The generation of free radicals during the lipid peroxidation of liposomes composed of 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC-liposome) in Fe2+/ascorbic acid (AsA) solution was studied by the ESR spin trapping technique. A carbon-centered radical adduct was observed using α-(4-pyridyl-1-oxide)-N-tert-butyl-nitorone (4-POBN) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO), but no oxygen-centered radicals such as ·OH, LO·, and LOO· were observed. The lipid peroxidation evaluated as 2-thiobarbituric acid reactive substances was inhibited by the addition of 4-POBN. The intensity of this inhibitory effect was dependent on the time when 4-POBN was added to the mixture of PAPC-liposomes and Fe2+/AsA solution, and no inhibitory effect could be observed after 4 min. The signal intensity of the carbon-centered radical adduct was dependent on the lipid concentration of PAPC-liposomes. These results suggest that the alkyl radicals generated from PAPC-liposome peroxidation induced by Fe2+/AsA were trapped by DMPO or 4-POBN at an earlier stage of lipid peroxidation.
A new method was developed to estimate the content of Trichosanthes root (TR) component in two Chinese traditional medicines. Characteristic antigens of TR were separated from TR extract using rabbit antiserum specific for TR, a dried root tuber of Trichosanthes kirilowii. Two selected antibody enzyme immunoassay (SAEIA) methods, the SAEIA A for assay of TR extract and the SAEIA B for assay of karasurin A were used as detection methods for the separation of TR antigens. Using several column chromatographies, two kinds of TR antigens, a protein component and a glycan component, were separated from TR extract. A SAEIA C method for assay of TR glycan component was developed using biotinylated second antibody and peroxidase-labeled avidin as the detection method. The SAEIA C was also found applicable for specific assay of TR extract as well as for the contents of TR component present in two Chinese traditional medicines, prescriptions of which contained TR. Content of trichosantin, an abortifacient protein component of T. kirilowii, in both the medicines were also measured by applying the SAEIA B for assay of karasurin A. Little trichosantin was found in either medicine and the reason for this was determined.
We examined effect of iridoid glucosides, aucubin, catalpol, geniposide and gardenoside, and their enzymic hydrolysates on neurite outgrowth of PC12h cells. Except for aucubin, these glucosides induced neurite outgrowth at 0.1 μg/ml and above in medium after 3 d of treatment. Hydrolysates of the four glucosides all caused neuritogenesis. Geniposide hydrolysate enhanced responses of cells to carbachol and KCl-induced depolarization in terms of cytoplasmic free-calcium concentration. The aglucone of geniposide, genipin, also promoted neurite outgrowth in a dose-dependent manner (ED50=0.7 μM). The neuritogenic effect of genipin was partially or considerably inhibited in the presence of H-89 and genistein. All the results presented suggest that certain iridoid compounds can induce neuronal differentiation in PC12h cells through activation of components of the intracellular signal transduction pathway.
The cDNA encoding for human liver isozyme I of biliverdin-IXβ reductase was cloned from human liver cDNA libraries. The constructed cDNA of 853 bp in length contained an entire reading frame coding 206 amino acid residues. It was found that isozyme I of biliverdin-IXβ reductase is identical to human erythrocyte NADPH-flavin reductase recently reported in a communication by Chikuba et al., Biochem. Biophys. Res. Commun., 198, 1170-1176 (1994). We, further, characterized this mRNA by Northern blot analyses of poly(A) RNA from four different human fetal tissues and eight different human adult tissues showed hybridization mainly to liver RNA in fetal tissues and to skeletal muscle and liver RNA in adult tissues. Sourthern blot analysis indicated that isozyme I of biliverdin-IXβ reductase appeared to be a single copy gene. Insertion of the enzyme-coding sequence into an expression vector pET-3c yielded relatively high amounts of the active enzyme in E. coli. The amino terminal sequence of the recombinant protein was identical to that of native enzyme, indicating that E. coli also removed the N-terminal methionine to produce the mature form. The recombinant and native biliverdin-IXβ reductases were indistinguishable as far as their mobility on SDS-PAGE gel, immunoreactivity and specific activity were concerned.
As previously reported, the cytotoxicity of bleomycin (BLM) toward cultured mammalian cells was remarkably potentiated by vortex-stirring this agent with the cells for a few seconds in the presence of high molecular weight polyacrylic acid (PAA). This paper describes that the enhancement of BLM cytotoxicity was also achieved by a sequential BLM-treatment immediately after vortex-stirring cells with PAA only. It was suggested that this enhancement resulted from the transient permeabilization of the plasma membrane induced during vortex-stirring in the presence of PAA, leading to an increase in BLM incorporation. This mechanism was supported by the finding of an appreciable uptake by vortex-stirred cells of Lucifer Yellow, a known non-permeant material. This procedure, which did not affect any cell viability, might be a beneficial method for transient permeabilization of cultured mammalian cells to facilitate the internalization of non-permeant materials.
The effects of M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin), an antidiabetic agent and aldose reductase inhibitor, on glycolysis were studied in rat and human erythrocytes in vitro. M16209 increased lactate production from glucose when incubated with rat and human erythrocytes, and also increased glucose consumption in rat erythrocytes. The rates of production of lactate in rat erythrocytes treated with M16209 at 10, 25 and 50 μM were 113, 118 and 123%, respectively, of those in vehicle treated cells. Sorbinil (aldose reductase inhibitor), tolbutamide (sulfonylurea), and buformine (biguanide) did not increase lactate production in rat erythrocytes when tested at 50 μM. On the other hand, M16209 did not affect lactate production from D-glyceraldehyde in rat erythrocytes. At 100 μM the agent decreased both glucose-6-phosphate and fructose-6-phosphate in rat erythrocytes, and increased fructose-1, 6-bisphosphate; at 10 μM it also increased 6-phosphofructokinase activity in rat hemolysates. These findings suggest that M16209 accelerates glycolysis in erythrocytes via activation of 6-phosphofructokinase.
To clarify the physiological roles of muscarinic acetylcholine (mACh) receptor subtypes, M1 and M2, on learning and memory, we examined the effects of three antagonists, atropine (non-selective), pirenzepine (M1 selective) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2, 3-b][1, 4]benzodiazepine-6-one, AF-DX 116 (M2 selective), on step-through passive avoidance tasks in mice. During acquisition tests, mice were trained repeatedly until they achieved criterion latency (300 s). In all experiments, drugs or vehicles were intracerebroventricularly administered. Pre-training (5 min before) administration of atropine (1-40 nmol) and pirenzepine(10 and 40 nmol) shortened the response latency in retention tests at 14 d after acquisition training. Pre-test (5 min before) and post-training (immediately after the acquisition training) administration of atropine slightly but not significantly impaired retention scores. The administration of AF-DX 116 did not apparently affect the scores in any of tests. Thus, the M1 receptor subtype coupling systems seem to be more important in the acquisition-consolidation process rather than in the retrieval process.
In pregnant stroke-prone spontaneously hypertensive rats, salt-loading causes symptoms similar to those of human preeclampsia, such as hypertension and proteinuria. To seek evidence of the therapeutic potential in preeclampsia of antithrombin III (AT III), which is a serine protease inhibitor active on various enzymes of the coagulation cascade, we examined the effect of consecutive treatment with AT III on hypertension and proteinuria in this animal model. Salt-loading (2% NaCl diet) caused a significant elevation of systolic blood pressure on day 15-17 and of urinary protein excretion on day 17-19 of gestation, as compared with animals fed a normal diet. AT III, administered i.v. at a dose of 60 or 300 U/kg/d for 10 d from day 9-11 to 18-20, attenuated these pathological changes in a dose-dependent manner. Histological examination of the kidney revealed that AT III prevented the occurrence of arteriosclerosis and thickening of the capillary basement membrane. However, the pathological changes induced by salt-loading were not attributable to activation of the blood coagulation system. These results demonstrate that AT III has preventive action against salt-induced hypertension and proteinuria in pregnancy through a mechanism largely independent of its anticoagulant action. AT III may thus be beneficial for the treatment of clinical symptoms of preeclampsia.
There are some highly cytotoxic anticancer compounds inducing differentiation of cancer cells to normal cells at below highly cytotoxic concentration. Naphthoquinone derivatives having cytotoxic effects on cancer cell were tested to learn whether they have differentiation inducing activity in human leukemia HL-60 cells or not. When HL-60 cells were treated with 2-chloro-3-amino-1, 4-naphthoquinone (CANQ) for four days, differentiation-related phenotypes such as nitrobluetetrazolium (NBT)-reducing ability and phagocytosis were induced. These differentiation markers were increased by cotreatment with 1, 25-dihydroxyvitamin D3 which is a well-known inducer of differentiation in HL-60 cells. To evaluate the route of differentiation induced by CANQ, we examined naphthol AS-D chloroacetate esterase and α-naphthylacetate esterase activities and changes in cellular size and granulation. Treatment of HL-60 cells with CANQ for four days resulted in an 82.4% increase in α-naphthylacetate esterase activity in spite of a 0.2% increase in naphthol AS-D chloroacetate esterase activity. The size of cells in cell mass was larger and granulation was more decreased than untreated cells. These results indicate that HL-60 cells were induced to differentiate into macrophage-like cells.
Satigrel (E5510, 4-cyano-5, 5-bis(4-methoxyphenyl)-4-pentenoic acid) is a potent inhibitor of platelet aggregation. Like cyclooxygenase/prostaglandin H synthase (PGHS) inhibitors such as aspirin, which suppress platelet aggregation by inhibiting thromboxane A2 production, satigrel inhibits collagen- and arachidonic acid-induced aggregation of human platelets. In contrast to other PGHS inhibitors, satigrel, like cyclic nucleotide phosphodiesterase (PDE)inhibitors such as cilostazol, shows inhibitory activity against thrombin-induced platelet aggregation. To investigate the mechanism of the anti-platelet activity of satigrel, we examined the selectivity and potency of satigrel against PGHS isozyme activities and PDE isoform activities. Two isozymes of PGHS are known; constitutive enzyme(PGHS1) and inducible enzyme (PGHS2). Satigrel showed inhibitory activity against PGHS1 (IC50 : 0.081 μM)and PGHS2 (IC50 : 5.9 μM), suggesting the selective inhibition of PGHS1. Indomethacin, which is a selective inhibitor of PGHS1, showed similar selectivity against PGHS isozymes (IC50 : 0.12 μM and 1.4 μM, respectively). These results support that satigrel suppresses thromboxane A2 production by inhibiting PGHS1. It is known that three isozymes of PDE exist in human platelets : Type V, which specifically hydrolyzes guanosine 3', 5'-cyclic monophosphate(cGMP), Type III, which mainly hydrolyzes cAMP, and Type II, which hydrolyzes both cGMP and cAMP. We separated these three isozymes from human platelets and examined the inhibitory activity of satigrel against each enzyme. Of the three isozymes, the inhibitory activity of satigrel was the most potent against Type III PDE (IC50 : 15.7 μM). The IC50 value for Type III corresponded with that for thrombin-induced platelet aggregation. Type V and Type II were also inhibited by satigrel (IC50 : 39.8 and 62.4 μM, respectively). In human platelets, satigrel increased both cAMP and cGMP levels in a dose-dependent manner (100, 300 μM). In conclusion, satigrel inhibits collagen- and arachidonic acid-induced platelet aggregation through preventing thromboxane A2 synthesis by selective inhibition of the target enzyme, PGHS1, which exists in platelets. The anti-aggregating activity of satigrel against thrombin-induced aggregation may be due to elevation of the cyclic nucleotide levels through the inhibition of PDE isozymes.
We investigated the effects of ginsenoside Rb1 (G-Rb1), a major saponin from Panax ginseng C. A. MEYER, on rat liver protein phosphorylation after intraperitoneal administration of CCl4 alone or together with G-Rb1. We found that 118, 63, and 34 kDa proteins were prominently phosphorylated in liver homogenates prepared from CCl4-administered rats, while these protein-phosphorylations were inhibited in the homogenate prepared from the G-Rb1 plus CCl4-administration group. When inhibitors of protein kinases were exogenously added to the homogenates from either the CCl4-administered group or the G-Rb1 plus CCl4-administered group, their phosphorylations were inhibited much more by W-7, an inhibitor of Ca2+/calmodulin-dependent protein kinase(CaM-PK), than by H-7, an inhibitor of protein kinase C (C-kinase). Interestingly, only 34 kDa was phosphorylated in homogenates prepared from the corn oil-, G-Rb1-, and G-Rb1 plus CCl4-administered groups by the exogenous addition of sodium fluoride (NaF), an inhibitor of glycogen synthase. Additionally, G-Rb1 inhibited the Ca2+-accumulation induced by CCl4 both in liver homogenates and microsomes. The above results imply that G-Rb1 inhibits the CCl4-induced protein phosphorylations by modulating CaM-PK rather than C-kinase, and that 34 kDa protein may play a different biological role in cellular environment from 118 and 63 kDa proteins. Therefore, a study in which G-Rb1 is employed as a modulator of critical CCl4-induced phenomena ranging from the disturbance of Ca2+ concentration to protein phosphorylation may be successfully applicable to investigate the diverse physiological functions of liver.
In the present study we evaluated the relationship between the cumulative amount of propranolol permeating through the stratum corneum and the formation of erythema, a skin irritation reaction, after transdermal application of adhesive patches containing propranolol to the skin of guinea pigs. The intensity of erythema was expressed in terms of a* values measured with a chromameter. The a* values increased in guinea pigs after application of the adhesive patches containing 0.4 mg/cm2 of propranolol to the skin. Since the adhesive patches showed good adhesion to the skin (propranolol content is less than the saturated concentration in the adhesive base) and the cumulative amount of propranolol permeating through the stratum corneum is small, the development of erythema was considered to be mainly due to physical factors such as peeling. Even in adhesive patches containing 0.8 mg/cm2 or 1.2 mg/cm2 of propranolol, a* values increased, although adhesion to the skin is low because of crystallization of propranolol in the adhesive base. On the other hand, in these two adhesive patches, the cumulative amount of propranolol permeating through the stratum corneum increased up to 24 h after application. These findings suggest that the skin irritation reaction is due to propranolol mainly absorbed transdermally, because there is a high correlation between the cumulative amount of propranolol permeating through the stratum corneum and the a* values (r=0.928).
N-Cyanomethylmethamphetamine (CMMA), N-formylmethamphetamine (FMA) and methamphetamine (MA)were given intraperitoneally to mouse and rat in doses of 3 mg/kg. The major matabolites of CMMA, FMA and MA in plasma were determined at short intervals after administration by GC-MS to obtain the area under the concentration-time curve (AUC).Regarding the plasma concentration of FMA after CMMA administration, a definite species difference was observed between mouse and rat. In rats given CMMA, FMA was the major component, followed by MA, amphetamine(AP), CMMA and N-formylamphetamine (FAP). In mice given CMMA, MA was major component, followed by AP, FMA, CMMA and FAP. However, it was demonstrated that MA is also non-enzymatically produced from CMMA in plasma. Following FMA administration to rats, FMA was the major component in the plasma, showing the largest AUC value of the four metabolites, FMA, FAP, MA and AP. Following FMA administration to mice, MA showed the largest AUC value, followed by FMA, FAP and AP which were present at low levels even 5 min after injection and were scarcely detectable at 60 min. These results suggest two main mechanisms involved in the metabolism of the N-cyanomethyl group, one of which is the formation of MA by elimination of cyanoformaldehyde from N-α-hydroxylated CMMA and the other which is the formation of FMA by elimination of hydrogen cyanide from N-α-hydroxylated CMMA. The formation of FMA from CMMA was the predominant pathway in rats but not in mice.
The hypoglycemic activity of 80% methanol extract of Polichia campestris (PM) was investigated in both normal and streptozotocin-induced diabetic mice, one of the insulin-dependent diabetic mellitus. PM (300 mg/kg) reduced the blood glucose of normal mice from 2 to 7 h after oral administration. It also decreased hyperglycemia from 594±46 to 472±31 mg/dl 4 h after oral administration in streptozotocin-induced diabetic mice (p<0.05). PM strongly increased glucose uptake in adipose tissues. In addition, the petroleum ether fraction of PM inhibited aldose reductase activity. These experimental results suggest that the antidiabetic activity of Polichia campestris supports its use in traditional medicine.
We examined the protective effect of Astragali Radix extracts (AE) by intraperitoneal injection against Japanese encephalitis virus (JEV) infection in mice. A protective effect was observed by all four samples of AE used. However, the degree of effectiveness for each AE was different. The observed survival rates of the groups injected with sample A (from Shanhsi, Japanese name Sansei-syo) and sample D (from Hokkaido) extracts were higher than 80% at 21 d after JEV inoculation. The groups injected with sample B (from Hopei, Japanese name Kahoku-syo) and sample C (from Hsiahsi, Japanese name Sensei-syo) extracts had a 60% survival rate. The increase in hemagglutination inhibition antibody titer was negligible in mice that survived 21 d after JEV inoculation. The antiviral effect of AE was examined by plaque assay in vitro, but no antiviral effect was shown. In mice injected with AE, the peritoneal exudate cell (PEC) numbers increased significantly, compared to the control. In these PEC, active oxygen production was also high. Also the group as a whole displayed a high survival rate against JEV infection, these were so strong.From these results, we propose that the protective effect of AE is dependent on a non-specific mechanism during the early stage of infection, before it shifts to antibody production, and that PEC plays an important role.
A cationic peptide amphiphile comprising an L-alanine residue interposed between a charged head group and a double-chain segment, N, N-dihexadecyl-Nα-[6-(trimethylammonio)-hexanoyl]-L-alaninamide bromide (NC5-Ala2C16), was synthesized and used to prepare sonicated liposomes. We examined the efficiency of this liposome in gene transfer according to the transient expression of chloramphenicol acetyltransferase (CAT). This cationic liposome reagent facilitates efficient DNA transfection in COS-7 cells. We determined the optimum conditions for NC5Ala2C16 liposome-mediated transfection. The optimal amounts of the amphiphile and plasmid DNA were determined to be about 100 μg and 10 μg per 35-mm dish, respectively. The activity of this liposome was greater than that of commercial reagents, lipofectin, and N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethyl-ammonium methylsulfate (DOTAP), and it was less toxic than lipofectin and DOTAP in COS-7 cells.
The uptake of low molecular weight fractionated [3H]heparin (LMWFH, 10000 Da) was compared with that of high molecular weight fractionated [3H]heparin (HMWFH, 23000 Da) in isolated rat Kupffer cells. Several heparin analogs, including HMWFH and ligands of scavenger receptors, inhibited both the surface binding and internalization of LMWFH, suggesting the involvement of scavenger receptors in the uptake of LMWFH in isolated rat Kupffer cells as well as HMWFH, in spite of a large difference in molecular weight. Metabolic inhibitors(2, 4-dinitrophenol and rotenone), receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of LMWFH. These results suggest that the scavenger receptor-mediated uptake of LMWFH is ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, in agreement with our previous results for HMWFH. The equilibrium binding of LMWFH to Kupffer cells was concentration-dependent with the dissociation constant (Kd) of 50 nM and maximum binding capacity (Bmax) of 2.3 pmol/106 cells. The dissociation constant of LMWFH was an order of magnitude larger than that of HMWFH (5.7 nM), suggesting a decrease in binding affinity to scavenger receptors with a decrease in the molecular weight of fractionated heparin. It was also shown that LMWFH is internalized by scavenger receptors according to first-order kinetics with an apparent internalization rate constant (kint, app) of 0.0053 min-1, which is about half that for HMWFH (0.0118 min-1). Molecular weight thus appears to be one of dominant factors determining the uptake of fractionated heparin by scavenger receptors in Kupffer cells, and may partly explain the reported lower hepatic uptake of low molecular weight heparin than that of unfractionated heparin.
To evaluate the risk of neurotoxicity induced by theophylline and its main metabolites, 1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 1, 3-dimethyluric acid (1, 3-DMUA) and 1-methyluric acid (1-MUA), we compared their convulsive potency to central nervous system (CNS) after intracerebral administration to mice. All compounds studied induced clonic convulsion in a dose-dependent manner, and the ED50 values for convulsion were 490, 546, 1107, 360 and 620 nmol/kg for theophylline, 1-MX, 3-MX, 1, 3-DMUA and 1-MUA, respectively. These compounds were also administered intravenously to mice by constant rate infusion until the onset of convulsion. Clonic convulsion was induced by i.v. infusion of theophylline, 1-MX and 3-MX, while convulsion was not observed during 1, 3-DMUA or 1-MUA infusion for 60 min. This finding may be due to the poor blood-brain barrier permeability of both 1-MUA and 1, 3-DMUA as compared with theophylline, 1-MX and 3-MX. However, it may be also necessary to consider the possibility of 1, 3-DMUA-induced-neurotoxicity judging from its intrinsic convulsive potency.
A bacterium isolated as the contaminant of a batch of commercial benzalkonium chloride (BAC) solution (10% (w/v)) stored in a loosely capped bottle in the Department of Pharmacy Shinshu University Hospital was identified as Pseudomonas fluorescens belonging to biotype G of Stanier, et al. The strain was highly resistant to BAC, and the lowest concentration of BAC that inhibited visible growth of the strain as measured on nutrient agar plates was ≥5000 μg/ml. BAC is a typical quaternary ammonium detergent. Thus we examined the tolerable growth concentration of various strains on surfactants. We were able to confirm growth of P. fluorescens of BAC resistance strain (PFRB) in 5% concentration, but the other strains were not able to grow in 0.1% concentration. We investigated the relationship between biotype and resistance to BAC. PFRB and three clinical isolated strains were found to be the same biotype G. However, no apparent correlation was found between the same biotypes and minimum inhibitory concentration (MIC) of disinfectant or growth permissible concentration on surfactants. The strain was unable to decompose BAC, as no growth occurred in the minimum medium containing BAC as the sole source of carbon, nitrogen or both. Our finding caused us to realize that P. fluorescens might also be a contaminant of disinfectants, as we have seen in Pseudomonas cepacia.
The effect of a cytotoxic substance (bis(2-hydroxyetyl) trisulfide; BS-1), isolated from Bacillus stearo-thermophilus UK563, on messenger ribonucleic acid (mRNA) level in mouse macrophage-like J774A.1 cells was investigated by the method of differential display. The treatment of J774A.1 cells with BS-1 led us to detect the inducible gene. The sequence analysis revealed that the gene was identical to mouse mitochondrial cytochrome b. In fact, Northern blot analysis showed that cytochrome b mRNA in J774A.1 cells was increased by BS-1.
Recently we have found that only cytokine-induced neutrophil chemoattractant (CINC)-3 induces an increase in intracellular [Ca2+] in rat neutrophils stimulated first with CINC-1, CINC-2α or CINC-2β, and hypothesized that rat neutrophils have another receptor specific for CINC-3 in addition to a common receptor for all CINCs[Shibata F., et al., Eur. J. Biochem., 231, 306 (1995)]. To evaluate this hypothesis, chemotactic activity of these CINCs in vitro was determined by using rat neutrophils together with each CINC. In the presence of CINC-1, migration of neutrophils toward CINC-1, CINC-2α and CINC-2βwas inhibited, but that toward CINC-3 was not.Similar results were obtained when CINC-2α or CINC-2β was added to neutrophils. However, addition of CINC-3 to neutrophil suspension in the upper chamber resulted in the complete inhibition of the neutrophil migration toward each CINC. The results support out hypothesis and indicate both the putative receptors can mediate chemotaxis of rat neutrophils.
We have purified bovine brain Zn2+-dependent acid phosphatase (Zn2+-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn2+-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1-and-2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the α- and β-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg2+-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn2+-dependent p-nitrophenyl phosphatase activity and Mg2+-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn2+-APase also exhibits Mg2+-dependent myo-inositol-1-phosphatase activity under physiological conditions.
The effects of an isoquinolinesulfonamide compound, H-87, on naturally acquired multidrug-resistance (MDR) in rat hepatoma AH66 cells were examined. AH66 cells were highly resistant to vinblastine, SN-38, an active camptothecin analog, adriamycin, and etoposide, compared with the sensitive variant AH66F cells. Although H-87 hardly affected the sensitivities to antitumor agents of AH66F cells, this compound completely inhibited the resistance to vinblastine, moderately inhibited the resistance to SN-38 and adriamycin and had little effect on etoposide, mitomycin C, cisplatin, and 5-fluorouracil. H-87 significantly decreased the efflux of vinblastine from the resistant cells and increased the drug accumulation. SN-38 and adriamycin also exhibited a weak but significant increase in vinblastine accumulation in AH66 cells. H-87 inhibited [3H]azidopine-photolabeling to 160 kDa P-glycoprotein in the plasma membrane of AH66 cells, as reported in acquired MDR leukemic cells. Consequently, the MDR-overcoming effect of H-87 seems to be due to its direct inhibition of the binding of antitumor agents on P-glycoprotein in the plasma membrane.
Gln-Val-Val-Ala-Gly is a sequence which is common in cystetine protease inhibitors. Poly(ethylene glycol)hybrids of Gln-Val-Val-Ala-Gly analogs were prepared by the solid phase method by fluorenylmethyloxycarbonyl chemistry. The hybrid formation of Gln-Val-Val-Ala-Gly resulted in improvement of the solubility and also in enhancement of the inhibitory effect of the peptide on papain. In addition, the hybrid formation of Gln-Val-Val-Ala-Gly analogs resulted in the enhancement of the inhibitory effect of the peptide analogs.
The effects of a series of polyol fatty acid esters (sefsols) on diclofenac permeation through rat skin were investigated using in vitro and in vivo methods. Four monoesters and one diester of sefsol were selected as a vehicle components. The effects of each sefsol on in vitro diclofenac permeation were compared at a concentration of 5% sefsol in water. Monoesters of sefsol, except the glyceryl monoester, enhanced the percutaneous permeation of diclofenac. The highest enhancement was observed in propylene glycol monocaprylate. The plasma concentration of diclofenac was increased dramatically by the addition of 10% propylene glycol monocaprylate when applying the diclofeanc sodium suspension to abdominal rat skin in vivo. These results suggest that the monoesters of polyol fatty acid are potential candidates to enhance the transdermal absorption of diclofenac sodium.
The oral administration of insulin in poly(vinyl alcohol)-gel spheres (PVA-GS), an oral dosage form with prolonged residence time in the ileum, was examined in streptozotocin-induced diabetic rats. Intragastric administration of PVA-GS containing insulin and a protease inhibitor, aprotinin or bacitracin, caused a significant and prolonged reduction of blood glucose levels, suggesting insulin absorption. The bioavailability of insulin estimated from the hypoglycemic effect was about 2% in the presence of either protease inhibitor. The release profiles of insulin and the protease inhibitors from the PVA-GS could be explained by Higuchi's plot, and the rates were similar to each other. The site dependency of insulin absorption in the intestinal tract was examined by an in situ loop method. Insulin absorption estimated by plasma insulin levels was larger in the ileum and the large intestine than in the jejunum. The prolonged residence time of PVA-GS in the absorption site, the lower intestine, and the synchronous release of insulin and the protease inhibitors from the PVA-GS are the two major explanations for the improved bioavailability of insulin administered as PVA-GS containing a protease inhibitor.
The pharmacokinetic behavior of glycyrrhizin (GZ) was examined in D-galactosamine-intoxicated (GAL) rats. When GZ was administrated intravenously, the apparent volume of distribution (Vdss) and the total body clearance (CLtotal) were more significantly decreased in GAL rats than those in normal rats. When GZ was administered orally, the area under the plasma concentration-time curve (AUC), the mean residence time (MRT) and the time to reach the maximum plasma concentration (Tmax) for GZ were higher, but the maximum plasma concentration (Cpmax) in GAL rats was lower than that in normal rats. The bioavailability of GZ, however, was not significantly changed. On the other hand, the AUC for glycyrrhetic acid (GA), a main metabolite of GZ, after oral administration of GZ was higher in GAL rats than in normal rats, although there was no significant difference in MRT or Tmax, Cpmax or the bioavailability for GA between GAL and normal rats. The reasons for these differences in GAL rats would be changes in the absorption rate (reduced gastric emptying rate) and reduction of the hepatic elimination rates (biliary excretion of GZ and hepatic metabolism of GA).
A ligand blotting experiment using [125I]epidermal growth factor (EGF) showed that there are EGF binding proteins in rice leaves. EGF binding proteins have isoelectric points and relative molecular masses of 5.5/35 kDa, 7.5/50 kDa and 8.3/40 kDa, respectively. The amino acid sequences of the N-terminal and internal regions of the EGF binding proteins were determined. The amino acid sequences of 35 kDa protein and 40 kDa protein were found to be homologous with those of the photosystem II oxygen-evolving complex. The amino acid sequence of the 50 kDa protein was homologous with that of riburose bisphosphate carboxylase/oxygenase. The presence of proteins capable of binding to EGF suggests the possibility of EGF-like regulation in plants.