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An isopeptide of amyloid β peptide 1–42 (isoAβ42) was considered as a non-aggregative precursor molecule for the highly aggregative Aβ42. It has been applied to biological studies after several pretreatments. Here we report that isoAβ42 is monomeric with a random coil structure at 40 μM without any pretreatment. But we also found that isoAβ42 retains a slight aggregative nature, which is significantly weaker than that of the native Aβ42.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The optimal conditions were determined for oxidative folding of the reduced human α-defensins, HNP1, HNP2, HNP3 and HD5, preferentially into their native disulfide structures. Since the human α-defensin-molecule in both reduced and oxidized forms raised a solubility problem arising from its basic and hydrophobic compositions, buffer concentration had to be lowered and cosolvent, such as CH3CN, had to be added to the folding medium in the presence of reduced and oxidized gluthathione (GSH/GSSG) to prevent aggregation and also to realize predominant formation of the native conformer. The four synthetic human α-defensins of high homogeneity were confirmed to exhibit the same antimicrobial potencies against E. coli as those reported for the natural products. All these peptides were shown to possess the native disulfide structure by sequence analyses and mass measurements with cystine segments obtained by enzymatic digestion. Edman degradation allowed for disulfide assignment of cystine segments involving adjacent Cys residues composed of three peptide chains, for which two possible disulfide modes could be considered, with the guidance of the cycles detecting diPTH cystine. As for HNP1, HNP2 and HNP3, however, diPTH cystine was expected at the same cycles in both structures, which would have resulted in not being able to distinguish between the two alternative modes. To avoid this, it was necessary to provide an acetyl tag for the specific peptide chain originating from the N-terminus. Edman degradation of cystine segments tagged with the acetyl group would be a practical procedure for analyzing disulfide structures involving adjacent Cys residues.

Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.

The peptide GsMTx4 from the tarantula venom (Grammostola spatulata) inhibits mechanosensitive ion channels. In this work, we report the cDNA sequence encoding GsMTx4. The gene is translated as a precursor protein of 80 amino acids. The first 21 amino acids are a predicted signal sequence and the C-terminal residues are a signal for amidation. An arginine residue adjacent to the N-terminal glycine of GsMTx4 is the cleavage site for release. The resulting peptide is 34 amino acids in length with a C-terminal phenylalanine and not a serine-alanine previously identified [J. Gen. Physiol. 115 (2000) 583]. We chemically synthesized this peptide and folded it in 0.1 M Tris, pH 7.9 with oxidized/reduced glutathione (1/10). Properties of the synthetic peptide were identical to the wild type for high performance liquid chromatography (HPLC), mass spectrometry, CD, and NMR. We also cloned GsMTx4 in a thioredoxin fusion protein system containing six histidines. Nickel affinity columns allowed rapid purification and folding occurred in conditions described above with 0.5 M guanidiniumHCl present. Thrombin cleavage liberated GsMTx4 with three extra amino acids at the N-terminus. The retention time in HPLC analysis and the CD spectrum was similar to wild type. Both the synthetic and cloned peptides were active in the patch clamp assay.

Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically by applying Fmoc/tBu strategy. An ester linkage between octanoic acid and the hydroxyl function of Ser3 was found to be maintained without serious damage during the final deprotection with trifluoroacetic acid (TFA). The most notable finding was the counter-ion-dependent stability change of the octanoyl moiety in the molecule. After consolidation of the counter-ion to TFA (TFA form), the octanoyl group persisted stably upon dissolution in water, whereas in the case of the acetate-form peptide, both de-octanoylation and dehydration (formation of the dehydro-Ala residue) occurred in aqueous solution at the same Ser3 residue. The amounts of these degraded products varied with factors such as solvent, temperature and times of lyophilization. These experimental findings lay the basis for performing the bioassay of ghrelin, which has an octanoyl moiety involved in its numerous biological activities thus far revealed.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John WileySons, Ltd.

Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.

The chimeric peptide [Lys(-2)-Arg(-1)]-sarafotoxin-S6b (KR-SRTb) designed from the Lys-2-Arg-1 dipeptide of the endothelin pro-sequence and the sarafotoxin-S6b sequence was synthesized. Its contractile activity was found to be decreased markedly when compared with that of the parent SRTb. In contrast, the extension by the Lys-Arg dipeptide was found to increase the formation of the native disulfide isomer (82/18 versus 96/4) when the reaction was carried out in the presence of redox reagents. The solution structure of KR-SRTb was determined by NMR as a function of pH. In the carboxylic acid state, the structure consists of the cystine-stabilized alpha-helical motif, with the alpha-helical part spanning residues 9-15, and of an unstructured C-terminal tail. In the carboxylate state, the structure is characterized by a salt-bridge between Arg(-1) and Asp8, which we identified previously in the [Lys(-2)-Arg(-1)]-endothelin-1 peptide (KR-ET-1). The fact that this salt-bridge is commonly observed in KR-SRTb and KR-ET-1, despite the 33% sequence difference between the corresponding parental peptides, highlights the remarkable adaptability of the Lys-Arg extension for the formation of a special salt-bridge. As a consequence, this salt-bridge, which does not depend on either the 4-7 sequence of the loop or the C-terminal sequence, appears to be particularly well suited to improve the stability of the cystine-stabilized alpha-helical motif. Therefore, because of its high yield in the native disulfide arrangement and its high permissiveness for sequence mutation in the 4-7 loop, such a stabilized cystine-stabilized alpha-helical motif could be a valuable scaffold for the presentation of a library of constrained short peptides.

As part of studies on the effects, especially the preventive effects, of exercise and psychological factors on cardiovascular diseases, the association between psychological tendencies and immune response was evaluated in patients with coronary heart disease who were receiving exercise therapy. The Pearson's product-moment correlation coefficients between natural killer (NK) cell activity and various psychological scales were obtained. For the Moudsley Personality Inventory, NK cell activity had a significant positive correlation with the extraversion scale and a significant negative correlation with the neuroticism scale. NK cell activity also had a significant positive correlation with the playful humor scale and a significantly negative correlation with the Self-rating Depression Scale. The positive correlation of NK cell activity with the extraversion scale and the humor scale and its negative correlation with the neuroticism scale suggest an association between a positive-feeling tendency and high NK cell activity. The negative correlations of NK activity with the depression scale and neuroticism scale indicate that decreased or excessive expression of feelings inhibits NK cell activity. Thus, high NK activity appears to be associated with optimal expression of feelings. (Jpn Circ J 1999; 63: 704 - 709)

An ankle-foot orthosis (AFO) for hemiplegic patients called the DACS (dorsiflexion assist controlled by spring) AFO was developed. Previous studies have shown the DACS AFO to have the following desirable characteristics: 1) the magnitude of the dorsiflexion assist moment and the initial ankle angle of the AFO can be changed easily, and 2) no plantarflexion assist moment is generated. DACS AFOs were used daily by 5 hemiplegic patients, and their evaluations were favorable. In this paper, the structure and the mechanical characteristics of the DACS AFO and the gait improvement of hemiplegic patients with the DACS AFO are shown.

The [Lys-Arg]-endothelin-1 analogue (KR-ET-1) yields almost selectively the native disulfide pattern (96%), in contrast to endothelin-1 (ET-1) that gives at least 25% of the non-native disulfide pattern. We have previously shown that the carboxylate-state structure of KR-ET-1 is more constrained and stabilized by a salt bridge between Arg(-1) and the Asp8 or Glu10 side chain [Aumelas et al. (1995) Biochemistry 34, 4546-4561]. To identify this salt bridge and its potential involvement in the disulfide bond formation, [E10Q], [D18N], and [D8N] carboxamide analogues were studied, which led to the unambiguous identification of the Arg(-1)-Asp8 salt bridge. Furthermore, while [E10Q] and [D18N] analogues gave a high yield of the native isomer (/=90%), the [D8N] analogue afforded a ratio of the two isomers close to that observed for ET-1 (68%) [Kubo et al. (1997) Lett. Pept. Sci. 4, 185-192]. Assuming that the formation of disulfide bonds occurs in a thermodynamically controlled step, we have hypothesized that the Arg(-1)-Asp8 salt bridge and concomitant interactions could be responsible for the increase in yield of the native isomer of KR-ET-1. In the present work, we describe the structural studies of the carboxamide analogues and of the minor non-native KR-ET-1 isomer. On the basis of 1H NMR and CD spectra as a function of pH, [E10Q] and [D18N] analogues display a conformational change similar to that of the parent peptide, whereas the structure of the [D8N] analogue is unchanged. For the non-native isomer, we measured a lower helical content than for the native isomer and observed a marked difference in the orientation of the KRCSC backbone. In addition, no salt bridge was experimentally observed. Altogether, these results allow us to hypothesize that the salt bridge between two highly conserved residues, one belonging to the prosequence [Arg(-1)] and the other to the mature sequence [Asp8], is involved in the formation of the native disulfide isomer of ET-1. The involvement of the prosequence in the formation of the native disulfide isomer strongly suggests that, in the maturation pathway of ET-1, cleavage of the Arg52-Cys53 amide bond occurs after native disulfide bond formation.

An amino-terminal extension of endothelin-1 by the Lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu10 or Asp18. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-1 is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-1; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-1. From these results and our previous findings that the helical structure in KR-ET-1 is stabilized by a particular salt bridge between the extended Arg-1 basic moiety and either the Asp8 or Glu10 acidic side chain in ET-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg-1 and Asp8 in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.

Oxidative folding of o-conotoxin MVIIC, a highly basic 26-amino acid peptide with three disulfide bonds, predominantly gave two products with mismatched disulfide bonds in 0.1M NH4OAc buffer (pH 7.7) at 21°C both in the presence and absence of redox reagents such as reduced and oxidized glutathione. A low reaction temperature (5°C) and a high salt concentration in buffer such as 2M (NH4)2SO4 were necessary to obtain the correctly folded biologically active product. The folding reaction was found to proceed via a two-stage pathway of (I) the formation and (II) the rearrangement of the mismatched disulfide bonds. Both the reaction temperature and the salt strongly affected the equilibrium between mismatched and correctly formed disulfide bonds in the second stage. Such an effect of salts on the rearrangement reaction could be explained by anion binding at a low concentration and the salting out effect at a high concentration by analyzing the rank order of their effectiveness. The anion-binding effect was also confirmed by examining the folding of the tetra-acetylated peptide at the Lys side chains. CD study suggested that the yield of the biologically active product was correlated with its conformational change as functions of temperature and salt concentration. © 1996 John Wiley & Sons, Inc.

In a two-step selective disulfide-bond-forming reaction of human uroguanylin, a 16-residue peptide with two intramolecular disulfide bonds, two compounds (I and II) were formed, which could be detected by RP-HPLC after the second disulfide-bond-forming reaction and were isolated as single entities. Their primary structures, molecular weights, and disulfide connectivities proved to be identical, but their optical rotation values were different, suggesting that they are topological isomers. Only compound I was found to increase the cGMP levels in cultured T84 cells significantly. The ratio of these compounds was affected by the order of the disulfide-bond-forming reactions, but not by the solvent used. The presence of a carboxyl-terminal leucine residue seems to be crucial for stabilizing the conformation of the two isomers.

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Transesophageal echocardiography (TEE) was performed in 17 cases of aortic aneurysms referred to our hospital for further examination and treatment. All 17 cases were treated surgically and TEE was performed as a preoperative examination. In nine of the 17 cases, there were already some signs of bleeding upon admission and in all of these nine cases, rupture of the aneurysm was confirmed during surgery. Measurement on cross-sectional TEE imaging disclosed large aneurysmal diameters in eight of these nine cases, suggesting a close relationship between diameter and rupture. Moreover, observation of the lesions by TEE suggested a relationship between the risk of rupture and morphological characteristics of the thrombus. In seven of the nine bleeding cases, TEE imaging revealed destructive features of the aneurysmal thrombus, such as exfoliation from the aortic wall and/or tearing-off, suggesting expansion of the aortic diameter. Detailed findings of the aneurysm and thrombus on TEE corresponded with surgical findings. Thus, we concluded that TEE is a useful method of obtaining information about aortic aneurysms not only as a preoperative examination but also as an independent examination to determine treatment options and prognosis.

Addition of the Lys(-2)-Arg(-1) dipeptide, present in the precursor protein, to the N-terminus of endothelin-1 (ET-1), to form a 23-residue peptide (KR-ET-1) has been shown to greatly improve formation of native disulfide bridges and to dramatically decrease biological activity. Conformational analysis was carried out on this peptide. During protonation of the carboxyl groups, CD spectra showed a decrease in the helical contribution, and NMR spectra displayed strong chemical shift modifications, suggesting the importance of electrostatic interactions in the KR-ET-1 conformation. CD spectra and two-dimensional NMR experiments were performed to investigate the KR-ET-1 three-dimensional structure in water in the carboxylic acid and carboxylate states. Distance and angle constraints were used as input for distance geometry calculations. The KR-ET-1 carboxylic acid conformation was found to be very similar to ET-1, with a helix spanning residues 9-15 and an unconstrained C-terminal part. In contrast, in the carboxylate state, large changes in Arg(-1) and Phe14 chemical shifts and long-range NOEs were consistent with a conformation characterized by a helix extension to Leu17 and a stabilized C-terminal section folded back toward the N-terminus. In addition, thanks to NOEs with Cys11 and Phe14, the Arg(-1) side chain appeared well-defined. Simulated annealing and molecular dynamics calculations, supported an Arg(-1)-Glu10 salt bridge and an electrostatic network involving the charged groups of Trp21, Asp18, and Lys(-2). Moreover, stabilization of the KR-ET-1 C-terminal part is probably reinforced by hydrophobic interactions involving the Val12, Tyr13, Phe14, Leu17, Ile19, Ile20, and Trp21 side chains. In vitro, native disulfide bond formation improvement observed for KR-ET-1 could be ascribed to electrostatic interactions and more specifically to the Arg(-1)-Glu10 salt bridge. In vivo, similar interactions could play an important role in the native folding of the ET-1 precursor protein. On the other hand, modification in the environment and a reduced mobility of the KR-ET-1 Trp21 key residue, when compared to ET-1, could explain, at least in part, the strong decrease in biological activity.

The solution structure of the P- and Q-type Ca 2+ channel blocker, ω-conotoxin MVIIC (a peptidic neurotoxin composed of 26 amino acid residues),has been determined by 1 H-NMR and simulated annealing calculations. The resulting calculated structures converged very well to a conformation with an average value of pairwised RMSD for N, Cα and C′ of 0.62 A. Lys-25 is buried in the molecule and less flexible so that among the four Lys residues, its side chain provides the lowest reactivity on biotinylation and the mono-biotinylation in this residue less influences the biological activity.

A potent and selective P type calcium channel blocker isolated from the venom of the funnel web spider Agelenopsis aperta, omega-agatoxin IVA, and its related peptides were synthesized by the solution procedure. Synthetic omega-agatoxin IVA was found to block high-threshold P-type calcium current in rat Purkinje neuron with the same potency as that reported for the natural product. Its disulfide structure was determined by amino acid analysis, gas-phase sequencing and mass spectrometry of the proteolytic fragments. The N-terminus biotinylated and truncated peptides showed the same disulfide-bond-forming profile and the same activities as those of omega-agatoxin IVA, indicating that the N-terminal basic tripeptide, Lys-Lys-Lys, is not important for both the folding and the expression of the biological activity. However, the Trp residue in the molecule might be essential for the toxin to bind tightly with the channel pores.

The patient was a 66-year-old man who had advanced gastric cancer with metastasis to liver and lymph nodes. He received daily oral administration of 100 mg of TS-1 for 28 days followed by 14 days rest as one treatment course. After 2 coures, regression of the primary lesion and reduction in size of the liver and lymph metastases were observed.

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.

The [des(17-21)]-endothelin-1 (CSH-ET) and [Lys(-)(2)-Arg(-)(1)-des(17-21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys(-)(2)-Arg(-)(1)]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg(-)(1) and Asp(8) side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg(-)(1)-Asp(8) salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation-pi interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation-pi interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 series.

TS-1 is a novel oral anticancer drug that is a formation of 5-FU. It consists of tegafur, CDHP (which inhibits 5-FU degradation enzyme), and Oxo (which reduces gastrointestinal toxicities) for an increased anticancer effect. We applied individual TS-1 therapy in 22 cases (cs) of inoperable gastric cancer and studied the clinical and adverse effects. Patients were treated with daily oral administration of 80-100 mg TS-1 for 4 weeks, followed by a rest for 1 or 2 weeks. The response rate was found to be 27.3% (6/22) (PR: 6 cs, NC: 4 cs, PD: 10 cs, NE: 2 cs). Overall, the median survival time was 8.2 months and the one-year survival rate was 23.6%. By location, the response rate of the primary lesion was 27.3% (6/22), abdominal lymph node metastasis 18.8% (3/16), and liver metastasis 33.3% (4/12). There was no significant difference in the response rate by tissue type. A comparison by whether or not patients had undergone previous chemotherapy revealed a response rate of 37.5% (6/16) in patients who had undergone previous chemotherapy, and 0% (0/6) in those who had not. The prevalence of adverse effects was 68.2% (15/22), with the main adverse effects being myelosuppression, pigmentation and appetite loss. However, adverse effects with a grade of more than 3 occurred in only one case of neutropenia. We could observe the course of all patients on an outpatient basis.

Background: Endoscopic resection of pedunculated polyps with heads 1 cm or greater in diameter is technically complex. To facilitate removal of such polyps, we developed grasping forceps–assisted endoscopic resection in which we use a detachable snare to prevent polypectomy-related bleeding and evaluated the usefulness and safety of the procedure. Methods: Ten patients with pedunculated polyps with heads 1 cm or greater in diameter were treated with this technique. A two-channel endoscope, grasping forceps, electrosurgical snare, and detachable snare are needed for the procedure. Results: All lesions were easily and safely resected. During this procedure, a two-channel endoscope with grasping forceps proved to be satisfactory for handling the detachable snare and the electrosurgical snare and for accurate recognition of the stalk under good visual control. No hemorrhage, perforation, or other complication occurred as a result of use of this new technique. Conclusions: Grasping forceps–assisted endoscopic resection of polyps with a detachable snare is an effective method for the prevention of polypectomy-associated bleeding. This technique makes it technically easier to resect large pedunculated polypoid lesions of the GI tract.

In conclusion, to evaluate the efficacy and safety of PTMC for mitral stenosis patients with markedly severe valve deformity, we performed PTMC in 17 patients with severe mitral stenosis assessed by echocardiography (echo score ≥12). This study demonstrates that PTMC can be performed safely and is clinically useful in treating the mitral stenosis patient with a markedly severe valve deformity.

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.

To understand the effects of high-altitude hypoxia on cardiac function and the change in cardiac function during high-altitude acclimatization, precise studies were first performed at greater than 5,000 m of altitude in Himalaya by 2-dimensional echocardiography. In addition to examining well-known indexes of cardiac function, the centerline method was used to assess regional wall motion, which has not been examined under conditions of high-altitude hypoxia. The subjects were 11 climbing members (aged 21 to 43 years) of the Kyoto University Medical Research Expedition of Xixabangma (8,027 m) in 1990. Examinations were performed at sea level, at the base camp (5,020 m), and twice at the advanced base camp (5,650 m). Heart rate, left ventricular (LV) end-diastolic volume, cardiac output, mean rate of circumferential fiber shortening, ejection fraction, % fractional shortening, and regional LV wall motion were measured. At high altitude, heart rate increased to 136% of the sea level value, but gradually decreased in the degree of increment at the early and late advanced base camp measurements. LV end-diastolic volume decreased significantly by 70%. At base camp there were significant increases in ejection fraction, mean rate of circumferential fiber shortening, and % fractional shortening, which showed little change during the long-term stay at high altitude. Regional wall motion at high altitude (measured by the center-line method) decreased at the septal wall and increased at the posterolateral wall. These results demonstrate that: (1) LV cardiac performance at high altitude is enhanced significantly in spite of reduced preload. After good acclimatization, cardiac performance remains augmented, but there is a tendency to decrease the degree of augmentation. (2) In regional LV wall motion, septal wall motion is impaired, but LV posterolateral wall motion shows a compensatory increase.

A series of studies has been done to establish an objective and quantitative method to determine the optimum dorsi-lplantarflexibility of an ankle-foot orthosis (AFO) for various patients.

Ankle-foot orthoses (AFOs) are categorized four ways with respect to their shape: posterior spring, anterior spring, side-stay and spiral. In this article the flexibility of these types of AFOs fitted to the limb is measured using a muscle training machine. A comparison of the flexibility of

Endothelin (ET)-related peptides including ET-1 (1–39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at position 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.

To evaluate the noninvasive detection of shunt flow using a newly developed real-time 2-dimensional color-coded Doppler flow imaging system (D-2DE), 20 patients were examined, including 10 with secundum atrial septal defect (ASD) and 10 control subjects. These results were compared with contrast 2-dimensional echocardiography (C-2DE). Doppler 2DE displayed the blood flow toward the transducer as red and the blood flow away from the transducer as blue in 8 shades, each shade adding green according to the degree of variance in Doppler frequency. In the patients with ASD, D-2DE clearly visualized left-to-right shunt flow in 7 of 10 patients. In 5 of these 7 patients, C-2DE showed a negative contrast effect in the same area of the right atrium. Thus, D-2DE increased the sensitivity over C-2DE for detecting left-to-right shunt flow (from 50% to 70%). However, the specificity was slightly less in D-2DE (90%) than C-2DE (100%). Doppler 2DE could not visualize right-to-left shunt flow in all patients with ASD, though C-2DE showed a positive contrast effect in the left-sided heart in 9 of 10 patients with ASD. Thus, D-2DE is clinically useful for detecting left-to-right shunt flow in patients with ASD.