In vitro release of paclitaxel derivative liposome by paddle membrane binding assay / 中国药科大学学报

@#The in vitro release is an important index to evaluate the quality of liposome formulation.Currently, there is no evaluation method for the in vitro release of liposome in pharmacopoeia of various countries, which leads to the lack of unified standard and safety guarantee for the quality evaluation of liposome formulation.Taking the self-made paclitaxel derivative liposomes as an example, the paddle membrane binding method established by optimizing external release conditions was used to simulate the complete release of paclitaxel derivative drugs in 12 hours under physiological conditions.The results showed that using 0.5% SDS-HEPES as the release medium and a dialysis bag with a molecular weight cutoff of 1 000 kD to release the liposome solution met the requirements and had discrimination ability, providing a reference for the development of drug-loaded liposomes release methods in vitro.

The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey / 药学学报

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H2SO4 terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

Physicochemical and immunological characterization of two forms of recombinant norovirus GⅡ.4 virus-like particles assembled in Hansenula polymorpha / 中华微生物学和免疫学杂志

Objective To investigate the physicochemical properties and immunogenicity of virus like particles(VLPs) in two different conformations assembled from the essential capsid protein VP1 of GⅡ.4 norovirus(NoV) in Hansenula polymorpha. Methods NoV GⅡ.4 VLPs in two different conforma-tions were prepared from high-density fermentation of recombinant engineered strains and VLPs purification. Physicochemical properties of the two forms of VLPs were identified by Western blot,size-exclusion high per-formance liquid chromatography (SEC-HPLC), dynamic light scattering(DLS) and transmission electron microscopy. Serum VLPs binding activities and blocking activities against VLPs binding to histo-blood group antigen(HBGA-VLPs) were evaluated after immunization of BALB/c mice with the two forms of VLPs. Re-sults VLPs of two different diameters with high homogeneity were obtained after purification. DLS results showed that particle sizes of two VLPs were 53.98 nm and 45.18 nm,respectively. The two VLPs were sim-ilar in binding abilities to HBGA receptors. Serum VLPs binding activities and blocking activities against HBGA-VLPs were found higher in NoV-VLP-L than NoV-VLP-S,but the difference was not statistically sig-nificant (P>0.05). Conclusion VLPs in two different conformations were obtained by expressing NoV GⅡ.4 VP1 proteins in Hansenula polymorpha. Though they were similar in physicochemical properties and immunogenicity,the NoV-VLP-L might be potential antigen candidates for the development of recombinant human norovirus vaccine.

G-protein activation revealed by [35S]-GTPγS binding assay is involved on the antidepressant-like effect of Hyperi cum caprifoliatum and Hypericum polyanthemumcyclohexane extracts

AbstractPrevious studies by us demonstrated the antidepressant-like and antinociceptive effects of lipophilic extracts and dimeric acyl-phloroglucinols from species of the genus Hypericum native to Southern Brazil. Uliginosin B and HC1 (an enriched phloroglucinol fraction from Hypericum caprifoliatum) are able to inhibit monoamine synaptosomal uptake without binding to the monoaminergic sites on neuronal transporters, unlike classical antidepressants. The current study aimed at investigating the action of H. caprifoliatum Cham. & Schltdl. and Hypericum polyanthemum Klotzsch ex Reichardt, Hypericaceae, cyclohexane extracts and their main component, HC1 and uliginosin B, on G protein coupled receptors by using the [35S]-guanosine-5′-O-(3-thio)triphosphate ([35S]-GTPγS) binding assay, which reveals the G protein activity. The antidepressant-like effect of acute (one or three treatments within 24 h) and repeated (five days with and without a three day wash-out) treatments with the cyclohexane extracts was evaluated using the rat forced swimming test. The [35S]-GTPγS binding to monoamines and opioid receptors stimulated by agonists was performed ex vivo in brain membranes of rats acutely or repeatedly treated with the cyclohexane extracts. The effect of HC1 and Uliginosin B on [35S]-GTPγS binding assay was performed by direct incubation with brain membranes in the absence of agonists. Their antidepressant-like effect was evaluated through the mice forced swimming test. The extracts, HC1 and Uliginosin B showed antidepressant-like effect in the forced swimming test. The acute treatments with extracts increased the [35S]-GTPγS binding stimulated by the monoamines, while after five days of treatment the [35S]-GTPγS binding was reduced even after three day wash-out. These effects are not due to HC1 or Uliginosin B interaction with the receptors, since direct incubation with these phloroglucinols did not affect [35S]-GTPγS binding to membranes. Our findings indicate that H. caprifoliatum and H. polyanthemumextracts bring about adaptive changes in monoamine receptors, which reinforces their antidepressant-like profile.

Identification of chikungunya virus interacting proteins in mammalian cells.

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.

Identification of chikungunya virus interacting proteins in mammalian cells.

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.

White Spot Syndrome Virus infection in Penaeus monodon is facilitated by housekeeping molecules.

White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp aquaculture, and its rampant spread has resulted in great economic loss. Identification of host cellular proteins interacting with WSSV will help in unravelling the repertoire of host proteins involved in WSSV infection. In this study, we have employed one-dimensional and twodimension virus overlay protein binding assay (VOPBA) followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the host proteins of Penaeus monodon that could interact with WSSV. The VOPBA results suggest that WSSV interacted with housekeeping proteins such as heat shock protein 70, ATP synthase subunit β, phosphopyruvate hydratase, allergen Pen m 2, glyceraldehyde-3-phosphate dehydrogenase, sarcoplasmic calcium-binding protein, actin and 14-3-3-like protein. Our findings suggest that WSSV exploits an array of housekeeping proteins for its transmission and propagation in P. monodon.

Research progress in receptor binding assay by mass spectrometry / 中国药学杂志

OBJECTIVE: To explain that receptor-ligand binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide information about the relative binding affinity of ligand-receptor. METHODS: The correlative literature and abroad were collected and analyzed. RESULTS AND CONCLUSION: The concept of MS binding assay based on mass spectrometric quantification of nonlabeled markers represents a promising alternative to conventional radioligands binding without inherent drawbacks. The technique will play an important role in the design and screening of receptor-targeting pharmaceuticals.

Changes of memory and M receptor injection of A?_(25-35) into basal ganglion region of forebrain and effect of catalpol / 上海第二医科大学学报

Objective To establish mouse model of dementia by intracranial injection of A?25-35 and small amount of ibotenic acid(IBO) and to explore whether the effects of catalpol can affect the brain M receptor density and the short term memory. Methods The mice were randomly divided into three groups: control group,model group,treat group which were given orally for 2 months with 50 mg?kg-1?d-1 of catalpol.Dementia model was developed by single unilateral injection of 0.3 ?L of a solution of A?(1?L normal saline containing 4 ?g of A?25-35 and 1 ?g of ibotenic acid) into right basal ganglion region according the atlas of mouse brain with the aid of a stereotaxic equipment.The track of injection was observed by HE staining.The learning/memory ability was measured by Y-maze perfor-mance.The brain muscarinic receptor density was analyzed with single-site binding assay using 3H-quinuclidinyl benzilae(QNB).Results Two months after model development,the learning ability as well as the density of muscarinic receptor in brain were significantly decreased in model mice compared with those in control mice.Parallel models treated with daily oral administration of Catalpol for two months improved the learning ability and increased the brain muscarinic receptor density when compared with model mice.The correlation coefficient between total M receptor densities and the learning/memory ability was significant when examined with linear regresion.Conclusion A dementia model was established in mice.Dementia model was developed by single unilateral injection of 0.3 ?L of a solution of A?(1 ?L normal saline containing 4 ?g of A?25-35 and 1 ?g of ibotenic acid) into right basal ganglion region was established in mice.Catalpol can significantly improve the learning and increase the brain muscarinic receptor density of the model.

Determination and clinical significance of the binding capacity of glucocorticoid receptor on peripher alleukocytes from patients with Graves disease / 重庆医科大学学报

Objective:To observe the change and clinical significance of glucocorticoid receptor on peripher alleukocytes frompatients with Graves disease.Methods:By radioligand binding assay,GR contents of peripher alleukocytes were determinated in 61 patients with Graves disease,20 healthy volunteers as controls.Results:The GR contents in Graves disease group were significantly lower than that of the normal control group(t = 7.389,P = 0.000 1).In 61 patients with graves disease,GR contents gradually decreased(F = 7.389,P = 0.000 1)with increased Graves disease severity.GR contents were negatively correlated with FT3,FT4 and TSH,correlation coefficients were 0.741(t = 5.643,P = 0.013),0.689(t = 5.119,P=0.025)and 0.755(t =6.391,P=0.009),respectively.GR contents were decreased(t = 4.274,P= 0.000)while patients complicated bythyrotoxic exophthalmos,but GR contents were not statisticallysignificantlydeclined while patients complicated by hyperthyroid heart disease(t = 0.273,P = 0.786) or thyrotoxic periodic paralysis were(t =0.961,P = 0.336).Conclusion:The binding capacity of GR on peripheral leukocytes in the Graves disease patients is lower than normal.This change may play roles in the pathogenic process of Graves disease and probably relate to the disease severity,thyrotoxic exophthalmos complicated.

Tetramethylpyrazine inhibition on binding of radiolabeled ligand to VEGFR / 中国药理学通报

Aim To study the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor. Methods The mice sera were collected after peritoneal injection with big-dose TMP,low-dose TMP,protamine and NS. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum. The culture medium of ECV304 was treated with the mice sera in different groups. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(B_ max) and dissociation constant(K_d).Results The sera of big-dose TMP inhibited 125I-VEGF binding to its receptor, K_d=343.30?36.64 pmol?L-1,B_ max=46.26?5.85 fmol/2?10~5 cells(P0.05),but B_ max decreased(P

The effect of three steroidal saponins from Yin-tonic herbal medicince on muscarinic cholinergic receptors of cultured rat myocardiac cells / 中国药理学通报

Aim To observe the effect of three steroidal saponins on the M-cholinoceptor density of cultured rat myocardiac cells. Methods The time course of M-cholinoceptor density was observed and diosgenin (DIO),timosaponin aglycone (ZMS,S-configuration) and XMS,a stereoisomeric compound of ZMS in C-25 methyl group,R-configuration) were added to the culture medium from the 12th day of culture at three final concentration of 10~(-7),10~(-6) and 10~(-5) mol?L~(-1),and the M-cholinoceptor density was measured on the 16th day with radioligand binding assay. Results The density of M-cholinoceptor increased gradually at the beginning of culture,reached a plateau at 4～10 days,and then dropped gradually. On the 16th day of culture,the M-cholinoceptor density was about 60% of the plateau value.The up-regulation effect of ZMS on the density of cultured rat myocardiac cells on the 16 th day was only significant at a final concentration of 10~(-5) mol?L~(-1). On the contrary,XMS was effective even when its final concentration was as low as 10~(-7) mol?L~(-1). DIO showed no effect on the M-cholinoceptor density at any of its three concentration. Conclusion The above results indicate that XMS with lower concentration showed similar effect on the M-cholinoceptor density of cultured mylcardiac cells as that of ZMS with more higher concentration.

Establishment and clinical application of collagen binding assay for von Willebrand factor / 中华检验医学杂志

Objective To establish a new methA of detecting vWF function. Methas The capability of vWF to bind collagen was evaluated with ELISA. Results The assay′s sensitivity was 0.001 U/ml. Coefficient of variation for inner-batch and inter-batch were 3.34 and 6.70 respectively.The vWF:CBA value of plasma was(90.24?22.87)% in 20 normal subjects. The vWF:CBA value was (31.94?27.36)% in 54 vWD, (35.22?20.02)% in 10 type 1 vWD, (8.74?6.38)% in 10 type 2A vWD and (0.70?0.58)% in 6 type 3 vWD,the values of all four vWD groups were lower than that of normal group( P

Comparison of the binding characteristics of insulin receptors on ovary, adrenal and hepatic plasma membrane from rats / 中国糖尿病杂志

Objective To compare the binding characteristics of insulin receptors (IR) on ovary,adrenal and hepatic plasma membrane from rats. Methods The number and affinity of IR was detected by radioligand binding assay. Results Scatchard plot analyses showed that the receptor numbers for high affinity sites are 7. 359?10/mg protein, 8. 029? 10/mg protein and 6. 440?10/mg protein of liver,ovarian and adrenal plasma membrane with KD of 6.147?107 M-1, 1. 528?10, M-1, and 1. 010?107 M-1 that. The receptor numbers for low affinity sites are 2. 403?10/mg protein,2. 212?10/mg protein,and 2. 257?10/mg protein respectively with KD of 2. 920?10M-1,2. 008?10 M-1 and 0. 433?10sM-1. Conclusion There are abundent IR on ovary and adrenal just as many as those on liver, although the KD are smaller. It suggests that insulin may play an important regulatory role in ovary and adrenal.

Density and Affinity of IL-6 Receptors in Human Leukemic Cells / 中国肿瘤生物治疗杂志

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

Analysis of endothelin receptor and its subtypes in the left ventricle of rats with dilated cardiomyopathy / 中国临床药理学与治疗学

Aim To observe the changes of endothelin receptors and their subtypes of left ventricules in normal SD rats and dilated cardiomyopathy rats. Methods To establish the best conditions of the binding experiment, different protein concentrations, incubation temperature ?and?incubating?time?were?tested? with 125 I-ET-1 ligand respectively. With the selected conditions, saturation binding experiments were performed to determine the amount of endothelin receptor and its subtypes in normal SD rats and in dilated cardiomyopathy ones. Results (1) The optimal incubating temperature was 37 ℃. Under this condition, the binding amount of 125 I-ET-1 increased rapidly in 0～30 minutes, and reached to saturation point at 60 minutes, and there was a linear correlation between 125 I-ET-1 binding amount and cell membrane protein concentration. (2) Endothelin-1, bosentan,BQ123,BQ788 etc. could competitively suppress the bound of 125 I-ET-1 to endothelin receptors. (3) The amount of endothelin receptor in left ventricle of dilated cardiomyopathy rats was ( 92.21? 34.34) nmol?kg -1 protein, which was significantly low than that in normal SD ones. There was no change on the ratio of endothelin receptor subtypes A and B. Conclusion 125I-ET-1 can be used to determine the amount of endothelin receptor and its subtypes in varied tissues specifically. The amount of endothelin receptor in left ventricle of dilated cardiomyopathy rats is down regulated, but the ratio of endothelin receptor subtype A vs B remains to be 21.

Characteristics of A|1 and A|2 adenosine receptors upon the acetylcholine release in the rat hippocampus

As it has been reported that the depolarization induced acetylcholine (ACh) release is modulated by activation of presynaptic A1 adenosine heteroreceptor and various lines of evidence suggest the A2 adenosine receptor is present in the hippocampus. The present study was undertaken to delineate the role of adenosine receptors on the hippocampal ACh release. Slices from the rat hippocampus were equilibrated with (3H)choline and then the release amount of the labelled product, (3H)ACh, which was evoked by electrical stimulation (rectangular pulses, 3 Hz, 2 ms, 24 mA, 5 V/cm-1, 2 min), was measured, and the influence of various adenosine receptor-related agents on the evoked tritium outflow was investigated. And also, the drug-receptor binding assay was performed in order to confirm the presence of A1 and A2 adenosine receptors in the rat hippocampus. N-ethylcarboxamidoadenosine (NECA), a potent adenosine receptor agonist with nearly equal affinity at A1 and A2 adenosine receptors, in concentrations ranging from 1apprx30 muM, decreased the electrically-evoked (3H)ACh release in a concentration-dependent manner without affecting the basal rate of release. And the effect of NECA was significantly inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 micrometer), a selective A1 adenosine receptor antagonist, but was not influenced by 3,7-dimethyl-1-propargylxanthine (DMPX, 5 micrometer, a specific A2 adenosine receptor antagonist. N6-Cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist, in doses ranging from 0.1 to 10 micrometer, reduced evoked (3H)ACh release in a dose-dependent manner without the change of the basal release. And the effect of CPA was significantly inhibited by 2 micrometer DPCPX treatment. 2-P-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680C), a potent A2 adenosine receptor agonist, in concentrations ranging from 0.1 to 10 micrometer, did not alter the evoked ACh release. In the drug-receptor binding assay, the binding of (3H)2-chloro-N6-Cyclopentyladenosine ((3H)CCPA) to the- A1 adenosine receptor of rat hippocampal membranes was inhibited by CPA (Ki = 1.22 nM), NECA (Ki=10.17 nM) and DPCPX (Ki-161.86 nM), but not by CGS-21680C (Ki=2,380 nM) and DMPX (Ki-22,367 nM). However, the specific binding of (3H)CGS-21680C to the A2 adenosine receptor was not observed. These results suggest that the A1 adenosine heteroreceptor play an important role in evoked ACh release, but the presence of A2 adenosine receptor is not confirmed in this study.

Estrogen Receptor Analysis on Fine Needle Aspirates and Biopsies from Palpable Breast Carcinoma

The estrogen hormone receptor (ER) content of human breast cancer has assumed an important role as a predictor of hormone therapy response and as a prognostic indicator. The conventional technique is the dextran-coated charcoal (DCC) method or a ligand-binding assay (LBA) based on the measurement of radiolabeled steroids in cytosolic extracts of tissue homogenate. The recent introduction of monoclonal antibodies with high specificity for human ERs has allowed the application of immunocytochemical assays (ICA) in human cancer tissue. An extension of the ICA technique to cytologic specimens is also widely used. Our aim was to evaluate the reliability of ER-ICAs on fine needle aspirates(FNA) from breast cancer patients by comparing it with ER-ICAs and ER-LBAs performed on surgically removed tissues. During a recent 6-month period, ER-ICAs and ER-LBAs were performed in 83 cases. Among these 83 cases, only the 40 cases for which the ER-ICA and the ER-LBA were performed simultaneously ere included in this study. As positive cutoff values, we assumed 10 fmol/mg protein for the ER-LBAs and a semiquantitative score of 4 for the ER-ICAs. The results were as follows : 1) The ER positive rate was 55% (22/40) for ICAs and 47.5% (19/40) for LBAs. The concordance rate between the ER of ICAs and that of LBAs was 82.5% (33/40). 2) The Pearson correlation coefficient between ER-ICAs of fine needle aspirates and that of surgically removed tissue was good (r=0.94, p<0.005) 3) The Spearman correlation coefficient between ER-ICAs of fine needle aspirates and ER-LBAs of surgically removed tissue was good (r=0.57, p=0.0001) In conclusion, ER determination by using the fine needle aspirate is a reliable method in palpable breast cancer. FNA-ER may be a useful method when it is difficult to take sufficient breast cancer tissue, i.e., in cases of diffusely recurrent cancer, liver metastasis, malignant pleural effusion, etc.

A Study of Estrogen and Progesterone Receptor Expression in Neuroepithelial Tumors

There exist many controversial debates on the presence of estrogen and progesterone receptor in neuroepithelial tumors. The receptors of these two female sex steroid hormones, i.e; estrogen and progesterone receptors, were examined in 24 neuroepithelial tumors. Using a dextran-coated chacoal(DCC) assay, high affinity binding sites were found in the cytosolic fraction with mean capacities of 3.42 and 4.71fmol/mg cytosol protein, respectively for progesterone receptors with stained positively for estrogen receptors and only 2 cases of tumor were stained positively for progesterone receptors with immunohistochemical technique. In addition, the most convincing evidence for the absence of estrogen and progesterone receptors was obtained by reverse transcription polymerase chain reaction(RT-PCR) and Southern blot hybridization using oligo nucleotide probes which is complementary to the fragments of the human estrogen and progesteone recepotrs messenger ribo nucleic acid(mRNA). In 24 neuroepithelial tumors, we were not able to find any expression of mRNAs coding for the estrogen and progesteone receptors. From the present study, it is concluded that estrogen and progesteone receptors are geneally absent in neuoepithelial tumors. This study suggests that estrogen and progesteone receptors would not be involved in neuroepithelial tumors and therefore have no current significance as makers for adjuvant medical therapy of most neuroepithelial tumors.