Unsteady flow field around a human hand and propulsive force in swimming.

Much effort has been undertaken for the estimation of propulsive force of swimmers in the front crawl. Estimation is typically based on steady flow theory: the so-called quasi-steady analysis. Flow fields around a swimmer, however, are extremely unsteady because the change direction of hand produces unsteady vortex motions. To evaluate the force correctly, it is necessary to know the unsteady properties determined from the vortex dynamics because that unsteadiness is known to make the force greater. Unsteady flow measurements were made for this study using a sophisticated technique called particle image velocimetry (PIV) in several horizontal planes for subjects swimming in a flume. Using that method, a 100 time-sequential flow fields are obtainable simultaneously. Each flow field was calculated from two particle images using the cross-correlation method. The intensity of vortices and their locations were identified. A strong vortex was generated near the hand and then shed by directional change of the hand in the transition phase from in-sweep to out-sweep. When the vortex was shed, a new vortex rotating in the opposite direction around the hand was created. The pair of vortices induced the velocity component in the direction opposite to the swimming. Results of this study show that the momentum change attributable to the increase in this velocity component is the origin of thrust force by the hand.

Studies on chemical modification of monensin IX. Synthesis of 26-substituted monensins and their Na+ ion transport activity.

The C-26 modified monensin derivatives, 26-O-benzoylmonensin (3), 26-O-benzylmonensin (4) and 26-phenylaminomonensin (5) were prepared from monensin (1). Na+ ion transport activity through biological membrane and antibacterial activity of 3-5 were evaluated and compared with the activities reported for a 26-phenylurethane derivative (2). Among these compounds, 5 showed the largest Na+ ion transport and antibacterial activities. In these compounds, the formation of head-to-tail hydrogen bonds was suggested to be an important factor for Na+ ion transport and antibacterial activities.

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase.

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. V. Drug-induced gastrointestinal system disorders].

OBJECTIVES: The purpose of this study was to develop, implement, and assess an estimation procedure for preventing adverse drug reaction by subjective symptoms (complaints) of patients. This time, we focused and studied on drug-induced gastrointestinal system disorders. METHODS: We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information System) since 1987. We studied 224 cases of drug-induced Gastrointestinal System Disorders (stomach or colon disorders: 148, esophagus disorders: 31, pancreas disorders: 45) cumulated in the CARPIS database. The evaluation scores were created based on the subjective symptoms and backgrounds of the patients. We estimated 224 cases using these evaluation scores. RESULTS: We could estimate 137 cases (92.6%) in 148 cases to be stomach or colon disorders by the use of these evaluation scores. The validity of this evaluation scores was estimated to be as follows: Sensitivity = 92.6%, Specificity = 95.0% and Predictive Value of Positive Test (PVP) = 96.5%. The positive likelihood ratio (LR) was 18.5 and negative likelihood ratio was 0.08. On the other hand, in the case of esophagus disorders, PVP was 84.8% and LR was 18.1. In the case of pancreas disorders, PVP was 90.7% and LR was 21.7. CONCLUSIONS: In this study, PVP and LR values were good. We thought that these evaluation scores could pick up the drug-induced gastrointestinal system disorders efficiently. We reported previously the evaluation scores about drug-induced liver disorders, extra-pyramidal symptoms, leukopenia and eruption before. In order to apply these evaluation scores onto the clinical practice, we prepared an evaluation form for subjective symptoms and backgrounds of the patients with adverse drug reactions. As a result, the adverse reactions symptoms of each one defined more.

[Application study of drug information to proper use of drugs--estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients].

We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information System) since 1987, and the case reports of adverse drug reactions accumulated in the CARPIS database to be total about 20,000. The purpose of our study is to develop, implement, and assess an estimation procedure for preventing the adverse drug reactions by subjective symptoms (complaints) of patients using this CARPIS. The case reports of the adverse reactions are not a systematical study and are elapse reports by the case, which encountered on the way of the cure. Therefore, it might be possible to use as the data if not gaining enough evaluation. Especially, the abstracts in the annual meeting of the society are fragmentary and does not often have objective evidence. Therefore, we contrived an original evaluation method and carefully selected the case reports for our study. Also, the adverse reactions are very colorful and it is difficult to handle them at once. Therefore, we study every organ and want to aim to make a finally all-round format. We reported on the Liver disorders, the Extra-pyramidal symptoms, the Drug eruption, the Leukopenia and so on. We discovered that there was an adverse reaction which shows very peculiar subjective symptoms (ex. Extra-pyramidal symptoms) and unpeculiar subjective symptoms (ex. Leukopenia). We want to show our point of view and the future of our study below.

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. IV. Drug eruptions].

OBJECTIVES: The purpose of this study is to develop, implement, and assess an estimation procedure for preventing adverse drug reaction by subjective symptoms (complaints) of patients. This time, we focused and studied on drug eruptions. METHODS: We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information System) since 1987, and the case reports of adverse drug reactions accumulated in the CARPIS database to be total about 20,000. We studied 1473 cases of drug eruptions cumulated in CARPIS database. The evaluation scores were created based on the subjective symptoms and backgrounds of the patients. We estimated 1473 cases using this evaluation scores. RESULTS: We could estimate 1455 cases (98.8%) in 1473 cases to be drug eruptions using this evaluation scores. The validity of this evaluation scores were sensitivity = 98.8%, specificity = 91.0% and predictive value of positive test = 99.4%. The positive likelihood ratio was 11.0 and negative likelihood ratio was 0.01. CONCLUSIONS: This study confirmed the validity of our evaluation scores. We reported the evaluation scores about drug-induced liver diseases, drug-induced extra-pyramidal symptoms and drug-induced leukopenia before. In order to apply these evaluation scores to the clinical practice, we prepared an evaluation form for subjective symptoms and backgrounds of the patients with adverse drug reactions.

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. III. Drug-induced leucopenia].

OBJECTIVES: The purpose of this study was to develop, implement, and assess an estimation procedure for preventing adverse drug reaction by subjective symptoms (complaints) of patients. This time, we carried out this study focusing on drug-induced leucopenia. METHODS: We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information System) since 1987, and the total number of case reports of adverse drug reaction accumulated in the CARPIS database amounts to about 16,000. Using the date obtained from 139 cases of drug-induced leucopenia accumulated in the CARPIS database, we investigated a relationship between leucopenia and patients. The evaluation scores were created based on the subjective symptoms and backgrounds of the patients. RESULTS: We could estimate 91 cases (65.5%) in 139 cases to be drug-induced leucopenia by the use of these evaluation scores. The validity of this evaluation scores was estimated to be as follows; sensitivity = 65.5%, specificity = 80.0% and predictive value of positive test (PVP) = 82.0%. The positive likelihood (LR) ratio was 3.3 and negative likelihood ratio 0.43. CONCLUSIONS: In this study, PVP and LR values were not good, because among the symptoms of leucopenia a very few specific symptoms could be detected. But we reported previous by the evaluation scores about drug-induced liver disorders. Therefore, in order to apply these evaluation scores to the clinical practice, we prepared an evaluation form for subjective symptoms and backgrounds of the patients with adverse drug reactions. In the future, we plan to examine other adverse reactions which adds the data to this form.

Green tea polyphenols: novel and potent inhibitors of squalene epoxidase.

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction.

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. II. Drug-induced extrapyramidal symptoms].

OBJECTIVES: The purpose of this study was to develop, implement, and assess an estimation procedure for preventing adverse drug reaction by subjective symptoms (complaints) of patients. This time, we focused and studied on drug-induced extrapyramidal symptoms. METHODS: We have built a database for CARPIS (Case Reports of Adverse Drug Reaction and Poisoning Information Systems) since 1987, and the case reports of adverse drug reaction accumulated in the CARPIS database to be total about 16,000. We studied for 180 cases of drug-induced extrapyramidal symptoms cumulated in CARPIS database. The evaluation scores were created based on the subjective symptoms and backgrounds of the patients. We estimated 180 cases using these evaluation scores. RESULTS: We could estimate 178 cases (98.9%) in 180 cases to be drug-induced extrapyramidal symptoms using these evaluation scores. The validity of these evaluation scores were sensitivity = 98.9%, specificity = 98.0% and predictive value of positive test = 98.9%. The positive likelihood ratio was 49.5 and the negative likelihood ratio was 0.01. CONCLUSIONS: This study confirmed the validity of our evaluation scores. We reported the evaluation scores about drug-induced liver diseases before. Therefore, to apply these evaluation scores onto the clinical practice, we prepared an evaluation form for subjective symptoms and backgrounds of the patients with adverse drug reactions.

Serotonin derivative, N-(p-Coumaroyl)serotonin, isolated from safflower (Carthamus tinctorius L.) oil cake augments the proliferation of normal human and mouse fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF).

N-(p-Coumaroyl)serotonin (CS) with antioxidative activity is present in safflower oil. We have reported that CS inhibits proinflammatory cytokine generation from human monocytes in vitro. As reactive oxygen species (ROS) affect cell proliferation, in this study the effect of CS on the proliferation of various cell types was examined. CS augments the proliferation of normal human and mouse fibroblast cells. The cells continue to proliferate in the presence of CS and form a transformed cell-like focus without transformation. CS, however, does not augment the proliferation of other cell types, either normal or tumor cells. CS augments the proliferation of fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), but not with acidic FGF(aFGF) or platelet-derived growth factor (PDGF). This study using synthesized derivatives of CS reveals that the growth-promoting activity is not due to antioxidative activity. These findings indicate that CS is a natural compound with unique growth-promoting activity for fibroblasts.

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. I. Drug-induced liver disorders].

The purpose of this study was to develop, implement, and assess an estimation procedure for preventing drug-induced liver disorders. We have built a database for CARPIS (case reports of adverse drug reaction and poisoning information services) since 1987, and the case reports of adverse drug reaction accumulated in the CARPIS database to be total about 11,000. We studied the estimation procedure by evaluating the subjective symptoms, backgrounds and laboratory data of patients in 199 cases cumulated in the CARPIS database. The evaluation scores were created on the basis of the subjective symptoms and backgrounds of the patients. Then, we re-estimated 199 cases with the evaluation scores, among which were 165 cases (82.9%) in 199 cases drug-induced liver disorders. For the rest of 34 cases (17.1%) drug-induced liver disorders could not be estimated from our evaluation scores. These 34 cases were either those which the subjective symptoms were not described in their reports, because the patients were hospitalized for other diseases and drug-induced liver disorders were discovered by a clinical examination, or cases of infants for whom it was difficult to confirm the subjective symptoms. The validity of this evaluation scores were sensitivity = 82.9%, specificity = 91.0% and predictive value of positive test = 94.8%. To apply this evaluation scores onto the clinical practice, we prepared an evaluation form for the subjective symptoms and backgrounds of the patients with drug suspecting-induced liver disorders.

Serotonin derivative, N-(p-coumaroyl) serotonin, inhibits the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 by endotoxin-stimulated human blood monocytes.

We have reported that N-(p-coumaroyl) serotonin (CS) and its derivatives with antioxidative activity are present in safflower seeds. As reactive oxygen species (ROS) are implicated in the signaling of lipopolysaccharide (LPS), we examined whether CS has a suppressive effect on inflammatory cytokine generation from human monocytes in vitro. CS at 50-200 microM reduced tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 activities in the culture supernatants from LPS-stimulated human blood monocytes without cytotoxicity. ELISA assay revealed that the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 was inhibited by CS. Northern blot analysis showed that LPS-induced expression of these cytokine mRNA in monocytes was suppressed by CS. NF-kappaB activation was also inhibited by CS. These findings indicate that CS has a suppressive effect on proinflammatory cytokine production from monocytes, and this effect is based in part on the suppression of cytokine mRNA expression through inhibition of NF-kappaB activation.

Inhibitory effects of fatty acid monoesters of 2-O-beta-D-glucosylglycerol on Epstein-Barr virus activation.

In a screening for cancer chemopreventing agents several glycosylglycerols were found to be active. In order to optimize the anti-tumor activity of this class of compounds, a series of 1-O-acyl-2-O-beta-D-glucopyranosyl-sn-glycerols differing in the acyl chain length, which varied from C4 to C18, were examined for their in vitro anti-tumor promoting effects on Epstein-Barr virus early antigen (EBV-BA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the compounds tested, the monohexanoyl derivative was the most active and, noteworthy, the most potent compound of the glycosylglycerol series hitherto known.

Contribution of cytosolic ionic and energetic milieu change to ischemia- and reperfusion-induced injury in guinea pig heart: fluorometry and nuclear magnetic resonance studies.

The contribution of cytosolic ion and energy milieu changes to ischemia/reperfusion injury was investigated in isolated guinea-pig hearts and mitochondria, with fluorometry and 31P nuclear magnetic resonance (NMR). The fura-2 Ca2+ signal during ischemia in the guinea-pig Langendorff heart changed triphasically (phases I, II, and III) and rapidly returned to the control level after the reperfusion. These triphasic changes during ischemia were affected by various agents that affect the cytosolic ion milieu: the combination of asebotoxin-III and dihydroouabain (which increase intracellular Na+) caused an increase in Ca2+ levels in the final stage (phase III) with a manifestation of contracture after the reperfusion of the heart. Inhibitors of the H+-Na+ exchange such as 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) produced a significant restorative effect on the contractility of the reperfused heart with increased proton and decreased Na+ and Ca2+ in the cytosol. The mitochondrial matrix Ca2+ ([Ca2+]m) preloaded with abnormally high Ca2+ levels was markedly increased by perfusion with either a physiologic concentration of Ca2+ or an acidified perfusate. These [Ca2+]m increases were reduced by the H+-Na+ and H+-K+ exchange inhibitor (EIPA; omeprazole), respectively. These findings will help to explain the Ca paradox at the mitochondria level (i.e., mitochondria for Ca2+ pumping play an essential role in the cellular homeostasis of Ca2+ for the maintenance of cell functions of the heart, acting like a Ca2+ scavenger in the cytosol). Factors that induce Ca2+ overload on mitochondria via sarcolemmal Ca2+ influx and any exchange mechanisms with Na+, K+, Ca2+, and H+ will lead to a loss of contractility, associated with the extremely reduced level of free energy change predicted from the reduced ATP x PCr/Pi ratio by 31P NMR.

[Transformation of natural products into more potent compounds: chemical modification of monensin].

Monensin (1) is a representative compound of polyether ionophore antibiotics, which selectively transport Na+ ions. In order to obtain potent Na+ ionophores, the modification of the carboxyl group of monensin was carried out to yield monensylamino acids (2) and monensylamino acid-1,29-lactones (3). The Na+ permeability of ion through the erythrocyte membrane of 2 and 3 was evaluated by the 23Na-NMR method. Compound 2 showed less Na+ ion transport activity than monensin, probably due to the lower lipophilicity caused by the conformational change of the chain moiety of the molecules. Although 3 showed higher lipophilisity than 1, 3 had no Na+ ion permeability, probably due to loss of the carboxyl group. As more lipophilic compounds possessing a carboxyl group was supposed to have more ion transport activity, 7-O-acylmonensins (8) and 7-O-alkylmonensins (11) were synthesized. Among these compounds, the value of Na+ ion permeability of 7-O-benzylmonensin (11c) was 1.4 time that of 1. Further investigation was carried out by preparing various 7-O-(substituted benzyl)monensins (13), and 7-O-(p-ethylbenzyl)monensin (13b) exhibited the largest Na+ ion permeability, about twice the value of 1. In order to convert monensin (1) to Ca2+ ionophore, 7-carboxylmethylmonensin (18) via protected 7-oxomonensin (15), and 25-carboxylmonensin (26) were prepared. In the course of the synthesis, 15 was clarified as a useful intermediate to give 7-amino and 7-alkyl derivatives. Ca2+ ion transport activities of 18 and 26 were determined by a CHCl3 liquid membrane system. 25-carboxylmonensin (26) showed 70% of the activity of Ca2+ ionophore, lasalocid A, and compound 26 could be the lead compound for the preparation of a new Ca2+ ionophore.

A simplified squalene epoxidase assay based on an HPLC separation and time-dependent UV/visible determination of squalene.

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible determination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer (> 15 min) than that for any other component required to prepare an enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.

Localization of the squalene epoxidase gene (SQLE) to human chromosome region 8q24.1.

Squalene epoxidase (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. We previously isolated the mammalian squalene epoxidase cDNAs and demonstrated the transcriptional regulation of human squalene epoxidase by sterols and inhibitors. The present study was undertaken to determine the chromosomal mapping of the human squalene epoxidase gene (SQLE). PCR evidence localizes human SQLE to chromosome 8 by using the NIGMS (National Institute of General Medical Sciences) Human/Rodent Somatic Cell Hybrid Mapping Panel 2 as template. To refine the localization of human SQLE further, PCR on the Stanford G3 Radiation Hybrid Panel was performed. The result shows that human SQLE is most tightly linked to D8S508, which is reported to be located at 8q24.13-qter (lod score 7.87). Moreover, fluorescence in situ hybridization also maps human SQLE to 8q24.1.

Unusual effects of some vegetable oils on the survival time of stroke-prone spontaneously hypertensive rats.

Preliminary experiments have shown that a diet containing 10% rapeseed oil (low-erucic acid) markedly shortens the survival time of stroke-prone spontaneously hypertensive (SHRSP) rats under 1% NaCl loading as compared with diets containing perilla oil or soybean oil. High-oleate safflower oil and high-oleate sunflower oil were found to have survival time-shortening activities comparable to that of rapeseed oil; olive oil had slightly less activity. A mixture was made of soybean oil, perilla oil, and triolein partially purified from high-oleate sunflower oil to adjust the fatty acid composition to that of rapeseed oil. The survival time of this triolein/mixed oil group was between those of the rapeseed oil and soybean oil groups. When 1% NaCl was replaced with tap water, the survival time was prolonged by approximately 80%. Under these conditions, the rapeseed oil and evening primrose oil shortened the survival time by approximately 40% as compared with n-3 fatty acid-rich perilla and fish oil; lard, soybean oil, and safflower oil with relatively high n-6/n-3 ratios shortened the survival time by roughly 10%. The observed unusual survival time-shortening activities of some vegetable oils (rapeseed, high-oleate safflower, high-oleate sunflower, olive, and evening primrose oil) may not be due to their unique fatty acid compositions, but these results suggest that these vegetable oils contain factor(s) which are detrimental to SHRSP rats.

Studies on chemical modification of monensin. VI. Preparation of C-7 modified monensins via protected 7-oxomonensin and evaluation of their ion transport activity.

7-Carboxymethylmonensin (3), 7-aminomonensin (4), and 7-oxomonensin (5) were synthesized via protected 7-oxomonensin (2), which is expected to be a useful intermediate for the preparation of various monensin derivatives. intermediate for the preparation of various monensin derivatives. Compounds 3-5 exhibited smaller ion transport activity than monensin (1).