A number of peptides with different lengths corresponding to various regions of human pulmonary surfactant protein SP-C were synthesized and their activity evaluated to improve in vitro surface activities and in situ lung pressure-volume characteristics of a ternary lipid mixture composed of dipalmitoylphosphatidylcholine, phosphatidylglycerol and palmitic acid (75 : 25 : 10, w/w). SP-C (1-35), a synthetic peptide with the entire length of human SP-C, and some other peptides with various lengths of its partial sequences were remarkably active. All of these peptides shared a common core sequence of (C)CPVHLKRLLIVVVVVVLIVVVIVGAL(L). Any deletion in this core sequence resulted in reduction of activity of the peptide. SP-C (5-31) and SP-C (6-32), the minimum peptides containing the core sequence, were combined with the ternary lipid mixture at the final peptide concentration of 2% (w/w) into synthetic surfactants which showed excellent properties comparable with those of Surfacten[○!R], a commercially available modified bovine lung surfactant. In a Langmuir-Wilhelmy surface balance, the synthetic surfactant containing SP-C (6-32) spread and adsorbed quickly to reach a surface tension of 30.8 mN/m at 30-s spreading time and 41.2 mN/m at 1-min adsorption time, respectively; the presence of SP-C (6-32) significantly prevented the decrease of surface activity of the ternary lipid mixture during dynamic compression-expansion cycles. Furthermore, tracheal instillation of the synthetic surfactant containing SP-C (6-32) at the dose of 50 mg of phospholipids/kg improved lug pressure-volume characteristics of immature rabbit neonates to a level similar to that of mature neonates at term.
The epitope chimeric antigen, CepCM, composed of the 9 selected major epitope regions (two in NS3, two each in the NS4 of two genotypes, two each in the core of two genotype, and one in the core in hepatitis C virus (HCV) polypeptide), was expressed as a fusion protein of the trpE peptide in E. coli. An ELISA test using this antigen produced the same judgements with most of the panel sera as a second generation HCV screening kit. Though discrepancies were found in twelve samples (5% of the samples), further analysis revealed that eleven samples were indeterminate sera as judged by an immunoblot test. The reactivity found in several seroconversion series sera suggested that CepCM has superior reactivity to HCV infected sera than some second generation kits. These data indicated that an epitope chimeric antigen with a man-made sequence will be a excellent tool for a diagnostic test kit.
The DNA strand-breaking activity of some dihydropyrazine derivatives, 2, 3-dihydro-5, 6-dimethylpyrazine (3), 2, 3-dihydro-2, 5, 6-trimethylpyrazine (4), 2, 3-dihydro-2, 2, 5, 6-tetramethylpyrazine (5), trans-2, 3-dimethyl-5, 6, 7, 8, 9, 10-hexahydroquinoxaline (6), its cis-compound (7) and the mixture of 6 and 7 (8) was tested by agarose gel electrophoresis using plasmide pBR322 ccc-DNA as a substrate. The order of DNA strand-breaking activity in the presence of Cu2+ was (7)>(8)≥(5)>(2)>(6)>(4)≥(1)≥(3). 2, 5-Bis(D-arabino-tetrahydroxybutyl)-2, 5-dihydropyrazine (1) and 2, 5-dihydro-3, 6-dimethylpyrazine (2) have already been described in terms of DNA breaking activity in a previous paper.The activity was suggested to be due to the dihydropyrazine skeleton, in addition to active oxygen radicals formed in an aqueous solutoin. The introduction of a methyl group to the dihydropyrazine skeleton enhanced the activities of dihydropyrazine derivatives. The possible chemical basis for DNA strand breakage by dihydropyrazine derivatives, especially in the presence of Cu2+, was discussed.
The effects of acetylshikonin (AS) on the activation of NADPH oxidase (EC 188.8.131.52) in guinea pig polymorphonuclear leukocytes (PMNs) in both whole cell and cell-free activation systems were investigated. When PMNs were treated with AS before exposure to phorbol myristate acetate (PMA), superoxide (O-2) generation in these cells was significantly reduced, but after exposure of PMNs to PMA, inhibition of O-2 generation by AS did not occur. Thiol compounds completely abolished the inhibitory effect of AS on the O-2 generating activity of PMNs. In the cell-free system, AS inhibited the activation of NADPH oxidase induced by myristate in a combination of cytosol and membrane fractions obtained from intact PMNs, but did not inhibit the activity of NADPH oxidase already induced. These results suggest that AS inhibits the generation of NADPH oxidase complex in the activation of respiratory burst of PMNs, but does not directly inhibit the activity of NADPH oxidase already generated.
To induce peptide-specific antibodies in mice, as a model for vaccination against human immunodeficiency virus (HIV), lipopeptide analogs conjugated with three repeating units (KAB-112; designated as gp120-peptide) of a part (Gly-Pro-Gly-Arg-Ala-Phe) of the amino acid sequences of the V3 loop region in gp120 of HIV were synthesized. The mitogenicity, production of nitric oxide (NO) and induction of peptide-specific antibodies in mice by synthetic lipopeptides were examined. Compounds, KAB-8 (diacylglycerol-tetrapeptide having a part of the amino acid sequence in Escherichia coli), KAB-116 (diacyglycerol-cysteine), KAB-117 (diacylglycerol with gp120-peptide) and KAB-121 (KAB-8 with gp120-peptide) were capable of increasing significantly the incorporation of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 12.5 to 100 μM, but KAB-112 (gp120-peptide) and KAB-115 (monoacylglycerol with gp120-peptide) did not show such activity. The compounds, KAB-8, KAB-117 and 121, exhibited NO production in murine macrophages. When 50 nmol of these compounds was administered intraperitoneally into BALB/c mice on days 0, 16 and 46, the peptide-specific antibody titers in their sera produced by each compound were determined with ELISA. The sera of KAB-117 and KAB-121, which were obtained on days 14, 30, 42, 57 and 70, had a higher titer than that of KAB-112 and KAB-115, suggesting that the diacylglycerol derivatives enhance the production of the peptide-specific antibodies.
We identified novel phenotypes of a pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol. The first phenotype is limited cell growth at a high temperature, under the condition of low salt. The phenotype was co-transduced with a phenotype lacking the potential to synthesize phosphatidylglycerol in a P1 transduction experiment, and was restored by transformation with a plasmid containing a wild type pgsA gene. The second phenotype of the pgsA3 mutant was resistant to growth in the presence of a low concentration of kanamycin (4 μg/ml). P1 transduction and transformation with the plasmid containing the wild-type pgsA gene revealed that the pgsA3 mutation was also responsible for the second phenotype.
As we previously reported, permeabilization of the plasma membrane was effectively achieved by vortex-stirring the mammalian cells in the presence of high molecular weight polyacrylic acid. Based on our present findings, as well as those of our previous studies, optimal conditions for permeabilization were considered in terms of the structure and dosage of a polymer, the constituent of the medium, the conditions of cells, vortex-stirring and post-vortex-stirring, etc. A standard procedure tentatively recommended is described.
We have previously reported that epidermal growth factor (EGF) augments the translation of pro-matrix metalloproteinase 3 (proMMP-3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs during the first 1-h treatment of human uterine cervical fibroblasts (Hosono, T. et al., FEBS Lett., 381, 115-118, (1996)). In this report, we have investigated the effect of interleukin 1α (IL-1α) and 12-O-tetradecanoylphorbol 13-acetate (TPA), potent stimulators of proMMPs and TIMP-1 production, on the translation of proMMP-3 and TIMP-1 mRNAs. When human uterine cervical fibroblasts were treated with IL-1α or TPA for 2 h, their translations were not augmented, whereas the steady-state levels of proMMP-3 and TIMP-1 mRNAs in the cells treated with these stimuli for 24 h were increased 13.3- and 1.3-fold by IL-1α and 52.5- and 5.7-fold by TPA, respectively. By contrast, transforming growth factor α (TGFα), which also binds to EGF-receptor, enhanced their production as early as 2 h after treatment, indicating that growth factors that bind to EGF-receptor are likely to be involved in the translational enhancement of proMMP-3 and TIMP-1 mRNAs. EGF partially translocated cytoplasmic protein kinase C (PKC) to plasma membrane, but hte PKC down-regulation induced by 100 nM TPA did not diminish the EGF-mediated translational augmentation of proMMP-3 and TIMP-1 mRNAs. In contrast, the PKC inhibitor of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) effectively suppressed the trahslational regulation of proMMP-3 and TIMP-1 in a dose-dependent manner during the first 2-h treatment with EGF. These results suggest that EGF and TGFα, but not IL-1α and TPA, specifically augment the translation of proMMP-3 and TIMP-1 mRNAs and accelerate their accumulation without modifying their transcripts during the first 1-2 h treatment of human uterine cervical fibroblasts. This translational augmentation is suggested to be mediated by a TPA-insensitive stypical PKC aubclass in the PKC family.
We showed that ratl liver lysosomes possess at least two types of ATPase besides H+-translocating ATPase (H+-ATPase); namely, N-ethylmaleimide (NEM)-sensitive and bafilomycin A1-insensitive Mg2+-ATPase [ATPase I] and NEM- and bafilomycin A1-insensitive (Ca2+-MG2+)-ATPase [ATPase II] [Hayashi H., et al. Chem. Pharm. Bull., 37, 2783-2786 (1992)]. Sobcellular fractionation showed the presence of similar activity of (Ca2+-Mg2+)-ATPase not only in the Golgi but also in the plasma membrane fractions, of which plasma membrane had the highest activity, most probably due to the ecto-ATPase. In this study, we partially purified the (Ca2+-Mg2+)-ATPases from both lysosomal and plasma membranes and compared their properties. Both enzymes had quite similar characteristics including : (1) broad pH-activity profiles with two pH optima at 4.5 and 7.0, (2) Km values for ATP being 21-27 μM (at pH 4.5) and 18-14 μM (at pH 7.0)(in the presence of CaCl2), (3) hydrolysis of both nucleoside triphosphates and diphosphate (ADP), (4) inhibition only by vanadate and 4, 4'-diisothiocyanatostilbene 2, 2'-disulfonic acid (DIDS) at pH 4.0 (but not at pH 7.0), (5) acidic pI values that were shifted by neuraminidase digestion, and (6) a positive reaction against an antibody to the N-terminal peptide of ecto-ATPase.
L-2, 4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1, 3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, wey also detected DABA DC activity in the species of Enterobacteriaceae : E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.
An aqueous water extract of Flos magnoliae, a Japanese Sino-medicine, inhibits angiogenesis in adjuvant-induced mouse pouch granuloma. Magnosalin (MSA) and magnoshinin(MSI), neolignans isolated from magnolia, have a crucial role in the anti-angiogenic effect of magnolia (Kimura et al., Int. Arch. Allergy Appl. Immunol., 93, 365 (1990); Phytother. Res., 6, 209 (1992)). We investigated the effects of these neolignans on tube formation of endothelial cells (EC) cultured in type I collagen gel during the angiogenic process. MSA (0.1-10 μM), MSI (0.23-7 μM) and corticosterone (CS : 0.3-30 μM) inhibited fetal bovine serum (FBS)-stimulated tube formation in a concentration-dependent manner. Their 30% inhibitory concentration (IC30, 95% confidence limits) values were 0.51 (0.20-1.27) for MSA, 8.14 (2.48-26.7) for MSI and 3.65 μM (2.47-5.40) for CS, respectively. MSA and MSI (1-3 μM) also inhibited interleukin (IL)-1α-stimulated tube formation in a concentration-dependent manner. Their IC50 values (95% confidence limits) were 1.22 (1.01-1.47) for MSA and 0.74 μM (0.24-2.31) for MSI against a submaximal concentration (69 pM) of IL-1α-stimulated tube formation. Their inhibitory effects on the action of IL-1α were non-competitive. These results demonstrate that MSA inhibited FBS-stimulated tube formation with a greater potency than MSI. The inhibitory effect of MSA on the action of FBS differed from that on the action of IL-1α.
Effects of optical isomers of ephedrine (EPH) and methylephedrine (MEP) on histamine H1-receptors and muscarinic receptors in guinea pig ileal muscle were investigated by radioligand binding assay and by measuring the mechanical response to histamine and acetylcholine. EPH and MEP inhibited the specific binding of [3H]mepyramine and [3H]quinuclidinyl benzilate (QNB) to microsomal fractions prepared from this tissue. The rank order of inhibitory potency of [3H]mepyramine and [3H]QNB binding was d->l-isomer and l->d-isomer, respectively. Furthermore, the rank order of antagonistic potency in the mechanical response was the same as that in the binding study. d-MEP competitively antagonized histamine-induced contration (pA2 value; 5.14). These results suggest that each isomer of EPH and MEP has a distinct affinity for histamine H1-receptors and muscarinic receptors in guinea pig ileal muscle. The antihistaminic and antimuscarinic activity of these compounds may be largely attributed to competition at receptor sites. In addition, it is suggested that d-MEP exhibits a competitive antagonist activity for the histamine H1-receptor.
The in vitro and in vivo antitumor activity of a novel nucleoside, 4'-thio-2'-deoxy-2'-methylidenecytidine (4'-thio-DMDC), was examined. 4'-Thio-DMDC inhibited the growth of various cell established from human solid cancers, particularly the growth of lung and bladder carcinomas and melanomas. 4'-Thio-DMDC exhibited potent antitumor activity against subcutaneously implanted murine sarcoma S-180 and human fibrosarcoma HT-1080 xenografts. It was shown that 4'-thio-DMDC suppressed cell growth by inhibiting cellular DNA synthesis, and phosphorylation of the 5'-position of its deoxyribose by deoxycytidine kinase was needed for its antitumor activity. 4'-Thio-DMDC is a poor substrate for cytidine deaminase, thus it is a promising agent for the treatment of malignant solid tumors because it will not be converted into its inactive uridine form.
The in vivo behavior of 20-[18F]fluoroarachidonic acid (18F-FAA) was investigated to evaluate its potential use as a radiotracer for studying the regional brain and heart lipid metabolism by positron emission tomography (PET). Tissue biodistribution studies in rats have revealed that 18F-FAA has a high uptake in the liver and lung, thus probably reflecting the metabolism, and is accompanied by both low in vivo defluorination and low blood levels. AT 30 min postinjection, the uptake in the brain and heart reached values of 0.26±0.02 and 1.22±0.58% dose/g, respectively, with ratios to the blood radioactivity of 1.04 and 4.88, respectively. Lipid extraction at 30 min postinjection showed that 39% of the brain radioactivity was in the organic phase whereas the organic phase from heart tissue contained 73% of the total radioactivity. A TLC analysis demonstrated that 18F-FAA was mainly bound to phospholipids in the brain and heart tissue as expected. Based on the findings of this study, the utility of 18F-FAA as an in vivo tracer for cerebral phospholipid studies appears to be limited because of its relatively high radioactivity in the aqueous brain fraction. However, our findings do suggest that this agent might be useful as a tool for studies of cardiac phospholipid turnover, even though it demonstrated a poor heart-to-lung and heart-to-liver contrast.
Retinoids, including all-trans-retinoic acid (ATRA), its isomers, and fifty synthetic retinoids (retinobenzoic acids), were tested for differentiation-inducing activity on human leukemia cell lines HL-60 and NB4. Binding activity of typical retinoids to nuclear retinoic acid receptors (RARs) was also investigated. A good linear correlation between the ED50 values of differentiation-inducing activity towards HL-60 cells and those towards NB4 cells was found. Binding activities of retinoids to RARα and RARβ also correlated well to the differentiation-inducing activities.
Ten dihydroxy- and trihydroxy triterpenes, viz., four taraxastanes : faradiol, heliantriol B0, heliantriol C and arnidiol; two lupanes : calenduladiol and heliantriol B2; two oleananes : maniladiol and longispinogenin; and two ursanes : brein and uvaol, isolated from the nonsaponifiable lipids of the flower extracts of Compositae plants were evaluated with respect to their anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice. All the triterpenes were found to possess marked inhibitory activity. The 50% inhibitory dose of these compounds with respect to TPA-inflammation (1 μg) was 0.03-0.2 mg/ear.
In order to investigate the fate of orally administered proteins, the absorption of ovalbumin (OVA) from the gastrointestinal tract into both the blood and lymph circulation was quantitatively evaluated. After oral administration, a significant amount of intact OVA was detected in both the plasma and the lymph fluid by means of a two-site enzyme immunoassay. The extent of absorption into the plasma, calculated from the area under the plasma concentration versus time curve of OVA after oral and intravenous administration, was only 0.007-0.008% of the dose. This value is extremely low compared to that after nasal administration, showing the stronger barrier function of the gastrointestinal tract against the invasion of macromolecular proteins into the body. The extent of absorption into the lymph was dose-dependent (0.0007-0.002% of dose), and a higher dose leads to a higher fraction of OVA absorbed into the lymph. Moreover, it was demonstrated that not only the small intestine but also the stomach can absorb OVA. OVA absorbed from the stomach was transferred almost exclusively to the blood circulation, which suggests different mechanisms and/or routes of absorption between the stomach and the small intestine. In order to improve the low oral absorption, OVA was incorporated in liposomes and administered orally. Although the effect of liposomes was not significant, it increased OVA absorption into both the plasma and lymph by about 2 to 3-fold. It was considered that the liposomes suppressed the enzymatic degradation of OVA and released it slowly in the gastrointestinal tract.
There are difficulties in drug absorption studies using dogs because of different gastrointestinal (GI) conditions between these animals and humans, including shorter intestinal transit time and strong agitation force in the GI tract. We attempted to modify the agitation force and GI transit time using atropine and loperamide. The agitation force was examined based on in vitro/in vivo correlation for two controlled release (CR) formulations of acetaminophen. Atropine treatment considerably reduced the agitating force whereas that of loperamide did not.
In a doping test for racing horses, it is useful for the elucidation of the illegal use of drugs if one can estimate the time at which the detected drug was administered. In order to estimate the time which has elapsed after the administration of caffeine (CA) into horses, the ratios of concentration for the respective metabolites to the unchanged CA in the plasma or the urine were determined. These ratios have been known to be independent of the dose of CA. The relationship between [plasma or urinary concentration of a metabolite]/[plasma or urinary concentration of the unchanged drug] and the post-administration time of CA was expressed in a theoretical equation using pharmacokinetic parameters. When CA was administered at 2.5 mg/kg intravenously, intramuscularly or orally, all of the experimentally observed values of plasma or urinary metabolites : theophylline, theobromine and paraxantine, agreed well with the theoretical formulas, indicating that there exists a theoretical relationship between the postadministration time and the concentration ratio of the respective metabolites to unchanged CA.
New lipid derivatives of polyethyleneglycol (PEG) have been synthesized and tested for the ability to allow liposomes to evade uptake by the reticuloendothelial system (RES) and to prolong the circulation time of liposomes in mice. Liposomes were prepared from distearoylphosphatidylcholine (DSPC) and cholesterol (CH) (1 : 1, m/m) containing 6 mol% of various PEG-derivatives. The activity of the CH derivative of PEG (CH-PEG) in prolonging the circulation time of liposomes was proportional to the average molecular weight of PEG, i.e., 4800>2600>1700>800. α-Methoxy-ω-(1, 2-dioctadecenoyloxy glyceryl)polyoxyethylene (DO-PEG) 1000 and α-methoxy-ω-(1, 2-ditetradecenoyloxy glyceryl) polyoxyethylene (DT-PEG) 1000, in which PEG is directly linked to glycerol, prolonged the circulation time as effectively as distearoylphosphatidyl-N-(methoxy polyoxyethylene succinyl)-ethanolamine (DSPE-PEG). PEG-derivatives with a functional group at the PEG terminal, such as distearoyl-phosphatidyl-N-(3-carboxypropionyl polyoxyethylene succinyl)ethanolamine (DSPE-PEG-COOH) and α-(dipalmi-toylphosphatidyl)-ω-hydroxypolyoxyethylene (DPP-PEG-OH), effectively prolonged the circulation time of liposomes. incorporation of PEG-derivatives did not change membrane fluidity even after treatment with serum. Furthermore, Incorporation of PEG-derivatives into liposomes decreased uptake by J774 cells, a murine macrophage-like cell line, in vitro. The newly synthesized PEG-derivatives seem to prevent or reduce the interactions of liposomes with serum protein and macrophages, resulting in enhanced stability and a prolonged circulation time.
Effects of plasma proteins such as α-globulin on the uptake of high molecular weight (HMWFH : 23000 Da) and low molecular weight fractionated [3H]heparin (LMWFH : 10000 Da) were examined in isolated rat Kupffer cells. α-Globulin (8 mg/ml) affected neither surface binding nor internalization of LMWFH by Kupffer cells, while it reduced both surface binding and internalization of HMWFH without affecting the fraction internalized, which was a ratio of internalized amount to the total association. The total associations of HMWFH were about four times larger than that predicted assuming only the unbound fraction is available for uptake, suggesting the participation of protein-mediated transport in the uptake of HMWFH in Kupffer cells. Based on the same assumption, the saturable initial uptake of HMWFH versus concentration profile in the presence of α-globulin (8 mg/ml) was also analyzed to further examine the suggested protein-mediated transport. The estimated dissociation constant of 487 nM was three times larger than that in in vitro binding experiments (168 nM) and the binding capacity of 0.155 was one third of the value in vitro (0.5), suggesting apparent reductions in both binding affinity and capacity. Thus, we demonstrated the involvement of protein-mediated transport in the uptake of fractionated heparin in Kupffer cells and kinetically characterized it as the apparent enhancement of dissociation.
We investigated the distribution of a novel angiotensin II type 1 (AT1) receptor antagonist, E4177 (4'-[2-cyclopropyl-7-methyl-3H-imidazo[5, 4-b]pyridine-3-yl]methyl-2-biphenylcarboxylic acid), in rat adrenal glomerulosa. In a binding assay of adrenal capsular tissue (mainly glomerulosa), E4177 exhibited a maximum displacement of approximately 80% of total 125I-labeled angiotensin II (125I-[Sal1, Ile8] Ang II) binding, and its IC50 value was 6.9±0.5 nM. This IC50 value indicated a slightly higher in vitro potency than that of losartan (21.0±0.6 nM). Also, in a receptor autoradiographic study, E4177 (10000 nM) displaced approximately 80% of radiolabeled 125I-[Sal1, Ile8] Ang II in rat adrenal glomerulosa and caused only slight displacement in rat adrenal medulla. Further, light and electron microscopic autoradiography of adrenal glomerulosa for 15 min after the intravenous administration of 1 mg/kg [14C]E4177, indicated the localization of 14C, possibly in the adrenal zona glomerulosa cell plasma membrane. It was strongly suggested that E4177 is a potent and selective antagonist of the AT1 receptor, and that it specifically binds to AT1 receptors in the adrenal zona glomerulosa.
To reduce the intestinal toxicity of orally administered 5'-deoxy-5-fluorouridine (5'-DFUR) in mice, we co-administered 5'-DFUR with acyclothymidine [AcyT, 5-methyl-(2'-hydroxyethoxymethyl) uracil], a potent inhibitor of pyrimidine nucleoside phosphorylase (PyNPase). Orally administered 5'-DFUR alone caused intestinal toxicity and severe damage to the intestinal villi, while 5'-DFUR with AcyT reduced the intestinal toxicity, and prevented damage to the intestinal villi. This toxicity arising from orally administered 5'-DFUR could not be reduced by intravenous administration of AcyT, but was alleviated by oral administration. Orally co-administered AcyT showed little effect on antitumor activity of 5'-DFUR toward subcutaneously implanted Lewis lung carcinoma, though the intestinal toxicity was reduced in the tumor-bearing mice. This finding suggests that orally co-administered AcyT may prevent the undesirable conversion of 5'-DFUR to 5-FU by PyNPase during the process of absorption in the intestinal tract.
We attempted to characterize the antidiarrheal action of Hange-shashin-to (TJ-14), a kampo medicine, by comparing its action with that of loperamide. The oral administration of TJ-14 caused the dose-dependent suppression of castor oil-induced diarrhea at 250 to 1000 mg/kg. No significant repression was noted by TJ-14 even at 1000 mg/kg, p.o. for diarrhea induced by pilocarpine, serotonin or barium chloride. Oral treatment with loperamide at 5 mg/kg markedly suppressed diarrhea induced by castor oil and barium chloride.Contractions of isolated guinea pig ileum in response to acetylcholine (1×10-7 g/ml), histamine (1×10-7 g/ml) or barium chloride (3×10-4 g/ml) were little affected by TJ-14 at 10-4 g/ml. The responses elicited by the three contractive drugs were dose-dependently suppressed by loperamide. TJ-14 did not affect the small intestinal transit even at an oral dose of 1000 mg/kg. On the other hand, the small intestinal transit was significantly suppressed by loperamide (5 mg/kg, p.o.). These results indicate that TJ-14 can effectively control castor oil-induced diarrhea, and that its antidiarrheal action was not based on the suppression of intestinal motility.
The nitric oxide-cyclic GMP pathway is still undefined regarding regulation of the release of hepatic lipase (HTGL). It was found that L-arginine (Arg) stimulated the release of HTGL activity from rat hepatocytes in a time- and dose-dependent manner. L-Arg-stimulated release of HTGL activity was inhibited by N-monomethyl-L-Arg, which is a nitric oxide synthase inhibitor. L-Arg markedly increased the cyclic GMP content of hepatocytes in the presence of a cyclic GMP phosphodiesterase inhibitor, zaprinast. The release of the enzyme activity was also suppressed by methylene blue (a guanly cyclase inhibitor) and KT5823 (a cyclic GMP-dependent protein kinase inhibitor). These results suggest that the stimulation of nitric oxide synthesis by L-Arg increases the release of HTGL activity due to processes associated with the elevation of cyclic GMP level, probably through an activation of protein kinase.
The nucleotide sequences of inducible nitric oxide synthase (iNOS) cDNA in rat kidney, lung, and uterus were determined. The amino acid sequences of iNOSs in various organs were highly homologous, and the important regions for the enzyme activity have been conserved.
We have reported previously that -gingerol increased Ca2+-ATPase activity. 1, 2)Here we synthesized gingerol related compounds (AP-004, AP-005 and AP-015) and investigated the effects of gingerols (-gingerol, -gingerol and -gingerol) and the synthesized compounds on the Ca2+-ATPase activity of sarcoplasmic reticulum (SR). The Ca2+-ATPase activity and the Ca2+-pumping activity increased by these compounds in a concentration-dependent manner. It is probable that both the o-methoxyphenol and hydrocarbon chain in the molecule of gingerol analogues are necessary for the activation of the Ca2+-pumping ATPase activity of SR.
Binding activity of Asp-hemolysin to low density lipoprotein (LDL) was measured. LDL binds to Asp-hemolysin with an affinity as high as the LDL receptor. The apparent Kd, determined by Scatchard plot analysis, was 8.9×10-9 M [125I]LDL. Asp-hemolysin is thus identified as a novel LDL-binding protein.
Deoxycytidine kinase (dCK) is a rate-limiting enzyme for the activation of ara-C. It is responsible for the phosphorylation of ara-C which is widely used in the treatment of leukemia. We examined the effect of etoposide on dCK in L1210 cells and found that incubation with 10 μM etoposide for 1 h increased dCK activity to 170% of the control. This effect of etoposide on dCK activity was concentration-dependent up to at least 100 μM of the substance.
The involvement of the peripheral serotonin2A (5-HT2A) receptor in α-methyl-5-HT-induced hyperglycemia was examined in rats. The 5-HT2A receptor antagonist, ketanserin, significantly inhibited α-methyl-5-HT-elicited hyperglycemia. Taken together with a previous report that 5-HT-induced hyperglycemia was prevented by ketanserin, it is suggested that the periperal 5-HT2A receptor participates in glucose regulation. As α-methyl-5-HT increased serum insulin but did not affect glucagon levels, it is indicated that these pancreatic hormones are probably not related to α-methyl-5-HT-induced hyperglycemia. Moreover, the peripheral 5-HT3 receptor agonist, 2-methyl-5-HT, did not affect blood glucose, insulin or glucagon levels. Our results therefore suggest that the peripheral 5-HT3 receptor is not involved in glucose regulation.
We have isolated two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase of Glycyrrhiza glabra L. by cross-hybridization with that of Arabidopsis thaliana squalene synthase under conditions of low stringency. Their nucleotide sequences contained an open reading frame for a polypeptide of 413 amino acids (GgSQS1) and 412 amino acids (GgSQS2), respectively. The deduced amino acid sequence of GgSQS1 exhibits 88%, 81%, 78%, 45-44%, and 45-41% identity to those of the GgSQS2, Nicotiana benthamiana, Arabidopsis thaliana, mammal, and yeast squalene synthases, respectively. To express the G. glabra enzymes in Escherichia coli, the entire coding region was subcloned into an expression vector. The cell-free extracts of E. coli transformed with the respective plasmid converted 3H-farnesyl diphosphate into squalene.