Cytokines of the transforming growth factor-β(TGF-β) superfamily are multifunctional peptides that regulate growth and differentiation of various types of cells. Members of the TGF-β superfamily bind to type II and type I serine/threonine kinase receptors, which mediate intracellular signals through SMAD proteins. Of 3 subtypes of SMADs, receptor-regulated SMADs are phosphorylated by the serine/threonine kinase receptors, form complexes with common-mediator SMAD, and move into the nucleus, where they act as components of transcription factor complexes. Abnormalities of the TGF-β receptors and SMADs have been detected in various tumors, including colorectal cancers and pancreatic cancers. Inhibitory SMADs and transcriptional co-repressors, including c-Ski and SnoN, repress the TGF-β/SMAD signaling. Perturbation of the TGF-β/SMAD signaling pathway may result in progression of tumors through resistance of the cells to the growth inhibition induced by TGF-β.

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We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes : CYP2C9 (CYP2C9^*2 and ^*3), CYP2C19(CYP2C19^*2 and ^*3), CYP2D6 (CYP2D6^*4, ^*10, ^*14, ^*18, and ^*21(C8)), N-acetyltransferase 2 (NAT2^*5B, ^*6A, and ^*7B), thiopurine methyltransferase (TPMT^*3C), and aldehyde dehydrogenase2 (ALDH2^*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.

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We examined the effect of murine kidney extract (MKE) on the clonal growth of highly purified CD34⁺ hematopoietic progenitor cells from human umbilical cord blood. MKE did not affect the total number of colonies of erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM) or granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-Mix/CFU-GEMM) in a methyl-cellulose culture with exogenous recombinant human granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor and erythropoietin. MKE significantly increased the proportion of BFU-E- or CFU-Mix-derived colonies, and suppressed the formation CFU-GM-derived colonies depending on the MKE dose. However, because of an increase in small megakaryocyte colonies derived from mature CFU-Meg MKE increased by approximately 40% the growth of megakaryocyte colony-forming units (CFU-Meg) in plasma clot culture stimulated by recombinant human thrombopoietin. Also MKE promoted an increase in hyperploid megakaryocytes, suggesting that the active factor(s) in MKE acts on the mature CFU-Meg and promotes the maturation of megakaryocytes. Gel-filtration high performance liquid chromatography of MKE showed that the promoting factor(s) in MKE was approximately 45 kDa. These results indicate that the factor(s) detected in MKE influence human hematopoiesis in vitro, especially thrombopoiesis.

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A study of the biliary bile acid composition in porcine fetus compared with that of the adult pig is described.Biles, collected during gestation (weeks 4, 15 to 17 and at birth), aged six months and two years old, were analyzed by gas-liquid chromatography and capillary GC-MS. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, piperidinohydroxypropyl Sephadex LH-20. All and one fourth of the total bile acids in the bile of weeks 4 and 15 of gestation, respectively, were present as unconjugated form, however, only a trace of unconjugated bile acids was present in bile of late gestation, the young and the adult pigs. The ratio of glycine/taurine (G/T) conjugates in the conjugated fraction of the fetal bile at 15 weeks gestation was less than 1, which markedly contrasted with the conjugation pattern for adult bile where the ratio of G/T conjugates was approximately more than 9.The predominant acids identified in porcine fetal bile of the 4 weeks gestation were cholic acid (3α, 7α, 12α-trihydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (3α, 7α-dihydroxy-5β-cholan-24-oic acid). However, cholic acid in late gestation, young, and adult bile was the smallest component, whereas chenodeoxycholic acid was still the major constituent of these biles. The presence of small but valuable amounts of allocholic acid (3α, 7α, 12α-trihydroxy-5α-cholan-24-oic acid) and cholic acid in early gestation suggested the presence of 12α-hydroxylase activity of steroid nucleus in fetal liver. Considerable amounts of glycine-conjugated hyodeoxycholic acid were found in the bile of the gestation periods, suggesting the placental transfer of this bile acid from maternal circulation.

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The solution structure of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), a protein of 155 residues, was determined. Three-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain 1424 distance constraints between individually assigned polypeptide chain hydrogen atoms. Supplemental geometric constraints of 90φ angles and 12_χ1 angles, and the distance constraints of 66 hydrogen bonds were experimentally derived. Using the DADAS90 program that calculates structures in dihedral angle space, 15 structures satisfying almost all constraints were obtained. The average root mean square deviation (RMSD) from the mean structure was 0.75Å for backbone atoms. The RMSD for backbone atoms between the representative NMR structure with the smallest constraint violation and crystal structures was within 1.2Å. Although the NMR and crystal structures thus resemble one another, a significant discrepancy was observed in a region termed 'basic protrusion.' The discrepancy observed in NMR experiments is explained by fluctuation in this region.

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We earlier demonstrated that gallic acid (3, 4, 5-trihydroxybenzoic acid) induced apoptosis in promyelocytic leukemia HL-60RG cells, which was inhibited by catalase and intracellular Ca²⁺ chelator. In this study, we further studied the involvement of reactive oxygen species (ROS) and intracellular Ca²⁺ in gallic acid-induced apoptosis. The enhancement of intracellular ROS in HL-60RG cells was detected dose-dependently as early as 5 min after stimulation with gallic acid by using 5, 6-carboxy-2', 7'-dichlorofluorescin diacetate (DCFH-DA). Further studies that used various antioxidants and ROS scavengers showed that the intracellular peroxide level was well correlated with the potency to induce apoptosis and that the increased intracellular peroxides after gallic acid treatment seemed likely to result from the influx of H₂O₂ derived from superoxide which were generated extracellularly. In addition, gallic acid, HX/XO, and H₂O₂-induced apoptosis was completely inhibited by pretreatment with intracellular Ca²⁺ chelator 1, 2-bis(2-aminophenoxyethane)-N, N, N, N'-tetraacetic acid tetrakis (acetoxymethyl ester)(BAPTA-AM), but increase of intracellular peroxide levels by gallic acid were suppressed only slightly. It is suggested that intracellular ROS induced by gallic acid plays an important role in eliciting an early signal in apoptosis. Especially, H₂O₂ which is derived from superoxide anion generated extracellularly may increase intracellular Ca²⁺ levels or cooperate with intracellular Ca²⁺, thus resulting in apoptosis induction.

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Raf-1 is a serine/threonine protein kinase that plays a critical role in mitogenic signal transduction. Raf-1 activation requires 14-3-3 binding to Raf-1 as an essential step. This binding is regulated through phosphorylation of Ser259 and Ser621 of Raf-1, each constituting part of the consensus motif for the binding of Raf-1 to 14-3-3. However, Raf-1 kinase kinase(s) that phosphorylates these sites remains unknown. In this report, we detected Raf-1 kinase kinase activity using recombinant glutathione-S-transferase-Raf-1 fusion proteins as substrate of in situ gel kinase assay. Ser259 was phosphorylated by a kinase with a molecular weight of 90 kDa, which was suggested to be Rsk judging from the molecular size, the time course of activation after EGF stimulation and the elution pattern from an anion-exchange column. The Raf-1 fragment containing Ser621 was phosphorylated by kinases with molecular weights of 85, 60, 50 and 48 kDa but not by the kinase that phosphorylates Ser259. These results suggest that although Ser259 and Ser621 lie in the same amino acid sequence motif for 14-3-3 binding, these two regulatory sites for this binding are phosphorylated by different protein kinases.

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Two types of phospholipase A₂ (PLA₂) inhibitory protein (PLIP-I, PLIP-II) were detected and isolated from human amniotic fluid by Sephacryl S300 gel filtration chromatography. The lower molecular weight-fraction (PLIP-II) was further purified by Sephadex G75 gel filtration and analyzed by SDS-PAGE. Its molecular weight was estimated to be approximately 18 kDa, and it was sensitive to heat treatment. Inhibition of phospholipase A₂ (sPLA₂ type IIA) by PLIP-II occurred in a dose-dependent manner (IC₅₀ about 0.82 μM), and the effect was stronger on sPLA₂ IIA than on pancreatic sPLA₂ (IC₅₀ about 3.11 μM). The ratio of the inhibitions of the sPLA₂ IIA by PLIP-II remained consistent over an entire range of substrate concentrations. Furthermore, addition of excess Ca²⁺ at concentrations of up to 10 mM did not antagonize the inhibitory activity of PLIP-II.

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The binding ability of bovine and human lactoferrins (bLF and hLF; LFs) to a glycyrrhizin (GL)-affinity column and their phosphorylation by casein kinase II (CK-II) in vitro were biochemically investigated. It was found that (i) both bLF and hLF are GL-binding proteins; (ii) purified both proteins function as phosphate acceptors of CK-II; and (iii) this phosphorylation is completely inhibited by two polyphenol-containing anti-oxidant compounds (quercetin and epigallocatechin gallate) at 1 μM, whereas a glycyrrhetinic acid derivative (oGA) inhibits it at one tenth the concentration of GL. The DNA-binding affinity of hLF was reduced by GL in a dose dependent manner. However, no significant effect of the CK-II-mediated hLF phosphorylation on its DNA-binding affinity was detected. These results suggest that the GL-induced inhibition of the DNA-binding affinity and the CK-II-mediated phosphorylation of hLF may be closely correlated with the anti-inflammatory effect of GL in the human body.

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The purpose of this study was to demonstrate that the histochemical staining reactivities of lectins in rat stomach actually represent the gastric mucins, and to estimate the utility of the lectins for mucin histochemistry.In this paper, the lectin histochemistry was compared with an enzyme-linked lectin binding assay (ELLA) of the mucins derived from distinct regions and layers of the Sprague-Dawley rat stomach and it was examined to determine the definite binding and problematic binding of the conventional lectin. Among the 10 different biotinylated lectins, Canavalia ensiformis (ConA), Griffonia simplicifolia II (GS-II), Triticum vulgaris (WGA), Ricinus communis I (RCA-I), Arachis hypogaea (PNA), Glycine max (SBA), Dolichos biflorus (DBA), Ulex europaeus I (UEA-I), Sambucus nigra (SNA) and Maackia amurensis II (MAL-II), examined in this study, GS-II, SBA, DBA, UEA-I, SNA and MAL-II bound clearly to the mucin of distinct regions and layers of the Sprague-Dawley rat stomach in agreement with the results of ELLA. Namely GS-II lectins preferentially bound to the mucin in the mucous neck cells of the corpus area. SBA and DBA clearly recognized the mucin in the covering epithelial mucous cells in the corpus and antral area. UEA-I was widely bound to all the mucin present in both the corpus and autrum. On the other hand, SNA and MAL-II could not react with the mucin obtained from the gastric mucosa but was specifically bound to the mucin purified from the mucous gel layer. These results suggested that the lectins described above are useful histochemical tools to recognize the mucus present in the different regions and layers of Sprague-Dawley rat gastric mucosa.

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The fibroblast-populated collagen gel culture method has been evaluated as a dermal model of wound contraction and granulation in tissues during the wound healing process and as an in vitro model of dermal tissue.We previously reported that an extract of Fucus vesiculosus promoted fibroblast-populated collagen gel contraction and that the promotion of the gel contraction was due to the increased expression of integrin α2β1 on the surface of the fibroblasts. In this study, we investigated the active component of the extract of this alga using extraction and fractionation techniques. Water extraction of the alga was followed by precipitation with excess ethanol and then gel filtration with the boundary molecular weight of 30000. The high molecular weight fraction obtained from gel filtration was fractionated by ion exchange chromatography on diethylaminoethyl cellulose column to give active fractions that have more polar properties. These polar, high molecular weight fractions which contained molecules with fucose and sulfate groups showed significant gel contraction-promoting activity and integrin expression-enhancing activity, and were estimated to be the sulfated-polysaccharide fucoidan. Commercially available fucoidan showed similar activities to the above-described fraction of this alga. Although it remains necessary to precisely identify the specific active component, the above results indicate that fucoidan is the active component which promotes collagen gel contraction, and also indicate the possibility that it dose so by enhancing the integrin α2β1 expression.

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We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [³H]Ang II binding to the membrane fractions exhibited both high (K_d=0.08 nM, B_max=2.19 fmol/mg protein) and low (K_d=4.19 nM, B_max=13.7 fmol/mg protein) affinity. Ang II, Ang III and saralasin completely displaced the [³H]Ang II binding, whereas CV-11974, an AT₁ receptor antagonist and PD123319, an AT₂ receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [³H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a K_d of 4.22 nM and B_max of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [³H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT₁ receptor subtype.

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The effect of long-term administration of amlodipine and cilnidipine was examined on the histopathology and 1, 4-dihydropyridine (DHP) calcium channel antagonist receptors in the left ventricle of BIO TO-2 hamsters, a model of dilated cardiomyopathy (DCM). Oral administration of amlodipine (3 and 10 mg/kg/d, 19 weeks) in 7 week-old BIO TO-2 hamsters produced a significant reduction in calcium deposition and necrosis with little change in the cavity area and fibrosis. A reduction of calcium deposition and necrosis in the myocardium of BIO TO-2 hamsters was also seen following similar administration of cilnidipine (10 mg/kg/d). The long-term administration of amlodipine (3 and 10 mg/kg/d) caused a significant increase (36.6 and 21.7%, respectively) in the B_max for specific (+)-[³H]PN 200-110 binding in the myocardium from BIO TO-2 hamsters, compared with that in control hamsters. In conclusion, the present study has shown that long-term administration of amlodipine and cilnidipine improves calcium deposition and necrosis in the myocardium from BIO TO-2 hamsters. Thus, these data suggest that both agents may be effective pharmacological treatments of DCM.

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A lipid fraction obtained by activity-guided fractionation from the hexane extract of Sideritis javalambrensis was evaluated for anti-inflammatory activity. This fraction significantly inhibited paw oedema induced by carrageenan as well as ear oedema induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in mice after oral or topical administration, respectively. Quantitation of the specific marker myeloperoxidase (MPO) demonstrated that its topical anti-inflammatory activity was associated with reduction in cell infiltration into inflamed tissues.The lipid fraction significantly decreased leukocyte granular enzyme release (β-glucuronidase), but failed to inhibit superoxide generation. Histamine release from mast cells was also supressed in a concentration-dependent manner. In addition, non-toxic concentrations of this fraction reduced nitric oxide (NO) generation in lipopolysaccharide (LPS)-treated J774 macrophages. Taken together, these results suggest that the lipid fraction exerts in vivo anti-inflammatory activity with the partial contribution of inhibitory actions on some inflammatory responses.

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We have previously shown that m1 and m2 muscarinic receptors were expressed on human peripheral blood lymphocytes (hPBL) and that pre-stimulation of these receptors enhanced phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) production. Possible intracellular signal pathways of muscarinic receptors to regulate IL-2 production were examined in human T cell line Jurkat cells. Pretreatment of the cells with muscarinic receptor agonist, oxotremorine M (Oxo-M), enhanced IL-2 production induced by phorbol 12-myristate 13-acetate (PMA)/A23187, while Oxo-M by itself did not affect IL-2 production. The enhancement of IL-2 production by Oxo-M was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) an m1/m3 receptor antagonist. When the cells were pretreated with AF-DX116, an m2 antagonist, the IL-2 production enhanced by Oxo-M was further stimulated. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that m1 and m2 muscarinic receptors exist on Jurkat cells.The stimulation of m1 receptors enhanced the PMA/A23187-induced binding activity to AP-1 consensus sequences in IL-2 promoter and production of c-Fos and c-Jun protein. The stimulation of m1 receptors did not modify the DNA binding of NF-κB, NF-AT or Oct-1. When m1 receptors were stimulated, activities of mitogen-activated protein kinase (MAPK)/extracellular signal regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were increased, while p38 MAPK was not affected. Incubation with Oxo-M induced a transient increase in [Ca²⁺]_i, which was abolished by pretreatment with 4-DAMP. Treatment with cyclosporin A markedly decreased the PMA/A23187-induced IL-2 promoter activity. This treatment, however, did not affect the enhancement of the promoter activity induced by m1 receptor stimulation.The results suggest that transcription factor AP-1 is involved in the m1 receptor-mediated enhancement of IL-2 transcript in Jurkat cells, and that pathways via MAPK/ERK and JNK, but not via p38 MAPK, are involved in the m1 receptor-mediated enhancement of IL-2 promoter activity.

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Biphenyl dimethyl dicarboxylate (PMC) has been reported to protect against chronic ethanol toxicity. The present study was conducted to evaluate whether PMC might be accompanied by a reduction of ethanol-induced cellular immunotoxicity. PMC at a dose of 6 mg/kg was orally administered to ICR mice daily for 28 consecutive days, and normal mice were given vehicle. Mice treated with ethanol were given free access to 20% w/v ethanol instead of water. Mice were immunized and challenged with sheep red blood cells (SRBC). Delayed-type hypersensitivity (DTH) reaction to SRBC was increased to normal levels by the combination of PMC and ethanol, compared with the treatment of ethanol alone. Splenic CD4⁺ cells were also greatly enhanced by PMC treatment as compared with the treatment of ethanol alone. In the case of CD8⁺ cells, however, a slight reduction was observed by the PMC or ethanol treatment. The natural killer (NK) cell and phagocytic activity used for evaluation of nonspecific immunocompetence were significantly augmented in PMC plus ethanol-treated mice when compared with the treatment of ethanol alone. The number of peripheral leukocytes was significantly decreased by the treatment of ethanol alone, then also restored to normal levels by PMC treatment. These findings indicate that cellular immunotoxicity caused by ethanol consumption is significantly restored or prevented by PMC treatment.

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Our previous studies have demonstrated that the oral administration of dimethylarsinic acid (DMA), a main metabolite of inorganics aresemics in mammals, in mice causes DNA damage in the lung as well as the promotion and progression of lung- and skin-tumorigenesis. Moreover, we indicated that 72-kDa stress protein (Hsp72) was induced in cultured human pulmonary (L-132) cells by exposure to DMA and was accumulated specifically in the cell nuclei. The present in vivo study reveals the induction of Hsp72 by intraperitoneal administration of DMA to A/J mice used previously as an animal model of dimethylarsenic-induced lung tumorigenesis. The Hsp72 was observed in the lung, a target organ for arsenic carcinogenesis in human, and in the kidney as well, but not in the liver and spleen. By immunohistochemical analysis, the Hsp72 in lungs was exhibited to exist in the nuclei of alveolar flat cells, including capillary endothelial cells, which were previously found to increase the clumping of heterochromatin, an early morphological change in the developmental process of pulmonary carcinomas, after administration of DMA to mice. These in vivo observations suggest that the increase and accumulation of Hsp72 by administration of DMA to mice may occur specifically in target organs for arsenic carcinogenesis.

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Three new phenanthrene derivatives, aristoliukine-C, aristofolin-E and aristolochic acid-Ia methyl ester, and one new sesquiterpene, madolin-P, together with 58 known compounds were isolated from the stem and root of Aristolochia kaempferi. The structures of these compounds were determined by spectral analysis. The cytotoxicity and antiplatelet activity of the isolated compounds are also discussed.

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The effects of poly vinyl alcohol (PVA)/Chitosan/Fibroin (PCF)-blended sponge on wound healing in rats were investigated. We excised the skin of a rat, including the dermis, approximately 2×2 cm in size. The wound was convered with PCF-blended spongy sheets. The spongy sheets absorbed the exudate, and gained flexibility and softness. Histopathological inspection of the wound 12 d later showed an increase of vascular ingrowth and the absence of inflammatory cells. Regeneration of the skin around the wound was faster than that of the control. We also tested wound healing effects of PVA, Chitosan and Fibroin, alone or in various combinations. Wound healing was accelerated in the order of PCF-blended sponge>Chitosan/Fibroin (CF)-blended sponge≥Fibroin (F) sponge>PVA/Chitosan-blended sponge (PC)>Chitosan (C) sponge.

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We investigated sorption and permeation of emedastine with 11 different vehicles, composed of single or binary solvents, in excised rat skin. In the sorption study, partition parameters of the drug with each vehicle were obtained by dividing the drug amount in skin at equilibrium by its donor concentration. When the logarithm of the partition parameters for the stratum corneum/vehicle partitioning (K_s') was plotted against the dielectric constants of the vehicles, a bi-liner relationship was obtained. The skin flux of emedastine largely differ among the vehicles. A quasi-steady-state flux of emedastine exhibited a good linear relationship with K_s', except for ethanol (EtOH)/isopropyl myristate (IPM) systems, indicating that the partitioning process is critical in determining the permeation rate. Delineation of the EtOH/IPM systems would be due to an increase in the diffusivity of the drug in the stratum corneum, as indicated by the analysis using a two-layer diffusion model. Thus, differential evaluation of partitioning and diffusion processes by both sorption and permeation studies would give further insights into the effects of vehicles on skin permeation of drugs.

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The application of fibroin, a major silk protein, to controlled release type dosage tablets was investigated in vitro and in vivo. Fibroin tablets containing theophylline were easily prepared by a direct compression method without additives. Five types of fibroin tablets with the same surface area and different amounts of theophylline were prepared. In an in vitro drug study, the drug release from the tablets was not affected by the pH of the release medium. The greater the fibroin content in the tablets, the lower the percentage released at time t. The Higuchi plots of the release data showed a linear release profile, indicating that the drug release from the fibroin tablets was diffusion-controlled through the matrix. Theophylline powder or a TF-41 tablet (theophylline : fibroin=4 : 1), or a commercial tablet, Uniphyl (once-a-day type) was administered to 5 healthy volunteers. The areas under the saliva theophylline concentration-time curve (AUC) of Uniphyl and TF-41 to that of powder were 85% and 70%, respectively (fasted). Conversely, the mean residence time and mean absorption time of TF-41 were long compared to Uniphyl (fasted). Therefore, the reduction of bioavailability in TF-41 was due to the delayed release from the tablets in vivo. Taken after a meal, the AUCs of TF-41 and Uniphyl increased and the absorption was completed. This suggested that the drug release from TF-41 may increase due to the stimulation of food on TF-41 itself and due to movement of the gastro-intestinal tract. In conclusion, fibroin could be used as the matrix in controlled-release tablets.

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Food products can be possible vectors of the agent responsible for cholera epidemics, because some of these products allow Vibrio cholerae O1 to develop to concentrations above the dangerous level. This study deals with the behaviour of essential oils, natural and concentrated lemon juice and fresh and dehydrated lemon peel against V. cholerae O1 biotype Eltor serotype Inaba tox⁺. Our aim was to evaluate whether these products, used at different dilutions, exhibit bactericidal or bacteriostatic activity against the microorganism, when present at concentrations of 10², 10⁴, 10⁶ and 10⁸ colony forming units (CFU) ml^-1, and after different exposure times. 10⁸ CFU ml^-1 was considered an infectious dose. Concentrated lemon juice and essential oils inhibited V. cholerae completely at all stuidied dilutions and exposure times. Fresh lemon peel and dehydrated lemon peel partially inhibited growth of V. cholerae. Freshly squeezed lemon juice, diluted to 10^-2, showed complete inhibition of V. cholerae at a concentration of 10⁸ CFU ml^-1 after 5 min of exposure time; a dilution of 2×10^-3 produced inhibition after 15 min and a dilution of 10^-3 after 30 min. It can be concluded that lemon, a natural product which is easily obtained, acts as a biocide against V. cholerae, and is, therefore, an efficient decontaminant, harmless to humans.

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To characterize the local kallikrein-kinin system, tissue distribution of mRNAs for kininogens, precursor proteins of kinins, such as high-molecular-weight (H-), low-molecular-weight (L-) and T-kininogens, were studied in the rat by means of reverse-transcription polymerase chain reaction (RT-PCR) using a fluorophore Cy5-labeled 5'-primer. High levels of these three kininogen mRNAs were present in the liver. Relatively high levels of H-kininogen mRNA were also detected in the skin, lung, kidney, and testis in a descending order, whereas L-kininogen mRNA was detectable in the lung and brain, but not in the kidney, skin testis, heart, adrenal gland, or skeletal muscle. T-Kininogen mRNA was present in these tissues, except for skeletal muscle. These findings suggest that the expression of each kininogen mRNA is regulated by the tissue-dependent mechanisms which is closely associated with functions of the kallikrein-kinin system in the tissue.

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The hepatoprotective effects of recombinant human epidermal growth factor (hEGF) on chemically and immunologically induced experimental liver injury models were examined. The hEGF clearly decreased serum transaminase levels in D-galactosamine (D-GalN) and D-GalN/lipopolysaccharide (LPS)-induced liver injury models under sub-lethal conditions. However, it has not significantly changed either serum or in vitro tumor necrosis factor (TNF)-α production or in vitro nitric oxide (NO) production, suggesting that the hepatoprotection by EGF is not mediated by inhibiting these pathological mediators produced in D-GalN and D-GalN/LPS-induced liver injury.

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From the butanol fraction of the starfish Asterina pectinifera MULER et TROSCHEL (Asteriidae), we have isolated a new component, 5α-cholest-7-en-3β-ol. Its antigenotoxic and antimutagenic activities were examined by the SOS chromotest with Escherichia coli PQ37 and by Ames test with Salmonella typhimurium TA1538, respectively. 5α-Cholest-7-en-3β-ol showed potent antigenotoxic activity against the mutagens, both MNNG and NQO. For 100% of antigenotoxicity, the concentration of the compound applied against MNNG and NQO were 10 μg and 5μg per reaction tube, respectively. Its antimutagenic activity with S. typhimurium TA1538 against the mutagen MNNG was very effective. When its concentrations were varied from 1 up to 10 μg dose per plate, the inhibition ratio of revertant CFU of TA1538 per plate was increased accordingly, from 25.2 to 99.2%. These results suggest that 5α-cholest-7-en-3β-ol possesses antigenotoxic and antimutagenic activity and might be useful as a chemopreventive agent.

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Screening of crude drugs for inhibitory activity on α-amylase in mouse plasma was performed. Hot water extracts and ethanolic extracts of Arecae Semen, Ephedrae Herba, Malloti Cortex, Rhei Rhizoma and ethanolic extract of Moutan Cortex inhibited enzyme activity in isolated mouse plasma strongly and dose-dependently. However, the effects of these 9 extracts were not observed in the plasma when they were administered intraperitoneally or orally. Ethanolic extract of Arecae Semen also showed a depressive effect on elevation of postprandial blood glucose.

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The effect of the l-menthol-ethanol-water system (MEW system), a skin penetration enhancer, on the systemic absorption of flurbiprofen (FP) after repeated topical applications was investigated. FP (1%) gel containing ethanol (25%) and l-menthol (3%) as penetration enhancers was applied to rabbit dorsal skin and the in vivo absorption rate of FP was compared with the in vivo penetration rate through excised skin. In vivo absorption rate of FP was initially high and decreased with time to a value approximately equal to the in vitro rate. The remaining FP in the gel 6 h after the application was 60% of the initial loading and the systemic bioavailability over the 6 h application was about 10%, suggesting that the rest (30%) had accumulated in the skin tissues. The gel was applied for 6 h on the same site or on a new site after the first 6 h-application to learn the effect of repeated applications on FP absorption. The maximum FP concentration after the second application on the virgin skin was slightly higher than that after the first application, as expected in a typical pharmacokinetic process. In contrast, the same site application induced remarkably lower plasma concentration and area under the curve (AUC). A drug-free gel was also utilized to evaluate the effects of the enhancer system. Pretreatment of the drug-free gel on the same site also decreased the FP absorption, whereas post-treatment increased the plasma level of FP, in spite of the removal of the drug gel. These phenomena could be explained by ethanol in the MEW system acting a local irritant and a drug carrier.

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A low level of hypergravity (1.5-2.0 G) stimulated the proliferation of ROS17/2.8 cells, whereas it inhibited the differentiated functions of alkaline phosphatase activity and osteocalcin synthesis. These results were just the opposite of our results obtained when the cells were exposed to a high level of hypergravity (40-80 G) : inhibition of cell growth and the stimulation of the differentiated functions. The direction of change in the cAMP contents of the cells was also reversed, with a low level of hypergravity causing a decrease in the cAMP content and a high level of hypergravity an increase in it. Therefore, bidirectional effects of hypergravity on the growth and differentiated functions exist in ROS17/2.8 cells according to the magnitude of the hypergravity.

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Significantly prolonged survival rate was obtained for the first time using carcinoma mice models after the administration of single-component liposomes of dimyristoylphosphatidylcholine(DMPC) without any drug. An increase in lymphocytes under optical microscope was observed without any increase in the neutrophils count, suggesting that the DMPC liposomes might inhibit the tumor growth as well as increase in lymphocytes in vivo.

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Cell invasion assay is important for studying various biological events, such as inflammation, cancer metastasis, and angiogenesis. In this study, we developed a simple method for the quantification of cell invasion by using a culture insert with fluorescence blocking micropore membrane (FBM). Fluorescence labeled cells were simply added to a culture insert with a 8 μm-pore FBM precoated with Matrigel and incubated for an appropriate duration. Then, the FBM was examined under a fluorescence microscope to count the invaded cell number. By this method, accurate invasion assay is easily performed without the steps of fixation and staining of cells and removal of cells which do not invade.

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