal deposits showed that the entity would be composed of xanthine crystals. The present study, therefore, showed that nephropathy in rats occurring after the administration of FYX-051 was a secondary change caused by xanthine crystals being deposited in the kidney, and no other causes could be implicated in this kidney lesion.

Animals↗
PubMed2005

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix.

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

Amino Acid Sequence↗
PubMed2005

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.

A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.

Animals↗
PubMed2005

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by Bächinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.

Collagen↗
PubMed2004

Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone.

We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.

Amino Acid Sequence↗
PubMed2003

Characterization of collagen model peptides containing 4-fluoroproline; (4(S)-fluoroproline-pro-gly)10 forms a triple helix, but (4(R)-fluoroproline-pro-gly)10 does not.

Collagen model peptide (Pro-Pro-Gly)10 has a triple helical structure and undergoes a thermal transition to a single random coil structure. The transition temperature of the analogous model peptides depends largely on amino acid substitution. Substitution of Pro by 4-hydroxyproline (Hyp) or 4-fluoroproline (fPro) has especially attracted attention because the position of substitution and chirality of the hydroxyl group or fluorine atom affect the transition temperatures. Here, we demonstrated that (4(S)-fPro-Pro-Gly)10 takes a triple helical structure, but (4(R)-fPro-Pro-Gly)10 exists in a single chain structure. This is not consistent with the case of Hyp substitution in our previous report where both (4(S)-Hyp-Pro-Gly)10 and (4(R)-Hyp-Pro-Gly)10 are in a single random coil state.

Collagen↗
PubMed2003

Specific increase in enzymatic activity of adenosine deaminase 1 in rheumatoid synovial fibroblasts.

OBJECTIVE: Adenosine deaminase (ADA; EC 3.5.4.4) activity is elevated in the synovial fluid (SF) of patients with rheumatoid arthritis (RA). Since the antiinflammatory effect of methotrexate is reportedly associated with increased levels of extracellular adenosine, the present study was undertaken to clarify the role of 2 ADA isozymes, ADA1 and ADA2, in the pathogenesis of RA. METHODS: The activities of ADA1 and ADA2 were measured in SF from RA and osteoarthritis (OA) patients, in sera from RA patients, and in lysates prepared from mononuclear and polymorphonuclear cells from SF from RA patients, peripheral blood from RA patients, and fibroblast-like synoviocytes (FLS) from RA and OA patients. Also measured were the effects of proinflammatory cytokines on ADA1 activity and ADA messenger RNA (mRNA) expression in RA FLS, as determined using real-time polymerase chain reaction. The adenosine concentration in RA SF was measured by radioimmunoassay. RESULTS: The adenosine concentration in RA SF ranged from 0.027 microM to 0.508 microM (mean +/- SD 0.156 +/- 0.132 microM). At those concentrations, ADA1 would be expected to be functionally dominant due to its higher affinity for adenosine. ADA1 activity in RA SF (mean +/- SD 14.4 +/- 8.5 IU/liter) was significantly higher than that in OA SF (3.0 +/- 1.1 IU/liter) or RA sera (3.0 +/- 0.6 IU/liter); moreover, ADA1 activity in RA FLS lysate was the highest among the cell lysates tested. Proinflammatory cytokines did not affect ADA1 activity or ADA mRNA expression in RA FLS. CONCLUSION: Elevated ADA1 activity is an intrinsic characteristic of RA FLS, which likely contributes to the pathogenesis of RA by neutralizing the antirheumatic properties of endogenous adenosine.

Adenosine↗
PubMed2003

Usefulness of postmortem ocular findings in forensic autopsy.

We report three autopsy cases in which the eyeballs were removed and examined macroscopically and microscopically after being cut using the alternate gross examination technique. The first case was a 67-year-old woman who was killed by ligature and manual strangulation. A postmortem opthalmological observation of the left eyeball revealed tiny hemorrhages and congestion in the pars plicae of the ciliary body. These changes were considered to arise by a mechanism similar to that by which facial hemorrhages arise during strangulation. The second case was a 54-year-old man who died from a cerebral contusion received during a traffic accident. A postmortem opthalmological observation of the eyeball using a surgical microscope revealed papilledema and retinal hemorrhages resulting from a rise in intracranial pressure. The third case was a 77-year-old woman who died from cardiac tamponade caused by a stab wound to the heart. A postmortem ophthalmological examination revealed that she had undergone cataract surgery and an intraocular lens implantation. However, her relatives did not know when and where she had received the surgery. An intraocular lens was removed and its material and shape was examined. Based on an examination of the lens, we were able to locate the hospital where she had received the surgery 4 years earlier. This suggests that postmortem opthalmological observations may be useful for forensic pathology and personal identification.

Aged↗
PubMed2003

Myocardial damage by resuscitation methods.

Medico-legal autopsy cases were reviewed to detect myocardial changes induced by resuscitation methods. Myofibrillar degeneration (MFD) induced by resuscitation methods was classified into two types according to Luxol fast blue staining: contraction band (CB) and diffuse staining (DS). In the cases in which cardiopulmonary resuscitation had been performed, myocytes showing CB or DS formed small foci and were distributed in the papillary muscles, septum, and inner to middle layers of the myocardium. MFD induced by vasopressors was characterized by solitary distribution of degenerating myocytes that mainly showed DS and sometimes CB. When direct current countershocks had been performed, focal MFD in the subepicardial zone appeared to be a characteristic feature.

Cardiopulmonary Resuscitation↗
PubMed2002

A pH-dependent variation in alpha-helix structure of the S-peptide of ribonuclease A studied by Monte Carlo simulated annealing.

Low-energy conformations of the S-peptide fragment (20 amino acid residues long) of ribonuclease A were studied by Monte Carlo simulated annealing. The obtained lowest-energy structures have alpha-helices with different size and location, depending distinctively on the ionizing states of acidic amino acid residues. The simulation started from completely random initial conformation and was performed without any bias toward a particular structure. The most conspicuous alpha-helices arose from the simulation when both Glu 9 and Asp 14 were assumed to be electrically neutral, whereas the resulting conformations became much less helical when Asp 14 rather than Glu 9 was allowed to have a negative charge. Together with experimental evidence that the alpha-helix in the S-peptide is most stable at pH 3.8, we consider the helix formation need the carboxyl group of Asp 14 to be electrically neutral in this weakly acidic condition. In contrast, a negative charge at Asp 14 appears to function in support of a view that this residue is crucial to helix termination owing to its possibility to form a salt bridge with His 12. These results indicate that the conformation of the S-peptide depends considerably on the ionizing state of Asp 14.

Amino Acid Sequence↗