The defense mechanism of the gastrointestinal mucosa against aggressive factors, such as hydrochloric acid, bile acid and non-steroidal anti-inflammatory drugs, mainly consists of functional, humoral and neuronal factors. Mucus-alkaline secretion, mucosal microcirculation, and motility act as functional factors, while prostaglandins and nitric oxide act as humoral factors, and capsaicin sensitive sensory neurons act as neuronal factors. All the above factors are known to contribute to mucosal protection. In recent years, heat shock proteins (HSPs), to include HSP70, have been implicated to be an additional factor utilized for the defense mechanisms of the gastrointestinal mucosa at the intracellular level. The expression of HSP70 and HSP47 markedly changes during the development and healing of chronic gastric ulcers in rats. It was revealed that HSC70 (a constitutive form of HSP70) is coprecipitated with cyclooxygenase-1 and the neuronal form of nitric oxide synthase after treatment with a mild irritant (20% ethanol). A positive relationship between enhanced interaction of HSC70 with either cyclooxygenase-1 or nitric oxide synthase and mucosal protection against a strong irritant (100% ethanol) was observed. It was concluded that HSPs might contribute to mucosal defense mechanisms and ulcer healing, most probably through protecting key enzymes related to cytoprotection.
Until recently, the determination of metallic elements concentrations in normal skin, in vivo, was rare due to the lack of non-invasive techniques. Microdialysis has the advantage of being slightly invasive when applied to the collection in vivo of endogenous or exogenous substances from the skin. Iron is an active element in different cutaneous disorders. The aim of this work was to assess iron by atomic absorption spectrometry (AAS) after the collection of samples by microdialysis from human dermis. A first essential step, before determining the in vivo iron concentration in human dermis, was to establish an experimental protocol applicable to ex vivo as well as in vivo conditions. For this reason, this work deals only with the assessment of iron in ex vivo human dermis. A skin microdialysis technique and a calibration method, the No Net Flux, were used to quantify basal iron concentrations in human dermis and the same method was also used to determine in vitro and ex vivo iron recoveries. No differences were detected between in vitro and ex vivo recoveries. Ex vivo basal iron dermis concentrations ranged from 3.6 to 7.7 μg/l. This study shows that non-invasive microdialysis is an efficient method for sampling iron from human skin. A sensitive and accurate AAS technique was able to assess low iron concentrations in human dermis. The strategy adopted for this work was efficient and appropriate for the determination of iron in human skin and experiments will be carried out in vivo.
The 32-kDa galectin (LEC-1) of the nematode Caenorhabditis elegans (C. elegans) is composed of two tandemly repeated homologous sequences, each containing a carbohydrate-recognition domain (CRD). Using the polymerase chain reaction (PCR) with LEC-1 cDNA as a template and “megaprimers”, we performed site-directed mutagenesis to substitute conserved amino acid residues in these domains. The resultant mutated LEC-1s were produced in E. coli, and their binding abilities were estimated by affinity chromatography. When one of the conserved amino acid residues in the first lectin domain was substituted, the binding ability of the mutant protein to asialofetuin-agarose was reduced but still remained. The binding ability of such mutants was similar to that of the recombinant half molecule containing the second lectin domain (Ch). However, when mutations were introduced into the second lectin domain, the binding ability of these mutant lectins to asialofetuin-agarose was significantly reduced just like the half recombinant molecule containing the first lectin domain (Nh). The different effects of the substitution of amino acid residues on the two lectin domains suggest that the binding properties of the two sites are different and that LEC-1 acts as a “heterobifunctional crosslinker.”
Mouse thioglycollate-induced peritoneal macrophages effectively, in the absence of serum, recognized mouse polymorphonuclear leukocytes (PMNs) mildly oxidized with diamide, superoxide (hypoxanthine/xanthine oxidase) or t-butylhydroperoxide, or modified with N-ethylmaleimide (NEM). The recognition reached a maximum when PMNs were treated with each of the reagents at relatively low concentrations, and the recognition was decreased on treatment with reagents at higher concentrations. Glutathione depletion in the diamide-oxidized PMNs may cause enhanced adhesion to macrophages. Sialylated sugar chains attached to a peptide chain in glycophorin A and sialylated poly-N-acetyllactosaminyl sugar chains in lactoferrin and band 3 glycoprotein effectively inhibited the increased adhesion of the diamide-oxidized PMNs. Enzymatic removal of sialyl residues and the degradation of poly-N-acetyllactosaminyl sugar chains by pretreatment of PMNs with neuraminidase or endo-β-galactosidase, respectively, lost their increasing ability for macrophage adhesion after oxidation with diamide, superoxide or t-butylhydroperoxide. Clustered sialylated poly-N-acetyllactosaminyl sugar chains on the cell surface may be involved in the increased adhesion of the oxidized PMNs to macrophages.
Proteoglycans (PGs) synthesized by chick corneal stromal cells in cell culture and organ culture were metabolically radiolabelled with [35S]sulfate (for glycosaminoglycans) and [3H]leucine (for core proteins). Media, cell extracts and organ extracts from cultures were chromatographed on DEAE-Sephacel columns and separated into three fractions: the pass-through fraction (Fraction 1: nonsulfated PGs, hardly sulfated PGs, or glycoproteins with some oligosaccharides), the fraction eluted with a low salt concentration (Fraction 2: undersulfated PGs), and the fraction eluted with a high salt concentration (Fraction 3: highly sulfated PGs). The PG compositions of each fraction of cell culture and organ culture were then compared. While the proportions of highly sulfated KSPG in Fractions 3 of medium and cell extract of cell culture were both very low compared with those of medium and organ extract of organ culture, respectively, the proportions of highly sulfated CS/DS PG in Fractions 3 of those of cell culture were higher than those of organ culture. On the other hand, the proportions in the 35S activities of nonsulfated or undersulfated KSPG in Fractions 1 and 2 of medium and cell extract of cell culture were comparable to those of organ culture. Furthermore, the proportions of core proteins of undersulfated KSPG in Fractions 2 were higher in cell culture than in organ culture. These results show that, when the cells are cell-cultured, the degree of sulfation of KS chains decreases markedly, but the syntheses of the glycosaminoglycan backbone and core protein are maintained.
Angiotensin-converting enzyme inhibitors prevent cardiac hypertrophy in vivo, and a component of this ameliorative effect has been attributed to accumulation of kinins in cardiac tissues. However, little is known regarding the levels of kallikrein-kinin components in the heart during the development of cardiac hypertrophy. The objectives of the present study were to define the effects of pressure-overload cardiac hypertrophy on cardiac levels of kininogen, kallikrein and bradykinin B2 receptor mRNAs. The pressure-overload induced by aortic constriction produced cardiac hypertrophy in mice after 14 and 28 d, assessed from the increased ratios of heart weight to body weight and elevation of brain natriuretic peptide mRNA in the heart. B2 receptor mRNA rapidly decreased in the heart within 7 d after the operation, subsequently returning to those of sham-operated animals. In contrast, levels of both low-molecular-weight kininogen and tissue kallikrein mRNAs were increased 7, 14 and 28 d after aortic constriction. These findings suggest that the mechanical load or stretch in cardiac tissue by pressue-overload rapidly produces the downregulation of B2 receptor expression during the initial stage which may allow the promotion of cardiac hypertrophy induced by a mediation of hypertophic factors such as angiotensin II, while upregulation of kininogen and kallikrein mRNAs during the chronic stage may lead to an enhancement of local kinin generation in the heart, from which further progression of cardiac hypertrophy during later stages may be regulated.
A complex of plasminogen activator inhibitor-1 (PAI-1) and PAI-1-binding protein (PAI-1-BP) contained S-protein (vitronectin), PAI-1 and unidentified 40-kDa protein on SDS-PAGE under reducing conditions. By Western-blot analysis, the 40-kDa protein was identified as SP-40, 40 using anti-SP-40, 40 antibody. Therefore, it was thought that PAI-1-BP consisted of S-protein and SP-40, 40. It is known that PAI-1 is a labile protein which becomes inactive during incubation at 37°C. However, after the incubation of PAI-1 with SP-40, 40 at 37°C for 1 h, PAI-1 could still form a complex with tissue plasminogen activator (tPA), and it inhibited plasmin formation in the mixture of plasminogen and urine plasminogen activator (uPA). The results clearly indicated that SP-40, 40 stabilized PAI-1 activity as well as S-protein did.
We sequenced and characterized the expression patterns of the genes (racG, racH, racI and racJ) in the Rho-family. The nucleotide sequences of these genes suggest that racI would be a pseudogene, while the other genes are likely to encode typical Rac proteins which contain either GTP-binding domain or CAAX prenylation motif as observed in other members of the family. The Northern blot analyses show that the expression patterns of these genes are distinctively regulated during development. The racG gene is expressed at almost the same level from the vegetative to the slug stage, but the amount of its transcript gradually decreases after culmination. Expression of the racJ gene is undetectable at the vegetative stage, becomes observable at the mound stage, reaches a peak at the slug stage and then suddenly disappears in the culmination stage. The racH gene is expressed in two forms of transcripts, both of which are undetectable at the vegetatively growing stage but abruptly increase in amount after starvation. Southern blot hybridization analysis demonstrates that these transcripts were derived from a single copy of the gene. Such distinct kinetics of the expression patterns suggests that these genes would have unique roles in Dictyostelium development.
The impairments of learning and memory function and of the cholinergic system were examined in rats with microsphere embolism. Microsphere embolism was induced by injection of 900 microspheres with a diameter of 48 μm into the right internal carotid artery. The retention latency of a passive avoidance test was shortened and the escape latency of a water maze test was prolonged, when the animals were tested on the 5th to 10th day after the embolism, suggesting learning and memory dysfunction. Cholinergic parameters of the striatum and hippocampus, such as acetylcholine (ACh) content (67 and 60% decrease, respectively), choline acetyltransferase (ChAT) activity (45 and 56% decrease, respectively), and Bmax of muscarinic acetylcholine M1-receptor (43 and 37% decrease, respectively), were reduced on the 11th day after the embolism, suggesting attenuation of ACh synthesis and a decrease in the number of muscarinic acetylcholine M1-receptors mainly in the striatum and hippocampus. Areas not stained with triphenyltetrazolium chloride, an indication of infarction, were detected mainly in the striatum and hippocampus and partly in the frontal cortex on the 11th day after the embolism. The results suggest that an animal with microsphere embolism may be a good ischemic model with relatively sustained impairments of learning and memory function and of the striatal and hippocampal cholinergic system.
We have recently found that L-glutamate suppresses astrocyte stellation induced by various stimuli, and that this effect of L-glutamate is mimicked by transportable glutamate uptake inhibitors. To test the possible role of the glutamate transporter in the regulation of astrocyte morphology, we investigated the Na+ and K+ dependence of this effect of L-glutamate. In astrocyte cultures obtained from the cerebral cortex of neonatal rats, the L-glutamate-induced suppression of astrocyte stellation was significantly attenuated in a low-Na+/high-K+ medium and by the Na+-K+ pump inhibitor ouabain. These results support that astrocyte morphology is affected by the activity of the Na+-dependent glutamate transporter.
Glycyrrhetinic acid (GA) is known to inhibit glucocorticoid metabolism inhibiting 11β-hydroxysteroid dehydrogenase (11β-HSD). Moreover, GA administration to mice has been shown to affect the lymphoid organs through elevation of endogenous corticosterone concentration. The effect of GA administration on thymus has been demonstrated to show that considerable amounts of thymocytes undergo apoptosis by elevated levels of corticosterone in systemic circulation. However, the effect of GA administration on peripheral lymphocytes has remained unknown. In our current study, we demonstrated that a significant involution of spleen as well as thymus occurred within 24 h of a single administration of GA in mice. In addition, a flow cytometric analysis of the splenocytes taken from mice treated with GA showed a significant increase in the number of apoptotic cells which exhibited translocated phosphatidylserine outside the plasma membrane. Furthermore, considerable inhibition of 11β-HSD activity in GA-treated mice was observed in liver and spleen, resulting in a significant increase in concentration of corticosterone in the blood. These facts showed that the apoptosis of splenocytes was the result of indirect effect of GA through elevated levels of corticosterone. We confirmed this using cultured splenocytes in vitro where no apoptotic effect of GA was observed. We concluded that GA administration induces cell death of not only thymocytes that are naive to corticosterone, but also splenocytes that are usually stable to its physiological concentrations.
Exercise decreases plasma total cholesterol and triglycerides, and simultaneously, increases high density lipoprotein (HDL) cholesterol. Furthermore, it has been reported that exercise improves insulin resistance. As a result, exercise is believed to aid in preventing atherosclerosis. However, we do not know whether exercise protects against the development of atherosclerosis in the elderly. The aim of this study was to ascertain whether exercise prevented atherosclerosis in aorta of old rats. Exercise for three months did not affect plasma lipid levels but decreased accumulation of visceral fat and body weight without the reduction of food intake in two year old rats. Exercise also decreased triglycerides in liver and gastrocnemius white muscle. Exercise resulted in an elevation of plasma lipid peroxide (LPO) levels without affecting superoxide dismutase (SOD). Exercise impaired the endothelium-dependent relaxation of the thoracic aorta caused by acetylcholine in old rats. In summary, the results of our study indicate that exercise induces the reduction of visceral fat, body weight and triglyceride contents in tissues in old rats. These results seems to show that exercise improves insulin resistance. However, exercise simultaneously may cause impaired endothelium-dependent relaxation by elevation of LPO in old rats.
The present study was carried out to clarify the effects of extracts of the leaves of Mentha piperita L. on experimental allergic rhinitis. The 50% EtOH extract of peppermint inhibited histamine release from rat peritoneal mast cells induced by compound 48/80. The effect was dose-dependent and significant inhibition was observed at a concentration of 3 μg/ml. In addition, the 50% EtOH eluate separated from the 50% EtOH extract of peppermint by column chromatography (DIAION HP-20) was also effective in inhibiting histamine release at a concentration of 1 μg/ml. Nasal symptoms, sneezing and nasal rubbing induced by antigen challenge in actively sensitized rats were inhibited by oral administration of the 50% EtOH eluate. Significant inhibition of sneezing and nasal rubbing was observed at doses of 300 and 1000 mg/kg, p.o., respectively. Furthermore, the 50% EtOH eluate inhibited dye leakage into the nasal cavity of rats induced by antigen in a dose-dependent manner. These results suggested that extracts of Mentha piperita L. may be clinically effective in alleviating the nasal symptoms of allergic rhinitis.
In order to establish an analytical method for the determination of phenobarbital (PB), phenytoin (PPH) and their hydroxylated metabolites in hair, animal model experiments were performed. Five male dark-agouti pigmented rats, aged 5 weeks, were intraperitoneally and orally administered PB or PPH independently at 25 mg/kg once a day for 5 successive days. The growing back hair was collected 15 d after the first administration. Four typical extraction methods, using NH4OH-methanol-acetone, TFA-methanol-acetone, 1 M sodium hydroxide and proteinase K, were evaluated using the rat hair samples containing PB or PPH. Methanol-acetone-NH4OH (10:10:1) was the best extraction method from all aspects, such as high extraction efficiency and low noise. The analytes in the extract were methylated in acetonitrile with 20% tetramethylammonium hydroxide and methyliodide at 70°C for 10 min. After purification with Bond Elut Certify, the methylated products were analyzed by GC-MS. From rat hair, PB, p-hydroxy PB, PPH and p-hydroxy PPH were detected at average concentrations of 26.9, trace, 4.2 and 0.4 ng/mg with an intraperitoneal (i.p.) injection, and at 30.9, trace, 4.0 and 0.4 ng/mg with oral administration, respectively. There was little difference in hair concentrations between i.p. injection and oral administration. This method was applied to the head hair of two patients who orally took toxic amounts of PB and two volunteers who orally took 100 mg of PPH daily for 5 d. The hair concentrations of PB in the two patients were 16.2 and 14.7 ng/mg, and those of PPH in the two volunteers were 3.3 and 0.1 ng/mg.
It is difficult to isolate derivatives of α-crystallin with only one type of post-translational modification, because this protein is subjected to several different types of modification. In the present study using bovine lens proteins, we isolated mono-phosphorylated αB-crystallin with no other post-translational modifications. Using this material, we demonstrated that mono-phosphorylation reduced the activity of αB-crystallin by approximately 30%.Our results confirmed that investigation of the correlation between chaperone-like activities of α-crystallin and post-translational modification is important to understand the mechanism of cataract formation.
A higher concentration of kinsenoside, 3-(R)-3-β-D-glucopyranosyloxybutanolide (1), was detected in the crude drug Anoectochilus formosanus, and A. koshunensis by HPLC analysis. A methylation reaction occurred to give methyl ester (4) when the lactone ring of 1 was cleaved by silica gel catalysis using methanol containing solvent used in the purification step resulting in difficulty to purify 1. To avoid the cleavage reaction, a reversed-phase or silica gel column without methanol was used to give a high yield of 1. In an anti-hyperliposis assay using high-fat diet rats, 1 significantly reduced the weights of body and liver, and also decreased the triglyceride level in the liver compared to those of control rats. On the other hand, the epimer of 1, 3-(S)-3-β-D-glucopyranosyloxybutanolide (2), trivially named goodyeroside A, which was isolated from Goodyera species, had no effect for anti-hyperliposis. In aurothioglucose-induced obese mouse, 1 suppressed the body and liver weight increase, significantly ameliorated the triglyceride level in the liver, and also reduced the deposition of uterine fat-pads.
Two quinoline alkaloids, japonine (1) and eduline (2) were isolated from the methanol extract of the leaves of Orixa japonica as relaxants against rat small intestine muscle. The activity of these alkaloids was comparable to that of the typical muscle relaxant papaverine (4). This is also the first report of the isolation of eduline (2) from O. japonica.
This paper presents a numerical integration method for estimating the area under the curve (AUC) over the infinite time interval. This method is based on the Gauss-Laguerre quadrature and produces AUC estimates over the infinite time interval without extrapolation in a usual sense. By contrast, in traditional schemes, piecewise interpolation is used to obtain the area up to the final sampling point, and the remaining portion is extrapolated using nonlinear regression. In this case, there is no theoretical consistency between the quadrature and extrapolation. The inconsistency may cause certain problems. For example, the optimal sampling criterion for the former is not necessarily optimal for the latter. Such inconsistency does not arise in the method of this work. The sampling points are placed near the zeros of Laguerre polynomials so as to directly estimate the AUC over the infinite time interval. The sampling design requires no particular prior information. This is also advantageous over the previous strategy, which worked by minimizing the variance of estimated AUC under the assumptions of particular pharmacokinetic and variance functions. The original Gaussian quadrature is belived to be inappropriate for numerical integration of data because of several restrictions. In this paper, it is shown that, using a simple strategy for managing errors due to these restrictions, the method produces an estimate of AUC with practically sufficient precision. The efficacy of this method is finally shown by numerical simulations in which the bias and variance of its estimate were compared with those of the previous methods such as the trapezoidal, log-trapezoidal, Lagrange, and parabolas-through-the-origin methods.
The release kinetics of dihydroetorphine (DHE) from pressure-sensitive adhesive (PSA) tape with an ethylene-vinyl acetate co-polymer (EVA) membrane as a diffusion-controlling membrane and its protective effect from an unpredictable increase in skin permeation of DHE caused by stratum corneum damage were investigated. The DHE flux through the EVA membrane was enhanced with the increase of vinyl acetate content. Although the DHE release from the PSA tape was proportional to the square root of the time, the release from the PSA tape covered with the EVA membrane was dominated by zero-order rate. The release rate increased by the addition of isopropyl myristate to the PSA layer, due to the increase of solubility and diffusivity of DHE in the PSA layer, and not a decrease of permeation resistance in the EVA membrane. When using the PSA tape with the EVA membrane, the steady-state flux of DHE through hairless rat skin with stratum corneum damage was not 2-fold more than that through non-damaged skin. Plasma DHE concentration rose promptly above 5 ng/ml after the application of the PSA tape onto the damaged skin in hairless rat. In contrast, when the PSA tape with the EVA membrane was applied onto the damaged or non-damaged skin, plasma concentrations in the both cases were maintained in the therapeutic range (0.2—1.2 ng/ml). These results suggest that the PSA tape with the EVA membrane can be used to protect from the unpredictable increase in skin permeation of DHE due to stratum corneum damage.
The inhibitory effects of ethaverine on dopamine content in PC12 cells were investigated. Ethaverine decreased dopamine content in a concentration-dependent manner in PC12 cells and showed 33.6% inhibition of dopamine content at a concentration of 1.0 μM for 24—48 h. The IC50 value of ethaverine was 1.4 μM. Dopamine content was lowered at 6 h and reached a minimal level at 12 h after exposure to ethaverine at 2.0 μM. The decreased dopamine level was maintained up to 48 h and then recovered to the control level at about 72 h. Tyrosine hydroxylase (TH) was inhibited at 6 h following treatment with ethaverine in PC12 cells and the activity was maintained at a reduced level up to 36 h (12—22% inhibition at 2.0 μM). These results indicate that ethaverine leads to a decrease in dopamine content by inhibition of TH activity.
To avoid the adverse effects of aprotinin (Apr) in autologous fibrin glue, we compared the inhibitory properties of four commercial anti-fibrinolytic agents (tranexamic acid (Tra), ε-aminocaproic acid (Ips), gabexate mesilate (Gab) and nafamostat mesilate (Naf)). The optimum conditions for the lysing of fibrin glue were an incubation temperature of 37°C, and incubation medium containing urokinase at 100 U/ml and plasminogen at 100 mU/ml (for the washed glue), or neither (for unwashed glue). Fibrin glues without an anti-fibrinolytic agent were quickly lysed in the incubation medium, while those with an anti-fibrinolytic agent were slowly lysed dose-dependently. Naf was the most potent inhibitor and had high affinity for the glue, but the other agents were poor inhibitors and had low affinity. The inhibition potency (IP) of each agent did not correlate with hydrophobicity, but a good correlation was obtained between the remaining coefficient (RC) and hydrophobicity. Naf did not affect the adhesive strength of the glue. These results indicate that Naf is the most suitable anti-fibrinolytic agent to replace Apr.