Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.
As described previously (Yamashita S. et al., Biol. Pharm. Bull., 23, 519-522 (2000)), high levels of a truncated actin with an N-terminus of Met-44 were detected in neutrophils of patients with Behçet’s disease. Since the increase of the truncated actin in neutrophils of patients may be important for understanding the pathology of Behçet’s disease, the mechanism of the truncated actin formation was studied. First, to investigate the presence of a specific protease, which cleaves the actin at the site between Val-43 and Met-44, a peptide with a partial amino acid sequence of actin from the N-terminal Pro-38 to Asp-51 was synthesized as the protease substrate. The synthesized peptide was digested with cytosolic fractions of neutrophils from patients and healthy volunteers, and digestion products were analyzed by C18-reverse phase HPLC. The chromatograms of these samples showed that an endoprotease, which cleaved the peptide at a specific site, was present in cytosolic fractions of neutrophils from patients with Behçet’s disease. Then, the effects of various kinds of protease inhibitors on the digestion of the peptide were investigated in order to identify the responsible endoprotease. The digestion of the peptide was suppressed by 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF, a serine protease inhibitor) and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone (CMK, a polymorphonuclear (PMN)-elastase inhibitor) in the presence of EDTA. Furthermore, PMN-elastase was found to cleave the substrate peptide and actin at the site between Val-43 and Met-44. These results lead to the conclusion that the PMN-elastase is responsible for cleavage of actin at the N-terminal site between Val-43 and Met-44 in neutrophils from patients with Behçet’s disease.
Fluvastatin, which is a synthetic 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase inhibitor, its metabolites (M2, M3 and M4) and trolox all inhibited the decrease of apolipoprotein B-100 (apoB) and α-tocopherol in a radical reaction of human plasma initiated by Cu2+. The concentrations of fluvastatin, M2, M3, M4 and trolox for 50% inhibition (IC50) of apoB fragmentation were 405, 8.55, 1.75, 305, and 43.4 μM, respectively. The IC50 value of pravastatin, which is another HMG-CoA reductase inhibitor, was 2880 μM, showing that pravastatin is not an effective antioxidant. Although fluvastatin, its metabolites and trolox inhibited the decrease of α-tocopherol in a similar manner to that of apoB, pravastatin did not significantly inhibit the decrease of α-tocopherol. Since oxidation of low density lipopotein (LDL) is an important step in the initiation and progression of atherosclerosis, fluvastatin may reduce the risk of atherosclerosis not only by lowering plasma cholesterol but also by protecting LDL from oxidation.
The effect of estrogen on the microbial colonization of the urogenital tract is widely discussed, mainly in regard to women with a high incidence of Urinary Tract Infections (UTI). The aim of this work was to study the effect of estradiol on the microbial colonization of lactobacilli and E. coli in mice. Female BALB/c mice were intramuscularly (i.m.) treated with β-estradiol (one or three doses). The next day, L. fermentum was inoculated intraurethrally with three doses of 107 CFU (Colony Forming Units). Later, mice were challenged with uropathogenic E. coli (1×108 CFU). The hormone levels in sera increased to values 10 times higher than in control animals. Increased differentiation of desquamated vaginal cells and keratinization of the vaginal surface were also observed. The hormonal treatment produced an increased E. coli colonization in the whole tract and a higher level of L. fermentum in kidneys on the 6th day. In mice treated with hormones and lactobacilli, one dose of estradiol was enough to protect animals against the challenge with E. coli. Three doses of estradiol produced a more pronounced protection with a lower number of E. coli. No histological modifications were produced by L. fermentum, while lymphocytic proliferation at submucosal level was observed in E. coli-challenged animals.
Trefoil peptides (TFFs) with a unique trefoil domain(s) are presumed to function in protection and repair of the gastrointestinal epithelial layer. Three peptide family members are differently distributed in the mouse gastrointestinal tract: TFF1/pS2 specifically in stomach, TFF2/SP mainly in stomach, pancreas and duodenum, and TFF3/ITF in intestine. We cloned and sequenced the mouse TFF1 gene 5'-upstream region by means of the genomic walking procedure. The cloned region was ligated to the luciferase reporter gene and then introduced into mouse gastric surface mucous GSM10 cells which express TFF1 and TFF2. The minimum promoter was located in the region containing the TATA-box between −39 and the transcriptional start site. Further upstream regions stimulated (−2192— −1630bp, −641— −243bp, −137— −39bp) and inhibited (−1630— −641bp, −243— −137bp) luciferase gene expression. These regions as well as short segments conserved in the mouse and human 5'-upstream sequences may be important for modulation of the mRNA level of the TFF1 gene.
Plasma hyaluronan biding protein (PHBP) is a novel serine protease, which has an amino acid sequence homology to that of hepatocyte growth factor activator (HGFA), and has a similar domain structure to that of urinary plasminogen activator (u-PA), found in human plasma. We searched the PHBP substrate in human plasma by measuring the digested protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that fibrinogen and fibronectin were the major substrates of PHBP. PHBP cleaved the α-chain at multiple sites and the β-chain between lysine53 and lysine54 but not the γ-chain of fibrinogen. Therefore, PHBP did not initiate the formation of the fibrin clot and did not cause the fibrinolysis directly. PHBP did not cleave (activate) prothrombin and plasminogen, but it converted the inactive single chain urinary plasminogen activator to the active two chain form.
The rat glutathione transferase P (GST-P) gene is strongly induced during chemical hepatocarcinogenesis, whereas mRNA of this gene is rarely expressed in normal rat liver. We previously identified a silencer region in the promoter of this gene. This silencer has several DNA binding sites and at least three proteins (Silencer factor-A, -B, and -C (SF-A, SF-B, and SF-C)) bind to these sites. We previously cloned and characterized the Nuclear Factor 1 (NF1) family and the CCAAT/enhancer-binding protein (C/EBP) family as SF-A and SF-B, respectively. However, SF-C which binds to GST-P silencer 2 (GPS2) remains to be cloned. By screening using yeast one-hybrid system, several zinc finger proteins were identified as a candidate of SF-C. The gel-mobility shift analyses showed that BTEB2, EZF, LKLF, TEIIIA, TIEG1, and novel zinc finger protein MZFP bound to GPS2 with different affinities. Several proteins of these are known to be transcriptional activators or repressors, suggesting that zinc finger proteins bind to GPS2 and regulate GST-P expression in the rat liver.
We used three human urological cancer cell lines, PC-3, LNCaP and SKRC-1, to investigate the effects of the extract from Serenoa repens (Palmae) on tumor cell invasion. The invasion activity of these cell lines was determined in vitro using a Transwell cell-culture chamber. The invasion activity of PC-3 cells into Matrigel was effectively suppressed by the extract at the concentration range of 1—10 μg/ml, while that of LNCaP and SKRC-1 cells was unaffected by the extract. The extract did not affect the viability, adhesion ability, or motility of the cell lines. uPA is more strongly expressed on the membrane fraction of PC-3 cells than that of LNCaP or SKRC-1 cells. The purified uPA activity is inhibited by the extract from S. repens in a dose-dependent manner, suggesting that the suppression of PC-3 cell invasion by the extract is based on an inhibition of the uPA activity which is necessary for tumor cell invasion. These data suggest that the extract from S. repens specifically inhibits the uPA activity and may therefore be useful for the therapeutic treatment of prostate cancer.
Extracellular-superoxide dismutase (EC-SOD) is one of the three SOD isozymes in humans and has affinity for heparin-like sulfated glycosaminoglycans in the extracellular matrix. The C-terminal portion of EC-SOD is responsible for the heparin-affinity of this enzyme, but this portion should be a target of proteinases. Recently, Oury et al. reported that EC-SOD is localized within the cytoplasm of neuronal cells, while this enzyme is found in the extracellular matrix in most other tissues. It has been speculated that the heparin-binding domain of EC-SOD in the brain is sensitive to proteolysis, which may result in a loss of extracellular immunoreactivity. This study was performed to investigate the heparin-affinity of EC-SOD in the human brain. We found that human EC-SOD in brain tissue has high heparin-affinity similar to that in the umbilical cord. Moreover, heparin-affinity of EC-SOD in brain homogenate was not decreased by incubation at 37°C for 72 h. The proteolytic activity in brain homogenate might be less than that in umbilical cord homogenates. The intracellular localization of EC-SOD could contribute to its slow clearance from the brain and maintenance of its physiological functions in the brain.
Several angiogenic inhibitors have been obtained from shark cartilage, some of these are currently in clinical trials for assessment of safety and therapeutic efficacy in humans. Still, shark cartilage taken orally is commonly used in alternative and complimentary medicine for various ailments including serious diseases such as cancer. However, only few studies of oral shark cartilage have demonstrated pharmacological effects in experimental animals or patients, to indicate safe doses with sufficient bioavailability. In the present study we demonstrated the antiangiogenic properties of oral shark cartilage in the rabbit cornea model. Slow-release, polymethylmetacrylate pellets containing basic fibroblast growth factor (bFGF) were surgically implanted in the rabbit cornea to stimulate neovascularization scored by stereo microscopy. Powdered shark cartilage (PSC; commercial product) was tested orally along with a water-soluble fraction (WSF) of this cartilage product which was tested by local application. Animals were treated with oral dosages of 100 mg/kg PSC or 200 mg/kg thalidomide as positive control. Pellets containing WSF (50, 100 or 200 μg/pellet) or bFGF-inhibitor pentosan polysulfate were implanted adjacent to the bFGF pellet. Oral shark cartilage inhibited bFGF-induced angiogenesis, as did oral thalidomide, in this in vivo model. WSF and pentosan polysulfate was shown to block neovascularization in the cornea when applied locally. This study demonstrates that in the rabbit, oral shark cartilage appears to produce systemic levels of angiogenesis inhibitors that can exert their effect at the cornea.
We studied the mechanism of the superoxide generation system in indomethacin-induced gastric mucosal injury. First, 10 mM indomethacin had no direct effect on xanthine oxidase (XOD) activity. Next, we found that NADPH oxidase activity in polymorphonuclear leukocytes (PMN) of peripheral blood was significantly increased 6 h after administration of indomethacin. This phenomenon was inhibited by the injection of the NADPH oxidase inhibitor, diphenylene iodonium chloride (DIC). Activation of NADPH oxidase caused the component, p47phox to be translocated to the plasma membrane. Since indomethacin did not directly activate NADPH oxidase, we sought another route of activation of PMN. As IL-1 and TNF α play in the inflammation, we examined these cytokines in this study. TNF α was not detected but IL-1 was increased significantly 30 min after administration of indomethacin.
Two saponins, methyl protodioscin and dioscin, were extracted from the root of Polygonatum Zanlanscianense Pamp. One of them, dioscin exerted significant inhibitory effects on the growth of the human leukemia cell HL-60, inducing differentiation and apoptosis. HL60 cells were induced mainly along the granulocytic lineage. In addition, we have found that dioscin affects many cancer cells. These studies may have important significance in treating related cancers.
We examined the effects of Ninjin-to, a traditional Chinese (Kampo) medicine, on the levels of brain-gut peptides (motilin, vasoactive intestinal peptide (VIP), gastrin, and somatostatin) in plasma from healthy subjects. A single oral administration of Ninjin-to, at a dose of 6.0 g, caused significant increases in plasma motilin levels at 40 to 90 min and somatostatin levels at 20 to 90 min, compared with a placebo treated group. Transient elevations of gastrin levels in the placebo group were inhibited by administration of Ninjin-to, but the medicine did not alter the levels of VIP. In conclusion, these results suggest that pharmacological effects of Ninjin-to on gastrointestinal functions closely relate to changes of motilin, gastrin, and somatostatin-immunoreactive substance levels in human plasma.
We have investigated the effects of thinner inhalation on serum LH, FSH and testosterone levels together with changes in hypothalamic catecholaminergic system in the male rat. A control group inhaled normal air ventilation. The remaining animals were divided into two groups and exposed to paint thinner in a glassy cage for 15 or 30 d. Toluene concentration (the largest constituent in thinner, 66%) was set at 3000 ppm in the inhalation air. At the end, all animals were decapitated and blood samples obtained. Serum LH and FSH levels were measured by RIA and testosterone by enzyme immunoassay. Following removal of brains on dry ice, medial preoptic area, suprachiasmatic nucleus, median eminence and arcuate nucleus were isolated by micropunch technique. Noradrenaline, 3,4-dihydroxyphenylglycol (DHPG) and dopamine concentrations of these hypothalamic areas were determined by HPLC-ECD. Fifteen-day thinner inhalation significantly suppressed serum LH and testosterone levels in parallel (p<0.001) compared to control group values (LH: 0.77±0.07; testosterone: 2.67±0.39). Thirty-day exposure markedly decreased LH levels (p<0.001), but surprisingly had no significant effect on testosterone. Serum FSH levels were not significantly altered in either group. Thinner inhalation for 15 or 30 d did not cause any significant change in noradrenaline, DHPG or dopamine concentrations in the hypothalamic regions examined (except in the arcuate nucleus). These results suggest that paint thinner has an anti-gonadotropic effect and may cause long-term endocrine disturbances in the male. It is thought that the hypothalamic catecholaminergic system is not involved in thinner inhibition of LH and testosterone secretion.
We investigated the expression of heat shock protein 27 (HSP27) at intermediate stages of a cutaneous tumor induced by UVB-irradiation stress (290—380 nm, max. 312 nm) using an immunostaining method. After 15—20 weeks of chronic exposure to UVB irradiation at a dose of 2 kJ/m2, HSP27 was found in the upper cell layers of bowenoid multilayers of epidermis, in areas of the lesions where normal stratification seems to be conserved. After 25 weeks, HSP27 was weakly expressed in squamous cell carcinoma (SCC). The HSP27 distribution patterns during cutaneous tumor progression resemble that of cytokeratin 10, a differentiation marker in keratinocytes. In SCC, a low degree of HSP27 expression was detected in the well-differentiated carcinomatous areas, but not in the poorly differentiated areas. These results indicate that the level of HSP27 decreases significantly as epithelial carcinoma growth progresses upon UVB-exposure. The expression of HSP27 may be associated with the onset of skin keratinocyte differentiation, but not with progression of SCC.
Two lignans were isolated from the heartwood of Pterocarpus santalinus by activity-guided fractionation and investigated for their biological properties and molecular mechanism of action. On the basis of their spectroscopic data, these compounds were identified as savinin (1) and calocedrin (2), dibenzyl butyrolactone-type lignan compounds having an α-arylidene γ-lactone structure. These lignans significantly inhibited tumor necrosis factor (TNF)-α production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and T cell proliferation elicited by concanavalin (Con A), without displaying cytotoxicity. The molecular inhibitory mechanism of compound 1 was confirmed to be mediated by the non-polar butyrolactone ring, according to a structure-relationship study with structurally related and unrelated compounds, such as arctigenin (a dibenzyl butyrolactone type lignan), eudesmin (a furofuran type lignan), isolariciresinol (a dibenzylbutane type lignan), and cynaropicrin (a sesquiterpene lactone). The results suggest that savinin may act as an active principle in the reported biological activities of P. santalinus, such as antiinflammatory effect, by mediation of the butyrolactone ring as a valuable pharmacophore.
Leaves of Perilla frutescens var. crispa DECNE. (perilla, Labiatae) are used as a garnishing vegetable in East Asian countries as well as an herbal medicine prescribed in Kampo medicines such as Saiboku-to. A previous in vitro study revealed that a decoction of perilla leaves inhibits the proliferation of murine-cultured mesangial cells. In the present study, we evaluated the in vivo anti-proliferative effects of a perilla decoction using rat mesangio-proliferative glomerulonephritis induced by an intravenous injection of rabbit anti-rat thymocyte serum (ATS). Leaves of perilla were boiled, and the decoction was orally administered to the rats as drinking water at doses of 100 and 500 mg/kg/d from the day of ATS-injection (day 0) to day 8, when rats were sacrificed. In the histological evaluation, the total number of glomerular cells, proliferating cell nuclear antigen (PCNA) positive cells, and macrophage/monocyte antigen-positive cells in the glomerulus, was significantly decreased in perilla-treated rats. A significantly lower level of proliferation was induced by the serum of the perilla-treated rats than by that of the controls. These results suggest that the perilla decoction suppresses the proliferation of mesangial cells in vivo by an inhibition of the glomerular infiltration of macrophage/monocytes and of the production of circulating growth factors.
By determining the hydrogen peroxide (H2O2) released in cultures of peritoneal macrophage cells from Swiss mice, we evaluated the action of 27 vegetable compounds (pristimerin, tingenone, jatrophone, palustric acid, lupeol, cladrastin, ocoteine, boldine, tomatine, yohimbine, reserpine, escopoletin, esculine, plumericin, diosgenin, deoxyschizandrin, p-arbutin, mangiferin, and others) using a 2 mg/ml solution of each compound (100 μg/well). Macrophages are cells responsible for the development of the immunological response reaction, liberating more than one hundred compounds into the extracellular environment. Among these are the various cytokines and the intermediate compounds of nitrogen (NO) and oxygen (H2O2). This coordinated sequence of biochemical reactions is known as the “oxidative burst.” When we compared the results with those obtained with zymosan (an important stimulator of H2O2) we observed that the compounds showing the highest activity were substances 2 (tingenone), 16 (reserpine) and 20. Other substances such as compounds 1, 4, 5, 6, 8, 12, 13, 14, 15, 17, 19, 23, 24, 26, and 27 also showed a certain activity, but with less intensity than the aforementioned ones. Compounds 3, 7, 9, 10, 11, 18, 21, 22 and 25 presented no activity. These results suggest that natural products (mainly tingenone and reserpine and others) with different chemical structures are strong immunological modulators. However, further tests are needed to determine the ‘oxidative burst’ in future studies.
A sensitive high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of lactone and total drug (lactone plus hydroxy-acid) of DX-8951 in mouse plasma. Solid-phase extraction by C18 cartridge separated lactone from total drug of DX-8951. Analysis was performed using a reverse-phase ODS column with a mobile phase consisting of acetonitrile/0.05 M potassium dihydrogen phosphate (pH 3)(18 : 82, v/v) at a flow rate of 1 ml/min. The limits of quantitation of lactone and total drug were 3 ng/ml in plasma and a linear range of determination were observed over the concentration of 3 to 500 ng/ml. This method was applied to pharmacokinetic study in male mice treated with a single intravenous administration of either lactone or hydroxy-acid of DX-8951. The plasma concentrations of lactone from 2 to 6 h after dosing were similar regardless of the form of DX-8951 administered.
Chitosan (CS) gel beads were prepared in 10% amino acid solution (pH 9) and implanted into air pouches (AP) prepared subcutaneously on the dorsal surface of mice. No inflammatory response was observed, and degradation of the beads in the AP increased as their degree of deacetylation decreased. Degradation could be altered by changing the nature of the CS or by increasing the CS concentration. The release of prednisolone (PS) in vivo from CS gel beads was similar to the release in vitro. When a suspension of PS was injected into the AP, the PS had almost completely disappeared 24 h after injection. Retention of PS in the AP was not increased by using a viscous CS solution. Alginate (Alg) gel beads, which were not degraded, released PS slowly into the AP over 3 d. The in vitro release profile of PS using 1% CS (deacetylation: 70% (7B) and 80% (8B)) and 1.5% CS (deacetylation: 90% (9B)) gel beads was similar to that with Alg gel beads. However, the in vivo release of PS was affected by the degradability of the gel beads. CS7B and 8B (1%) gel beads had released PS into the AP earlier than 3 d according to their rate of degradation. CS9B (1.5%) gel beads were not degraded after 3 d and went on to release PS into the AP for 3 d similar to the release profile of Alg gel beads. CS9B (2%) gel beads were also not degraded after 3 d and the release of PS from these beads into the AP was sustained; 76% and 27% of administered PS remained in the gel beads after 1 and 3 d, respectively. Therefore, degradation and drug release of CS gel beads can be controlled by changing the structure of the gel matrix, which appears to make these beads a promising biodegradable vehicle for sustained delivery.
Two antithrombotic polysaccharides with relatively high molecular weight (HMW) and low molecular weight (LMW) were isolated from the flower buds of Syzygium aromaticum (L.) MERR. & PERRY (clove) by anion-exchange chromatography, hydrophobic interaction column chromatography and size exclusion chromatography (LMW: EC-2B-IIIa-2, M.W. ca. 34000; HMW: EC-2C-Ia-2, M.W. ca. 103000). The LMW polysaccharide was mainly composed of Rha, Gal, GalA and Ara (molar %: 24.1, 18.9, 18.0 and 17.9, respectively) with 10.8% of sulfate and 18.2% of protein. The HMW fraction consisted of Ara, Gal, Glc and Rha (molar %: 26.0, 23.7, 17.5 and 12.4, respectively) with 15.4% of sulfate and 8.0% of protein. Both polysaccharides had the backbone of type I rhamnogalacturonan and the side chain of arabinan. Also, most of the sulfates were attached at the position 6 of 3-linked galactosyl residues. Compared to the antithrombotic activity of the HMW fraction (plasma clotting time of 145 s in APTT assay), the LMW fraction displayed a slightly low activity (90 s). However, animal studies indicated that crude LMW polysaccharide did not show acute toxicity, while the acute LD50 of the HMW fraction was approximately 2-fold lower than that of heparin.