An in vitro assay of fibronectin (FN) was established based on the adhesion of baby hamster kidney (BHK) cells through the cell-binding domain of FN.Each well of a microtiter plate was coated with samples or various concentrations of standard FN. Bovine serum albumin was further coated to prevent the non-specific adhesion of the cells. Various numbers of BHK cells were plated and incubated. After washing out the non-attached cells, the number of attached cells was measured using neutral red (NR)-staining.The conditions for the assay were optimal when 1×105 cells/well were plated and incubated for 90 min. The linear relationship between the concentration of FN coated and the absorbance of NR was observed in the range of 0.1-1.0 μg/ml of FN. The inhibition of cell binding by the peptides containing an Arg-Gly-Asp (RGD) sequence demonstrated that this assay system depended on FN-mediated cell adhesion through the major cell-binding domain.
We have developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all IgG1 sub-isotype mAbs) against Glycyrrhizae Radix protein (GRP), which was recently determined to be a marker protein of Glycyrrhizae Radix (GR). Among these, anti-GRP mAb-1 and 2 were found to recognize different epitopes on the GRP molecule, as demonstrated by ELISA analysis, and were used for the development of a sandwich enzyme immunoassay (SEIA) for GRP in traditional Chinese medicines (TCMs). The SEIA was based on the principle of binding an analyte to anti-GRP mAb2 coated on polystyrene microtiter wells, followed by immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was specific to GRP in GRs, and showed no cross-reaction with any Leguminosae crude drugs other than GRs. This SEIA detected GRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/well and a detection limit of 0.1ng/well. The present SEIA was about 10-times more sensitive in detecting GRP than the selected antibody enzyme immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself. Also, the SEIA has such a low assay background that it allowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-daiichi-kagen (KTIDK), a TCM consisting of only 2.7% GR. The GRP SEIA was simple, accurate, reproducible and may provide a general analytical method for the quality control of GR-based TCMs.
Nitric oxide (NO) has been reported to play various roles as a signal transmitter. However, detailed functions of NO have yet to be clarified. We have developed a fluorescent indicator for NO imaging in living cells. The N-nitrosation of newly designed and synthesized 4-((3-amino-2-naphthyl)aminomethyl)benzoic acid (DAN-1) by NO yielded the highly fluorescent triazole-form. The membrane permeable ester derivative of DAN-1 (DAN-1 EE) was applied to the imaging of NO produced in activated rat aortic smooth muscle cells. After DAN-1 EE has been loaded into cells, the ester bond is hydrolyzed by intracellular esterase, yielding original DAN-1 with less permeability. The fluorescence intensity of the cells loaded with DAN-1 EE increased according to NO production. The imaging method with fluorescent indicators will be significant for the functional clarification of NO in vivo.
The activity of mammalian topoisomerase I is inhibited by acidic phospholipids. We investigated the effect of psychotropic drugs and local anesthetics on cardiolipin-inhibited calf thymus DNA topoisomerase I activity. Chlorpromazine, promethazine, and imipramine, which interact with phospholipids, suppressed the inhibitory effect of cardiolipin. The present results suggest that relaxation of DNA supercoiling is involved in the cytotoxic action of these drugs.
Human liver contains high molecular weight-type Zn2+-dependent acid p-nitrophenylphosphatase (HMW-ZnAP). The enzyme was purified 1000-fold by a new procedure, including preparative isoelectrofocusing. The HMW-ZnAP was homogeneous in non-denaturing disk-gel electrophoresis with an MW of about 93 kDa determined by Sephadex G-100 chromatography. A single polypeptide chain of 43 KDa was detected on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting a homodimeric structure. The isoelectric point (pl) was 7.2-7.4. Human liver HMW-ZnAP requires Zn2+-ions for activity; other divalent cations are ineffective or act as inhibitors. It dephosphorylated p-nitrophenylphosphate (pNPP) (Km=0.24 mM), o-carboxyl phenylphosphate (oCPP) (Km=0.92 mM) and phenylphophate (PhP) (Km=1.42 mM). Other substrates including [32P]-labelled casein or phosvitin, adenyl nucleotides and myo-inositol-1-phosphate, were not dephosphorylated. Human liver HMW-ZnAP obeys Michaelis-Menten kinetics with pNPP as substrate; the enzyme was competitively inhibited by inorganic phosphate (Ki=0.55 mM), and by oCPP (Ki=0.65 mM) and PhP (Ki=1.16 mM). Adenosine monophosphate (AMP), adenosine diphosphate (ADP) and ATP displayed mixed-type inhibition. The enzyme was also inhibited by some modifiers such as EDTA, oxalate, p-chloromercurybenzoate, tartrate, imidazole, cyanide, cysteine, histidine and diethylpyrocarbonate, but not by fluoride or okadaic acid. Human liver HMW-ZnAP is sensitive to temperatures higher than 40°C. The pH-dependence of the steady-state kinetic parameters indicates the existence of an essential ionizable group with a pKa of 7.25-7.50, similar to that of histidine. However, diethylpyrocarbonate inactivation experiments suggest that other amino acid residues may also be involved in enzyme catalysis.
To investigate the role of constitutive hsp70 in protein folding and to probe the supplementation by other hsps in this folding, yeast cells expressing reduced constitutive hsp70 proteins, ssa1ssa2, were transformed with a plasmid expressing a bacterial luciferase protein. With several independent clone cells of transformants, the levels of luciferase activity and some hsps, such as hsp104, hsp90, hsp70 and hsp26, were examined. The luciferase activity was significantly lower in ssa1ssa2 transformants than in the wild type (wt) cell transformed with the same plasmid. Among several clone cells of ssa1ssa2, the cells with higher luciferase activities exhibited higher amounts of Ssa4 which is known to be expressed instead of lacking Ssa1 and Ssa2. The luciferase activity closely correlated with the amount of Ssa proteins, more than with the amount of other hsps. It is suggested that consitutional Ssa "chaperones" are needed for the folding of proteins and, in cells lacking Ssa1 and Ssa2, the increased Ssa4 is thought to partly compensate for their role in the folding of luciferase in vivo.
Glycyrrhetyl mono-glucuronide (GAMG) is an intermediate in the hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid (GA). An enzyme responsible for its hydrolysis, characterized as a GAMG β-D-glucuronidase of Eubacterium sp. (species) GLH, has been isolated from human intestinal bacteria. The pattern of GAMG β-D-glucuronidase activity was different from that of GL β-D-glucuronidase activity by Butyl-Toyopearl 650S column chromatography. Thus, these enzymes showed differences in the purification ratio and substrate specificity. After this step, GAMG β-D-glucuronidase was completely separated from GL β-D-glucuronidase by gel filtration through Toyopearl HW-55 S, indicating that the GAMG β-D-glucuronidase is a novel type of β-D-glucuronidase which hydrolyzes one glucuronic acid linkage of GA.
Sixteen naphthoquinones with various substituents were tested for cytotoxicity toward cultured resting leukemia L1210 cells. The cytotoxicity of all the naphthoquinones examined was not affected at all by the treatment in combination with diethyl maleate, a glutathione-specific SH-blocking agent in the cells. Depending on the characteristics of the response to the treatment in combination with iodoacetamide (IAA) and glutathione (GSH), these naphthoquinones were tentatively classified into three groups. The chemicals of Group I, which include naphthoquinones carrying an electron-donating group(s) other than an OH group on the quinone ring moiety, showed synergistic cytotoxicity with IAA and no reduction in cytotoxicity with GSH. Those of Group II, which include naphthoquinones without an electron-donating group on the quinone ring moiety, showed no synergy with IAA but appeciable reductions in cytotoxicity with GSH. Group III includes 2-hydroxylated naphthoquinones, which showed neither synergy with IAA nor cytotoxicity reduction with GSH. Cytotoxicity was discussed in terms of the electron-deficiency of the quinone ring moiety. It is suggested that the cytotoxicity of Group I quinones comes from the active oxygen generation which follows semiquinone formation, whereas that of the other groups of quinones is not likely caused simply by either SH-blocking of biomolecules or active oxygen-related mechanism.
The antitumor effects of toremifene were studied in vitro using 10 anaplastic thyroid carcinoma cell lines and were compared with those of tamoxifen. The antitumor effect of toremifene was dose-dependent and the IC50 values for these cell lines ranged 20.1-58.5 μM. Although the cell lines expressed multidrug resistance gene mRNA, multidrug resistance-associated protein gene mRNA and insulin-like growth factor-1 (IGF-1) receptor mRNA to various degrees, nine of them lacked estrogen receptor expression as evaluated by immunocytochemistry. These data indicate that toremifene, as well as tamoxifen, is cytolytic at relatively high doses for multidrug-resistant anaplastic thyroid carcinoma cell lines.
We investigated the selectivity of T-440 for the inhibition of phosphodiesterases (PDEs) in vitro and for bronchial anti-spasmogenic effects in vivo. Using a fast protein liquid chromatography system, five PDE isozymes, PDE 1, PDE 2, PDE 3, PDE 4 and PDE 5 were prepared from guinea pig and dog tissues. T-440 selectively inhibited PDE 4 with an IC50 of 0.071 μM and 0.13 μM for guinea pig lung and dog trachea, respectively. The IC50 values for all other PDE isozymes were over 20 μM. In contrast, theophylline nonselectively inhibited all the tested PDE isozymes, and the inhibition did not exceed 50%, even at 100 μM. T-440 inhibited the histamine-induced bronchoconstriction of anesthetized dogs in a dose-dependent manner with an ED50 of 0.029 mg/kg, indicating that T-440 is 600 times more potent than theophylline. Both T-440 and theophylline increased LV dp/dt/P (LVP; left ventricular pressure) in anesthetized dogs with ED50 values of 3.6 mg/kg and 4.4 mg/kg, respectively. This potency was 1/125 times the bronchial anti-spasmogenic effects for T-440 and 4.2 times that of theophylline. Rolipram, a PDE 4 inhibitor, also showed selective bronchial anti-spasmogenic effects in anesthetized dogs. These results suggest that T-440, which specifically inhibits PDE 4 activity, has potent and selective bronchial anti-spasmogenic effects.
Some chalcones are known to be phototransformed in a solution from trans into cis isomers. 3-Hydroxy-3'-methylchalcone (3'Me-3-C) has been found to be isomerized from trans into cis by irradiation of daylight in the methanolic solution. The presence of a hydroxyl in the 2'- or 4-position in the trans-chalcone structure prevents phototransformation into cis isomers. The feasibility of phototransformation of chalcones was discussed using UV-spectral analysis. The phototransformed cis-3'Me-3-C showed more potent antitumorigenic activity than the original trans form.The generally recognized parallelism between antitumorigenic and antiinflammatory, activities was not observed in trans and cis 3'-Me- and 4'-Me-3C, which are antitumorigenic but inactive in 12-O-tetradecanoylphorbol 13-acetate (TPA)- and arachidonic acid (AA)-induced mouse ear edema. However, inhibitory activity against ornithine-decarboxylase (ODC) was commonly observed in both naturally occurring and synthetic antitumorigenic chalcones.
The cardiotonic effect of the rhizome of Polygonatum sibiricum was investigated in the left atria of rats. The methanol extract of the rhizome of Polygonatum sibiricum (OM) (1-7 mg/ml) concentration-dependently increased the developed tension of the left atrium. It also strongly inhibited cAMP phosphodiesterase. The increase cAMP level correlated the increase in left atrial contraction. On the other hand, OM did not inhibit Na+, K+-ATPase. The cardiotonic effect of OM was strongly inhibited by reserpine, a sympatholytic agent. Futhermore, OM-treated left atria inhibited the tension produced by propranolol, a beta adrenocepter antagonist. These findings suggested that the cardiotonic effect is due to stimulating beta adrenoceptors through activation of sympathetic nerves.
From a human intestinal bacterium, Eubacterium sp. A-44, which is capable of hydrolyzing saikosaponins to saikogenins, two glycosidases, β-D-glucosidase and a novel type of β-D-fucosidase, were isolated and characterized as saikosaponin-hydrolyzing β-D-glucosidase and prosaikogenin-hydrolyzing β-D-fucosidase.Relative to the hydrolyzing activities toward saikosaponins a, b1 and b2, the β-D-glucosidase showed lower ability to hydrolyze saikosaponin d, but no ability to hydrolyze saikosaponin c or prosaikogenins.By Sephacryl S-300 column chromatography, the molecular weight of prosaikogenin-hydrolyzing β-D-fucosidase was estimated to be about 130 kDa. The β-D-fucosidase could hydrolyze prosaikogenins A and F, but not prosaikogenins D and G or saikosaponins. Relative to p-nitrophenyl β-D-fucoside-hydrolyzing activity, this enzyme had 32.0% and 22.2% of its hydrolyzing ability toward p-nitrophenyl β-D-glucoside and p-nitrophenyl β-D-galactoside, respectively. p-Nitrophenyl β-D-fucoside-hydrolyzing activity was inhibited by D-fucose, and was weakly inhibited by D-glucose, D-glucono δ-lactone, D-galactose and D-galactono δ-lactone.By combining these two glycosidases, saikosaponins a and b1 were converted to their saikogenins via the corresponding prosaikogenins.
The in vitro metabolism of pirmenol (cis-α-[3-(2, 6-dimethyl-1-piperidinyl)propyl]-α-phenyl-2-pyridinemethanol) and glucuronidation of its metabolites, a 4-hydroxylated derivative of pirmenol (M3) and 3-methylether of M3 (M5), were investigated using a hepatic 9000×g supernatant and microsomes, respectively, of female and male rats in order to elucidate the higher urinary excretion of M3G and M5G (glucuronides of M3 and M5, respectively) in females previously observed in in vivo metabolism. Pirmenol Δ1' iminium ion (M2) and M3 were formed by the oxidation of pirmenol in both sexes; however, M2 was the main metabolite in males, while M2 and M3 were formed at nearly the same level in females. On glucuronidation of M3 and M5, the Vmax values of both compounds were higher in female rats, consistent with the results in vivo. In addition, the sex difference in the urinary excretion ratio of M5G to M3G (1.1 in female, 2.5 in male) might reflect the lower availability of M3 for glucuronidation in male rats in vivo. The chromatographic separation of diastereomers of M5G was also described.
8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1, 5-a]-1, 3, 5-triazine-4(1H)-one (BOF-4272) blocks xanthine oxidase/xanthine dehydrogenase in the liver. BOF-4272 with a sulfoxide chiral center includes R(+)- and S(-)- enantiomers. The enantioselectivity in the global disposition of BOF-4272 can be attributed to that in the local disposition of organs, especially the liver. Thus, the enantioselectivity in the hepatic local disposition of BOF-4272 was compared between R- and S-enantiomers by a hepatic perfusion experiment with a pulse input into the portal vein. The influence of perfusate albumin on the enantioselective local disposition was also investigated. The elution time profile of each BOF-4272 enantiomer from the liver into the hepatic vein was measured at four different bovine serum albumin (BSA) concentrations (0, 0.25, 1.0 and 4.0%) in the perfusate at 37 and 4°C. A crossover test was carried out for R- and S-enantiomers using one rat liver. In the absence of perfusate BSA at 37°C, hepatic extraction ratios (EH) of R- and S-enantiomers of BOF-4272 were 75.6±4.3% and 71.7±3.3%, respectively, which were statistically the same. In the presence of 4.0% BSA at 37°C, EH values of R- and S-enantiomers were 31.7±4.6% and 19.6±3.8%, respectively, which demonstrated that EH of R-enantiomer was significantly greater than that of S-enantiomer (p<0.001). In the absence and presence of perfusate BSA at 4°C, there was no significant difference in EH between S- and R-enantiomers. An amplification of stereoselectivity with albumin was observed by the perfusion experiment using BOF-4272 enantiomers.
Free amino groups of chitosan, a substance which has previously been shown to be a good scaffold for hepatocyte attachment, were covalently modified with fructose. The modification significantly increased the number of cells that could be attached on the surface of chitosan gel. Rat hepatocytes cultivated on fructose-chitosan behaved similarly to those on unmodified chitosan, i.e., they retained the spherical shape they have in vivo, and released much less lactate dehydrogenase than cells attached on a collagen-coated surface. The modification with fructose did not alter the important characteristics of chitosan for hepatocyte culture : liver-specific functions such as urea synthesis and drug metabolism were stably maintained for 5 d in the hepatocytes cultured on fructose-chitosan. In sharp contrast, hepatocytes attached on a collagen-coated surface underwent a severe morphological change, from spherical to flat, and lost almost all their lidocaine-removal activity within 5 d. A very thin fructose-chitosan layer was also applied onto the collagen-coated surfaces of polystyrene plates and a dextran microcarrier by crosslinking free amino groups in the chitosan and collagen with glutaraldehyde to fix the thin layer. Hepatocytes on the fructose-chitosan-coated surface retained their spherical shape, masking the cell-flattening effect of the collagen layer. Perfusion culture was then carried out using a hollow-fiber cartridge in which hepatocytes attached on fructose-chitosan-coated microcarriers were suspended in the extracapillary space : the liver-specific functions were stably maintained during 4 d of the culture. A fructose-chitosan-coated surface thus appears to be a very promising scaffold for hepatocyte attachment which can be used in cellular biological studies of liver functions, especially in relation to cytochrome P450, as well as in bioartificial liver support systems.
Temporal changes of the serum levels of 16-hydroxypregnenolone (3β, 16α-dihydroxy-5-pregnen-20-one) 3-sulfate (16-OH-Preg S) and 16-hydroxydehydroepiandrosterone (3β, 16α-dihydroxy-5-androsten-17-one) 3-sulfate (16-OH-DHEA S) were investigated by analyzing the levels of their precursor steroids, pregnenolone (3β-hydroxy-5-pregnen-20-one) 3-sulfate (Preg S) and dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one) 3-sulfate (DHEA S), respectively, in the early neonatal period. The serum levels of these steroids were measured by GC-MS is full-term (gestational age : 37-41 weeks), pre-term (gestational age : 28-36 weeks) and extremely immature (gestational age : 24-27 weeks) infants. The changes in 16-hydroxysteroid production were also investigated by analyzing the ratios of the serum levels of 16-OH-Preg S and Preg S (16-OH-Preg S/Preg S ratio), and 16-OH-DHEA S and DHEA S (16-OH-DHEA S/DHEA S ratio).It was confirmed that the 16-hydroxylation of DHEA S and Preg S increased after birth, and the 16-OH-Preg S/Preg S ratio in full-term infants was significantly higher than in pre-term and extremely immature infants at days 0, 1-6 and 7-13. On the other hand, there were no significant differences between the 16-OH-DHEA S/DHEA S ratios of the three groups at days 0, 1-6 or 7-13.The mechanism of differences in the 16-hydroxylation of Preg S and DHEA S is also discussed.
Intracellular signal transduction for regulation of alkaline phosphatase (ALP) activity in renal epithelial cells treated with calcitonin is not yet completely understood, although it is known that calcitonin receptors couple to cyclic AMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Salmon calcitonin increased the cyclic AMP content in LLC-PK1 porcine kidney cells in a concentration-dependent manner. When the confluent cells were incubated for 47 h after a 1 h-pulse exposure or continuously exposed to calcitonin and forskolin for 48 h, ALP activity in the cells was increased by calcitonin about 2-fold compared with the basal acitivity at the maximum level but was not dependent on the exposure time; it was markedly increased by forskolin in parallel with the exposure time. The increase in activity produced by calcitonin was abolished by a PKA inhibitor H-89, and, in contrast, potentiated by a PKC inhibitor, NA-382 to near the forskolin-induced level. These results indicate that calcitonin exerts a dual-regulation of ALP activity in LLC-PK1 cells, positively through the PKA pathway and negatively through PKC.
The effect of the anti-tuberculosis drug rifampicin on pirarubicin activity was investigated in multidrug-resistant cells overexpressing P-glycoprotein. Rifampicin increased the sensitivity of pirarubicin to anthracycline-resistant mouse leukemic P388 cells and significantly enhanced the cytotoxicity and intracellular accumulation of pirarubicin in resistant cells, but had no effect in parent cells. By contrast, two other rifamycins, rifamycin B and SV, had no effect on pirarubicin accumulation in resistant cells. Rifampicin also enhanced pirarubicin-induced apoptosis and G2/M blockade on the cell cycle in resistant cells. These results show that rifampicin enhances the cytotoxic action of pirarubicin in resistant cells, at least partly via the inhibition of cellular pirarubicin efflux.
The inhibitory effects of a potent nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on 2-deoxy-D-glucose (2-DG)-induced hyperglycemia were investigated in rats. L-NAME significantly inhibited 2-DG-induced hyperglycemia, although NG-nitro-D-arginine methyl ester (D-NAME) did not affect it. A similar NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), also inhibited 2-DG-induced hyperglycemia. The antagonistic effects of L-NAME are unrelated to the cholinergic system, since the muscarinic receptor antagonist scopolamine did not affect 2-DG-induced hyperglycemia. The neuronal NO synthase inhibitor 7-nitroindazole (7-NI) did not reduce 2-DG-induced hyperglycemia, but rather enhanced it. Our results suggest that NO may be involved in glucose homeostasis and that the inhibitory effects of L-NAME on 2-DG-induced hyperglycemia are not related to muscarinic receptors or neuronal NO synthase.
The toxic interaction between disopyramide and propranolol were studied in chick embryos. Fertilized eggs of White Leghorns were incubated and investigated. Disopyramide with and without propranolol was injected into the air sac of a fertilized egg on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after the injection. After each drug injection alone, the heart rate was not different compared with control. However, the heart rate was significantly decreased by combination with disopyramide and propranolol. In addition, arrhythmia was produced by disopyramide 1.0 mg/egg alone and in combination with propranolol. These findings indicate that the interaction between disopyramide and propranolol has a marked influence on the heart rate in chick embryos.
The intestinal local absorption and the hepatic local disposition of 5-fluorouracil (5-FU) in a single conscious rat was investigated by the simultaneous sampling of portal and systemic bloods (PS method). The portal blood flow rate, measured using a compact electromagnetic flow-meter, was estimated to be 15.3±2.2ml/min per body weight (250g). The portal vein and the femoral artery of the rat were cannulated to simultaneously obtain blood samples from two sites. 5-FU (30mg/kg) was administered first intraarterially, and subsequently orally 90 min after intraarterial administration to a single conscious rat (short-period double dosing; DD). Concentrations of 5-FU in the portal and arterial bloods were determined by HPLC. The local absorption ratio (Fa) and the absolute bioavailability (F) were 71.2±15.4 and 25.1±13.2%, respectively. Consequently, the hepatic extraction ratio (FH=F/Fa) was estimated to be 34.9±14.4%. The mean local absorption time (ta) and the mean absorption time (MAT) were 37.5±15.5 and 31.4±13.7min, respectively and they were statistically the same. In conclusion, a PS method by short-period double dosing (PS-DD method) has been developed to evaluate the first-pass effect, separating the intestinal absorption and hepatic elimination of a drug in a single conscious rat. It was demonstrated by applying PS-DD method that the low bioavailability of 5-FU can be explained by the large hepatic first-pass extraction, and that the large inter-individual variation in bioavailability of 5-FU is caused mainly by a large variation in the hepatic first-pass effect. The large variation in ta (or MAT) was predicted to be due to a variation in the gastric emptying time.
The amount of human immunodeficiency virus type 1 (HIV-1) genomic RNA in sera is considered to be one of the most direct markers for patient prognosis and monitoring the efficacy of antiretroviral therapy. Quantitative detection of HIV-1 RNA was performed by the dilution endpoint method and competitive PCR. Conditions for detecting one copy of HIV-1 control DNA were examined to decide the dilution endpoint exactly. PCR of the gag region by SK145/SK39 primer pair and seminested PCR by SK145/SK101 primer pair gave the best result. Conditions for competitive PCR of HIV-1 cDNA, which was reverse transcripted from HIV-1 control RNA, were also studied using a SK38/SK39 primer pair in the presence of HIV-PCR MIMIC as a competitor DNA. The detection limit of HIV-1 control DNA by competitive PCR was 10 copies but that of HIV-1 control RNA was 50 copies. Time-course quantitation of HIV-1 RNA in frozen-stored sera from an AIDS patient was carried out and traced back to 1993. The amount of serum HIV-1 RNA markedly decreased when the treatment was changed but increased again before deterioration of his clinical status. It is considered that the quantitation of serum HIV-1 RNA is useful for the prognosis of HIV-infection and the evaluation of the antiretroviral therapy.