The primary structure of rat spermidine synthase having the N-terminal acetylated methionine and 98.7% homology with that of the mouse enzyme is presented using a limited amount of the homogeneous enzyme. The study strategy was principally to compare the molecular masses of liberated peptides determined by three specific cleavage methods with those expected from known cDNA-derived amino acid sequences of mouse and human enzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The cleavage methods involved two enzymatic methods using lysylendopeptidase and arginylendopeptidase, and a chemical method for cleaving at the cysteine residue using 2-nitro-5-thiocyanobenzoic acid. Their usefulness was clearly demonstrated. Column-switching semimicro reversed-phase HPLC, which permits application of the entire reaction mixture, was useful for collecting a small amount of peptides containing the N-terminal amino acid, to confirm acetylation of the N-terminal methionine by MALDI TOF-MS. It was necessary in this approach to examine the amino acid sequence of certain peptides. The Edman method was used for the sequence analysis, and this will be replaced by an improved MALDI TOF-MS now available in a few laboratories.
Anaphylactic reactions of mice sensitized percutaneously with 2, 4-dinitrofluorobenzene (DNFB) were investigated by the AW method assay, which is a mouse anaphylatic model using the abdominal wall as the site for induction with either 2, 4-dinitrophenyl (DNP)-human serum albumin or anti-mouse IgE antibody and then estimation of the response. DNP-specific and IgE-dependent anaphylatic reaction after contact sensitization with DNFB could be induced and detected by the abdominal wall (AW) method assay in both groups with and without previous ear challenge with DNFB. Thus, the anaphylatic reaction in the group of twice-contact with 0.5% DNFB was observed on the 9th day from the sensitization (5th day from the ear challenge), and the reaction in the group of a single contact with 0.5% DNFB was observed 10 d after sensitization. The DNP-specific anaphylactic reaction was observed earlier than the 10th day with higher doses of DNFB.As for the mice of the former twice-contact group, the first and second characteristic ear swelling resonses appeared within 1-6 h and 2 d of the ear challenge, respectively, and small swelling was observed 7 d after the challenge.It is suggested that Th1 and Th2 cells are activated at the almost same time, in other words, the preparation for both cell-mediated and humoral immunity could be accomplished to function, in vivo by a single percutaneous sensitization with DNFB.
Hydrogen peroxide was cytotoxic to the small intenstine epithelial cell line, IEC-6, as judged from an MTT assay and the release of lactate dehydrogenase. The glutathione S-transferase and thioredoxin reductase activities and SH content decreased dose-dependently with H2O2, but thioredoxin activity increased at low H2O2 concentrations. In addition, the increase in thioredoxin activity was time-dependent during the initial stages of oxidative stress. A reverse transcription-polymerase chain reaction (RT-PCR) amplification also showed that the mRNA content in IEC-6 cells increased time-dependepntly at 0.25 mM H2O2. These results indicate that cellular oxidative shock causes an increase in the activity of thioredoxin, which is involved in the defense mechanism against oxidative stress.
The induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells causes a lethal phenotype. Based on our results that the decreased ATPase activity of DnaA E204Q is activated to the level of a wild-type protein in the presence of partially purified stimulating factors for DnaA ATPase, we tested the hypothesis that genes encoding the stimulating factors can be cloned as high-copy-number plasmid suppressors for the lethality caused by DnaA E204Q. We isolated a number of high copy-number plasmids that suppress the lethal phenotype. The genes responsible for the suppression of the lethal phenotype were revealed to be dnaN, relA, pcnB and cyaA by subcloning, and a site-directed mutational analysis. The mechanisms by which these genes suppressed the lethal phenotype were examined in vivo and in vitro.
pMRB01 cloned from Pseudomonas K-62 plasmid pMR26 conferred bacterial hypersensitivity to organomercurials. DNA sequence analysis of a 2.3-kb SacI-Aor51HI fragment encompassing the whole region required for expression of the hypersensitive phenotype, revealed three open reading frames. The DNA sequence of these frames had 82.5%, 99.2% and 97.0% homology with the pDU1358 merR, merB and merD, respectively. The pMRB01 mer operon differs from the already known mer operon by the absence of the merT, merP and merA genes in this plasmid. An inverted repeat-like sequence upstream from the predicted merR was observed suggesting that this defective mer operon could be part of a transposon-like structure.Induction experiments and maxicell analysis of the mer-polypeptide showed that the lyase enzyme encoded by pMRB01 merB gene is mercurial-inducible and regulated by the transacting product of the merR gene. These results suggest that the hypersensitivity to organomercurials resulted from the expression of lyase activity encoded by the defective mer operon in the absence of reductase activity. The lyase enzyme encoded by pMRB01 merB catalyzes the protonolysis of the C-Hg bond of both arylmercury and alkylmercury compounds.
A homolog of oscillin, the Ca2+ oscillation-inducing factor of the hamster, was identified from the cellular slime mold Dictyostelium discoideum and designated Dd-oscillin. In the developmental stages of D. discoideum, the gene is expressed at the prestalk region which contains higher concentration of cytosolic Ca2+ than the prespore region. The Dd-oscillin null strain aggregated but did not develop further when the cells were plated on non-nutrient agar at a density of 1.5×106 cells/cm2, showing that the Dd-oscillin gene is important for development. Since the null cells carrying the hamster oscillin gene formed fruiting body, the hamster oscillin was the homolog of Dd-oscillin as far as function is concerned. In addition, the null cells formed fruiting body in the presence of 2, 5-di(tert-butyl)-1, 4-hydroquinone (BHQ : a specific inhibitor of Ca2+-ATPase activity in the endoplasmic reticulum). These results suggest that Dd-oscillin will increase cytosolic Ca2+ in the cells and promote further development.
Norzoanthamine is an alkaloid isolated from a colonial zoanthid. We examined the effects of norzanthamine hydrochloride on bone weight, strength and morphology in ovariectomized mice, a postmenopausal osteoporosis model. Norzoanthamine hydrochloride significantly suppressed the decrease in femoral weight and bone biomechanical parameters caused by ovariectomy without an increase in uterine weight. This means that norzoanthamine hydrochloride does not have an estrogen-like effect on reprodcutive organs. Morphological observations of longitudinally ground sections of the humeri showed that norzoanthamine hydrochloride administration (2 mg/kg/d, p.o.) completely suppressed the loss of trabecular bone. Furthermore, norzoanthamine hydrochloride thickened the cortical bone. Based on these results. norzoanthamine hydrochloride may act as both a suppressor of bone resorption and an enhancer of bone formation in vivo.
The role of the signal transmission pathway of thunberginol A (TA) in mast cell degranulation was examined using rat peritoneal mast cells. First of all, we investigated the cellular distribution of TA using fluoroscent microscopy. This indicated that TA is immediately incorporated into cells and distributes in cytosol around the nucleus. We then investigated the effect of TA on mast cell protein tyrosine phosphorylation, which is part of the signal transduction cascade for degranulation. TA non-specifically inhibited the tryosine phosphorylation in duced by compound 48/80 (Co. 48/80), at 10 to 100 μM, and orthovanadate/hydrogen peroxide at more than 50 μM in a dose-dependent manner. As far as the intracellular Ca2+ change in fluo-3-loaded cells was concerned, TA (10 μM) suppressed the rise in Ca2+ induced by antigens, ionomycin and thapsigargin, while TA did not suppress the rise induced by Co. 48/80. This evidence suggests that TA mainly inhibits extracellular Ca2+ influx, but TA does not act on the intracellular Ca2+ mobilization from the endoplasmic reticulum. We also investigated the influence of TA on the cytoskeleton and membrane changes using mast cells and erythrocytes. TA (10 μM) inhibited the cytoskeletal assembly formation in dicyanovinyl julolidin-loaded mast cells induced by Co. 48/80. Moreover, TA suppressed the hypotonic hemolysis of erythrocytes, from 3 to 1000 μM, in a dose-dependent manner. These results suggest that inhibition of protein tyrosine phosphorylation, extracellular Ca2+ influx and cytoskeletal assembly formation, and membrane stabilization are involved in the inhibitory effect of TA in mast cell degranulation.
The mechanism of Phagocytic activity of the ethyl alcohol fraction of Cervus nippon (CN-E) was investigated in vivo. The administration of CN-E (100 mg/kg, p.o.) enhanced lucigenin chemiluminescence and the engulfment of fluorescein-conjugated E. coil particles in murine peritoneal macrophages. Phagocytic activity was suppressed by the treatment of S-nitrosoglutathione (GSNO) which is an exogenous nitric oxide donor depending on the concentration of dose. CN-E suppressed the production of nitric oxide and enhanced the concentration in [Ca2+]i. The enhancement in [Ca2+]i was diminished by the treatment of EGTA. These results indicate that CN-E enhances the phagocytic activity of murine peritoneal macrophage via a suppression of nitric oxide production and an increase in [Ca2+]i.
Several L-arginine analogs are known as potent inhibitors of nitric oxide synthase (NOS). We recently synthesized dipeptides containing such amino acids, and found that they are potent and isozyme-selective NOS inhibitors. For example, S-methyl-L-isothiocitrullinyl-L-phenylalanine showed 66-fold selectivity for iNOS over nNOS, while S-methyl-L-isothiocitrullinyl-L-leucine and NG-nitro-L-argininyl-L-phenylalanine showed 20- and 14-fold selectivity, respectively. Interestingly, S-methyl-L-isothiocitrullinyl-D-phenylalanine showed no selectivity, and S-methyl-L-isothiocitrullinyl-L-phenylalanine showed compective inhibition. These results suggest that each NOS isozyme has a cavity of different size near the C-terminal of the L-arginine binding site, and that the selectivity of inhibitors is due to the differences in the size of the cavity.
We examined the effect of Eucommia ulmoides OLIVER leaf on rat skeletal muscles together with spontaneous running-training in terms of the isozyme profile and specific activity of lactate dehydrogenase (LDH; EC 220.127.116.11) and 3-hydroxyacetyl-CoA dehydrogenase (HAD; EC 18.104.22.168). On the twenty-ninth day of the experimental period, a mandatory endurance running exercise (treadmill, 7° grade) was conducted. Twenty-four hours later, the rats were sacrificed and the skeletal muscles and other organs were dissected. Due to the training, the HAD specific activity in the skeletal muscles had increased and a more oxidative metabolism had developed, which was further enhanced by the admistration of the leaf. In soleus (SOL) muscle in the Eucommia leaf treated running-training group (ET), the LDH specific activity in the skeletal muscle was significantly higher than in the sedentary control group (SC). The isozyme profile of the group ET was significantly different when compared with the group SC. The changes in the LDH isozyme profile were larger in the SOL than that in extensor digitorum longus (EDL) muscle. The results show that mechanical training and the use of the leaf cooperatively increase the ability to avoid lactate accumulation in skeletal muscle. This effect is supported by the group where 67% of rats accomplished the endurance running exercise. These results suggest that the administration of Eucommia ulmoides OLIVER leaf along with light intensity training enhances the ability of a muscle to resist fatigue.
Two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase have been isolated from licorice, Glycyrrhiza glabra L., and characterized. The deduced amino acid sequence of GgSQS1 was 88%, 81%, 78%, 45-44%, and 45-41% identical to those of GgSQS2, Nicotiana, Arabidopsis, mammal and yeast squalene synthases, respectively. Squalene synthase activity was found in the cell-free extracts of Escherichia coli transformed with the recombinant plasmids for GgSQS1 and GgSQS2, respectively. Genomic Southern blot hybridization indicated that there are three squalene synthase genes in the licorice genome. Northern blot analysis showed that GgSQS2 mRNA is mainly expressed during the exponential growth phase of the cultured licorice cells.
To determine the transminas-lowering action of glycyrrhizin (GL) immunologically, the effect of GL on tumor necrosis factor (TNF)-α- and Fas-mediated apoptosis was assessed using a human hepatoblastoma line, HepG2 cells. The HepG2 cells were resistant to TNF-α and anti-Fas antibody, but were rendered susceptible to TNF-α and anti-Fas antibody in the presence of antinomycin D (Act D), an inhibitor of RNA synthesis. The cytotoxicity induced by TNF-α/Act D or anti-Fas/Act D was accompanied by DNA fragmentation, indicating apoptotic death of HepG2 cells. GL partially prevented the apoptosis of HepG2 cells induced by TNF-α/Act D in a GL-dose dependent fashion. However, this protective effect of GL was not observed in the cytotoxicity of HepG2 caused by anti-Fas/Act D. Although the protection mechanism of GL, observed in a limited fashion against TNF-α-mediated apoptosis, is unclear, the present results provide an immunological explantation for the transaminase-lowering action of GL in the GL treatment of chronic liver diseases involving apoptotic hepatocyte death in their pathogenesis.
The purpose of this study was to investigate the absorption behavior of an ophthalmic drug injected in rabbit periocular tissues. After intracapsular, retrobulbar and palpebral conjunctival injections of 150 μl and 50 μl fluorescein isothiocyanate dextran (FITC-dextran, average molecular weight 11000), leakage of the dye into the tear fluid was dependent on the injection route and volume. After periocular injecitons (50 μl) of tilisolol, as a model beta-blocker, the concentrations in the tear fluid blood, aqueous humor and vitreous body were determined by HPLC. Slight drug leakage was observed in the tear fluid after injections. The periocular injections showed a faster absorption and a higher area under the concentration-time curve (AUC) in the plasma and a lower AUC in the aqueous humor than those observed in instillation. They also showed a higher ratio of AUC of tilisolol in the vitreous body to AUC in the aqueous humor than that observed in the instillation. Among the periocular injecitons, retrobulbar injection showed the highest concentrations in the plasma and the lowest in the aqueous humor and vitreous body, while intracapsular injeciton showed the lowest in the plasma and the highest in the aqueous humor and vitreous body. Although the periocular injections showed a rapid systemic absorption of drug by a rich topical vasculature, it might be an effective approach to deliver the drug to the periocular tissues and vitreous body.
The purpose of this study is to evaluate periocular injections with viscous solution as a topical delivery system of ophthalmic drugs. Tilisolol and carboxymethylcellulose (CMC) were used as a model beta-blocker and a viscous polymer, respectively. After intracapsular, retrobulbar and palpebral conjuctival injections (50 μl) of tilisolol with 3% CMC into rabbits, drug concentrations in the tear fluid, blood, aqueous humor and vitreous body were determined by HPLC. Periocular injection (50 μl) of tilisolol with 3% CMC showed slight leakage of the drug in the tear fluid from the injection site. The viscous vehicle decreased the absorption rate constant of the drug from the injection site to systemic circulation compared with the buffer solution. It suggests that the viscous solution improved the retention of drug at both the injection site and in periocular tissues. Although the periocular injections with viscous vehicle (3% CMC) showed lower AUC in the aqueous humor than that observed in instillation, they showed comparable AUC in the vitreous humor. Compared to the results after the periocular injections with buffer solution, CMC increased the AUCs in the vitreous body 3.1-fold with retrobulbar injection and 1.4-fold with palpebral conjunctival injeciton, respectively. As a result, periocular injections with 3% CMC showed higher delivery of tilisolol to the vitreous body against the aqueous humor than the instillation and periocular injections with buffer solution.
A polysaccharide (CS-F10) purified from a hot water extract of the cultured mycelium of Cordyceps sinensis was composed of galactose, glucose and mannose in a molar ratio of 43 : 33 : 24; its molecular weight was estimated to be about 15000. The results of chemical and spectroscopic investigations suggest that CS-F10 has a comb-type structure, and has α-D-glucopyranosyl residues on the terminal of the side-chains and characteristic sugar residues of C. sinensis i.e., 1, 5-linked β-D-galactofuranosyl residues. CS-F10 significantly lowered the plasma glucose level in normal, streptozotocin (STZ)-induced diabetic and epinephrine-induced hyperglycemic mice after intraperitoneal administration (50 mg/kg). Administration of CS-F10 to STZ-induced diabetic mice significantly increased the activity of hepatic glucokinase. A significant reduction in the hepatic glucose output was observed following the infusion of CS-F10 using the perfused rat liver. CS-F10 also significantly decreased protein content of facilitative glucose transporter isoform 2 (GLUT2) from rat liver following i.p. administration. These effects presumably contribute to the hypoglycemic activity.
Fluvastatin is a synthetic hypolipidemic drug which inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. We compared in vitro the antioxidative effects of two enantiomers (3R, 5S and 3S, 5R) of fluvastatin, which is clinically used as a racemic mixture, on copper ion-induced oxidation of human low-density lipoprotein (LDL). Although 3R, 5S-enantiomer of fluvastatin has 30-fold stronger inhibitory activity on HMG-CoA reductase than its optical counterpart, the antioxidative effects of these enantiomers on copper ion-induced LDL oxidation were similar. The antioxidative effects of the metabolites of fluvastatin (M2, M3, M4 and M7) on the copper ion-induced LDL oxidation were also investigated. All the metabolites tested showed an inhbitory effect on this system. Among them, the effects of M2 and M3, which have a phenolic hydroxyl group in each indole moiety, were strong and their potencies were 30-50 times greater than that of fluvastatin. We couclude that not only 3R, 5S-enantiomer of fluvastatin but also its optical counterpart and the metabolites also have a potential to show the anti-atherosclerotic effect through their antioxidative activities on lipid peroxidation.
A simple and highly sensitive high-performance liquid chromatographic method is described for the determination of valproic acid in human serum. The method is based on the direct derivatization of serum sample with 6, 7-methylenedioxy-1-methyl-2-oxo-1, 2-dihydroquinoxaline-3-ylpropionohydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37°C. The resulting derivatives were separated on a reversed-phase column (YMC Pack ODS-A) with isocratic elution and were fluorimetrically detected at 440 nm with excitation at 365 nm. The detection limit (signal-to-noise ratio=3) for valproic acid added to serum sample was 0.1 μg (700 fmol)/ml serum sample (2.3 fmol on column). The method was applied to determine the unbound- and total-valproic acid levels in the serum obtained from three healthy volunteers after oral administration of the drug (600 mg).
We found that calcein-loaded liposomes can be used to evaluate the sizes of channels in membranes by measuring changes in calcein release when the molecular size of the solute added to the outer suspension medium is changed. If the solute added to the outer medium can enter the inner aqueous phase through the channel, the osmotic pressure of the inner phase increases, causing bursting of the liposomes and the release of calcein. Thus, the size of the channel formed in the liposomal membrane can be determined by examining whether the solute added to the outer medium induces calcein release. We used a series of sugars as solutes and estimated the channel size formed by polyene antibiotics. The results agreed well with those conducted in a similar manner using erythrocytes, demonstrating that this method should be useful for examining channel sizes in membranes.
In order to investigate the physiological role of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 22.214.171.124), a cytosolic acetoacetate-activating enzyme, the effects of animal development on the activity and content of the enzyme were examined in rat liver. In male rats, the enzyme specific activity increased 21-fold at 4 weeks of age from that at 2 weeks of age, and then gradually decreased, while in female rats, it increased similarly to that of male rats, but further increased, reaching a maximum about 3-fold higher than that of male rats, at 6 weeks of age. The developmental patterns of the enzyme content correlated with that of the enzyme specific activity. These results indicate that changes in this enzyme activity and content during the developmental process might influence the rate of ketone body utilization for the formation of physiologically important lipidic substances in rat liver.
Qian-Hu is a well-known traditional Chinese medicine used for the treatment of respiratory disease and pulmonary hypertension. We compared the relaxant effects of pyranocoumarin compounds, including (+)-praruptorin A (Pra-C), Pd-Ia (=(±)-praeruptorin A), pteryxin, peucedanocoumarin II (P-II) and 8-methoxy-psoralen (8-MOP) purified from Bai-Hua Qian-Hu (BQ) in isolated rabbit tracheas and pulmonary arteries. Pra-C, pteryxin and Pd-Ia produced significant relaxant effects in tracheal preparations constricted with 40 mM KCl or 10 μM acetylcholine. The relazant response to Pra-C, pteryxin or Pd-Ia in preparations constricted with KCl was significantly more potent than that in preparations constricted with acetylcholine. Pra-C, pteryxin or Pd-Ia at a concentration of 30 μM completely relaxed tracheas constricted with 40 mM KCl, whereas P-II at the same concentration showed only partial relaxation. In pulmonary arterial preparations, 8-MOP produced a significant relaxant effect on contractions by 10 μM phenylephrine, without any effect on the contraction by 40 mM KCl. These results suggest that Pd-Ia, pteryxin and Pra-C for their calcium antagonistic action, and 8-MOP for its inhibitory effect on contraction induced by phenylephrine, may be the active principles of BQ for relazing the smooth muscle of tracheas and pulmonary arteries, and the principle may produce a synergistic effect.
This study was designed to clarify the relationship between streptozotocin (STZ) dosage (100, 150 and 200 mg/kg i.p.) and the resulting diabetogenic response in mice (8-week-old male ICR). In this experiment, we found that a single i.p. injeciton of 100 mg/kg STZ is able to induce progressive diabetes mellitus, in which non-fasting serum glucose levels begin to rise from 3 weeks and continue to rise throughout the experimental period until 9 weeks. The non-fasting serum insulin levels of 100 mg/kg STZ-treated mice were normal during the experimental period. In addition, the population of insulin-immunoreactive cells (beta cells) in the islets of pancreata was slightly less than in normal mice at 9 weeks. In 200 mg/kg STZ-treated mice, on the other hand, the insulin levels were below measurable values and insulin-immunoreactive cells were not observed. It is concluded from these results that the progressive diabetic mouse model induced by a single i.p. injection fo 100 mg/kg STZ, unlike 200 mg/kg STZ-induced diabetes which is insulin-dependent, is non-insulin-dependent.
γ-Thujaplicin, β-dolabrin and hinokitiol(β-thujaplicin), hinokitiol-related compounds isolated from the wood of Thujopsis dolabrata S. and Z. hondai MAK have antimicrobial activity. In particular, strong antibacterial activity of hinokitiol and β-dolabrin on Staphylococcus epidermidis IFO-12993 was found, with a minimum inhibitory concentration (MIC) of 0.2 μg/ml. This activity was higher than that of gentamicin, used as a positive control, and so the strong antibacterial activity of both compounds on this bacterium is of considerable interest. Of the three compounds, γ-thujaplicin showed the strongest antifungal activity and its MIC was found to be around 1.5 μg/ml. The three compounds also inhibited metalloproteases. The inhibitory activity of hinokitiol on carboxypeptidase A was especially strong, its 50%-inhibitory concentration (IC50) being 2.76×10-6 M. Considering that metaloproteases are involved in inflammation, the strong inhibitory activity of hinokitiol could be important. On the other hand, hinokitiol-acetate did not show any antimicrobial activity and metalloprotease inhibition, suggesting that at least part of the activity is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton.
Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK) is a traditional herbal medicine (Kampo medicine) which has been found to ameliorate hypercholesterolemia and hypertriglyceridemia in rats and rabbits. In the present study, the effect of OKGK on acyl-CoA : cholesterol acyltransferase (ACAT) was studied in order to elucidate the mechanism of its antihypercholesterolemic action. Oral administration of OKGK to rats fed a cholesterol-enriched diet for 4 weeks markedly repressed the increase in ACAT activity in the small intestine. In contrast, OKGK did not influence hepatic ACAT activity. These results indicate that OKGK selectively inhibits ACAT activity in the small intestine relative to that in the liver, resulting in a reduction of cholesterol absorption, followed by a decrease in serum cholesterol.
Taste acceptability of ground Polymyxin B sulfate and Bactramin C tablets was examined when flavored BMI-60, a food additive, was added. Both adult and child volunteers found the bitter taste of the two drugs markedly inhibited, making it clinically useful. Noncompliance, due to this bitterness, was improved using flavored BMI-60. The most striking characteristic of flavored BMI-60 is the ease of preparation compared with the manufacture of other hospital pharmaceuticals such as jelly, gummi and candy done to mask bitterness.
A tripeptide growth factor, glycyl-L-histidyl-L-lysine (GHK), was immobilized on the surface of poly(vinylalcohol)-quarternized stilbazole (PVA-SbQ) gel. The photoreactive substance, 4-(3-trifluoromethylazirino)benzoyl-N-hydroxysuccinimide (TDBA-OSu), was employed to link the gel and ligand GHK. The density of immobilized GHK was 70 nmol/cm2. Isolated rat hepatocytes were inoculated on the GHK-immobilized PVA-SbQ gel and cultured for 5 d. About 24 h after inoculation, hepatocytes started to aggregate and formed multicellular spheroids while almost no cells attached to GHK-non-immobilized PVA-SbQ gel. The formed spheroids attached firmly to the surface of PVA-SbQ gel for 5 d. GHK was, thus, shown to be an effective ligand for hepatocyte attachment. Dodecamethylenediamine was used to extend the length between the gel surface and GHK. Extension of the length significantly increaesd the number of attached hepatocytes.
We have previously reported that ligands of scavenger receptor such as acetylated low density lipoprotein (acetyl-LDL) and oxidized LDL induced growth of peripheral macrophages in vitro. This suggests the possibility that in addition to foam cell formation, modified or oxidized LDLs induce macrophage proliferation in atherosclerotic lesions. To learn further the physiological regulation of macrophage growth, we comparatively examined the effect of interleukin (IL)-4 and IL-10 which have been reported to be suppressive to various macrophage functions on macrophage growth-stimulating activities of the acetyl-LDL, oxidized LDL and macrophage colonystimulating factor (M-CSF). An in vitro study showed that the activity of M-CSF-containing L-cell-conditioned medium was the most sensitive to the suppressive effects of these cytokines. The growth-inducing activity of acetyl-LDL was significantly inhibited by both IL-4 and IL-10. On the other hand, the activity of oxidized LDL was not attenuated by IL-4 or IL-10. These data indicate that macrophage growth-stimulating activity of oxidized LDL, in contrast to that of M-CSF or acetyl-LDL, is refractory to these suppressive cytokines. Oxidized LDL may act as a potent macrophage growth-stimulating factor in atherosclerotic or other inflammatory sites, even when these cytokines are produced by inflammatory and immunological reactions in situ.
A fluorescent HPLC method for the assay of arginyl-tRNA-protein transferase (R-Transferase) activity was applied to obtain quantitative data of the enzyme activity in rat tissues for the first time. In this assay, the major problem was a significant hydrolysis of the substrate, N-aspartyl-N'-dansylamido-1, 4-butanediamine, and the product, N-arginylaspartyl-N'-dansylamido-1, 4-butanediamine (ArgAsp(4)DNA) by aminopeptidases in crude samples such as 105000 g supernatants (105S) of tissue homogenates. As bestatin inhibited the hydrolysis of ArgAsp(4)DNA, a standard-addition method in the presence of bestatin, using a partially purified R-Transferase preparation from hog kidney as a standard, made it possible to measure directly R-Transferase activities in 105S with a short incubation time and sufficient reliability. It was found by the established method that of 14 tissues examined, stomach was rich in the R-Transferase activity with the highest specific activity, suggesting a target tissue for the future studies on R-Transferase to elucidate its physiological significance.
Recently, we developed a series of novel and potent aminopeptidase inhibitors with a homophthalimide skeleton. Among them, N-(2, 6-diethylphenyl)homophthalimide (PIQ-22) possesses a specific aminopeptidase-inhibiting activity more potent than that of bestatin or actinonin, as assayed in terms of hydrolysis of L-alanine 4-methylcoumaryl-7-amide (Ala-AMC) by human acute lymphoblastic leukemia MOLT-4 cells. We show here that PIQ-22 and its 2, 6-dimethylphenyl derivative (PIQ-11) are more potent inhibitors of tumor cell invasion than bestatin and actinonin in a Matrigel assay using mouse melanoma B16F10/L5 cells.
The inhibitory effects of the hybrid liposomes on the growth of B-16 melanoma cells in vitro and in vivo were examined. The 50% inhibitory concentration of the hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylenedodecyl ether (C12(EO)10) was one-twelfth of that of DMPC liposomes. It was noteworthy that for the first time significantly prolonged survival was obtained using a mouce model of carcinoma after the administration of the hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n=10 or 23) without antitumor drugs.