OBJECTIVETo investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation.

METHODSViable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression.

RESULTSKd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01.

CONCLUSIONThere is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.

Adult ; Cell Membrane ; chemistry ; Humans ; Male ; Progesterone ; Radioligand Assay ; Receptors, Progesterone ; analysis ; Sperm Capacitation ; Spermatozoa ; chemistry

To explore novel antifatigue agents targeting with AMPA receptor, 10 compounds were synthesized and their structures were confirmed by 1H NMR, ESI-MS and elemental analysis. 1-BCP was treated as the leading compound. The antifatigue activities were evaluated by weight-loaded forced swimming test, and the AMPA receptor binding affinities were tested with radioligand receptor binding assays. The results unveiled that 5b appeared to possess potent antifatigue activities and high affinity with AMPA receptor, which deserved further studies.
Animals ; Benzamides ; chemistry ; pharmacology ; Dioxoles ; chemistry ; pharmacology ; Fatigue ; prevention & control ; Piperidines ; chemistry ; pharmacology ; Radioligand Assay ; Receptors, AMPA ; metabolism ; Swimming

OBJECTIVE: Oxytocin antagonists maybe useful in inhibiting the uterine contractions of preterm labor. One such compound is TT-235. The purpose of this study was to compare the resistance of TT-235 and oxytocin to enzymatic degradation by oxytocinase in pregnant human. METHODS: Blood samples from pregnant women not in labor were incubated in vitro with known amounts of oxytocin and TT-235. Samples were collected at 0, 15, 30, 45 and 60 minute intervals for oxytocin analysis and at 0, 10, 60 and 360 minutes for TT-235 analysis. Oxytocin was analyzed by radioimmunoassay after extraction while TT-235 was analyzed by radioreceptor assay. RESULTS: In human blood, oxytocin was readily metabolized with greater than 83% disappearance over the 60 minute incubation period. In contrast, TT-235 was stable up to 360 minutes of incubation. CONCLUSION: This study suggests that: (1) blood from pregnant human does contain oxytocinase at least in vitro; and (2) TT-235 was resistant to enzymatic degradation by human blood, implying that this oxytocin antagonist may have prolonged activity in vivo in humans.
Cystinyl Aminopeptidase ; Female ; Humans ; Metabolism* ; Obstetric Labor, Premature ; Oxytocin* ; Plasma* ; Pregnancy ; Pregnant Women* ; Radioimmunoassay ; Radioligand Assay ; Uterine Contraction ; Uterus

Background: A lifelong thyroxine therapy is indicated in all patients who have hypothyroidism as a result of autoimmune thyroiditis. However, it has been reported that some hypothyroid patients with autoimmune thyroiditis have spontaneous remission with restriction of iodine intake instead of thyroxine therapy. The purpose of study was to investigate how many hypothyroid patients with autoimmune thyroiditis can recover from hypothyroidism with restriction of iodine intake instead of thyroxine therapy and which factors predict recovery from hypothyroidism. Methods: We studied 64 patients with autoimmune thyroiditis(goitrous autoimmune thyroiditis 56, atrophic autoimmune thyroiditis 8). Thyroxine therapy was discontinued in patients with goitrous autoimmune thyroiditis on the way(group 1, n=32) or from the beginning(group 2, n=24) and atrophic autoimmune thyroiditis on the way(group 3, n-8). All patients were asked to avoid iodine-rich foods and thyroid function was monitored every one to two months for up to 35 months. Serum T3, T4, TSH concentrations, antithyroglobulin and antimicrosomal antibodies were measured by radioimmunoassay(RIA). TSH binding inhibitor immunoglobulin(TBII) was measured in serum using radioreceptor assay. Two hundred micrograms of thyrotropin releasing hormone (TRH) were given as intravenous bolus and TSH levels were measured in blood samples taken at 0, 30, and 60 minutes. All values were expressed as mean+-SEM. Statistical analysis was done with paired or non-paired t-test, ANOVA, and the Chi-square test. Statistical significance was defined as p-value below 0.05. Results: Thirteen(40.6%) of 32 patients in group 1 remained euthyroid after 12-35 months of discontinuation of thyroxine therapy. The other 19(59.4%) patients in group 1 had recurrences of hypothyroidism within 3 months after discontinuation of thyroxine therapy. In 11(45.8%) out of 24 patients in group 2, serum TSH concentrations declined below 5 mU/L within 3 months without thyroxine therapy. The other 13(54.2%) patients in group 2 remained hypothyroid till 2-16 months and the thyroxine was given. In contrast, all 8 patients in group 3 had recurrences of hypothy- roidism within 3 months after stopping thyroxine therapy. When we compared the recovered patients of goitrous autoimmune thyroiditis with the non-recovered patients of goitrous autoimmune thyroiditis, regardless of thyroxine therapy from the beginning, age at onset of disease of the 24 recovered patients was significantly younger than the 32 non-recovered patients(30.1+2.0 years vs. 40.2+ 2.4 years; p=0.004). Concl#usion: These findings suggest that 42.9% of hypothyroid patients with goitrous autoim- mune thyroiditis remain or become spontaneously euthyroid with restriction of iodine intake instead of thyroxine therapy. Young age may be a predicting factor of recovery from hypothyroidism in goitrous autoimmune thyroiditis.
Age of Onset ; Antibodies ; Humans ; Hypothyroidism ; Iodine ; Radioligand Assay ; Recurrence ; Remission, Spontaneous ; Thyroid Gland ; Thyroiditis ; Thyroiditis, Autoimmune ; Thyrotropin-Releasing Hormone ; Thyroxine

OBJECTIVETo investigate the binding characteristics of endothelial cell (EC) with LPS free from the participation of serum factors.

METHODSLaser confocal microscope was employed in the observation of the binding of EC with FITC-LPS. The KD and the binding sites of each EC were calculated by radioligand binding assay of receptors (RBA) using [(3)H]-LPS.

RESULTSThe binding of EC with LPS was saturable, time and concentration dependent and it could be competed with overdosed LPS of the same type. The fluorescence mainly distributed in cytoplasm, especially near the nucleus, which could also be stained.

CONCLUSIONSThere might be some specific LPS binding sites existing on ECs and LPS could function intracellularily.

Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Fluorescein-5-isothiocyanate ; Fluorescent Dyes ; Humans ; Lipopolysaccharides ; metabolism ; Microscopy, Confocal ; Radioligand Assay ; Umbilical Veins ; cytology

Radioiodinated oxytocin prepared by the lactoperoxidase method exhibited a substantial biologic activity in uterotonic assay of the rat uterus. 125I-oxytocin was bound to the uterine membrane particulate fraction, but the unlabelled oxytocin did not inhibit the binding of 125I oxytocin to the membrane fraction of rat uterus. Cold iodinated oxytocin, however, inhibited the 125I-oxytocin binding to the membrane fraction of rat uterus in proportion to its concentration. These results suggest that 125I-oxytocin is not a suitable radioligand for oxytocin receptor binding study.
Animal ; Binding Sites ; Cell Membrane/metabolism ; Female ; Iodine Radioisotopes/metabolism* ; Oxytocin/metabolism* ; Radioligand Assay ; Rats ; Receptors, Cell Surface/analysis* ; Uterus/metabolism*

OBJECTIVETo establish a protocol of automated synthesis of 1-(2-chlorophenyl)-N-[(11)C]methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ((11)C-PK11195) as the positron-emitter-labeled ligand for peripheral benzodiazepine receptor (PBR) using a commercial synthesizer and explore the quality control methods for the resulting product.

METHODS(11)C-methyl iodide ((11)C-CH(3)I) was synthesized via liquid-phase distillation approach using a (11)C-iodomethane synthesizer. (11)C-PK11195 was prepared by (11)C-methylation of 1-(2-chlorophenyl)-N-(1-methylpropyl)-3-isoquinoline carboxamide (N-demethyl-PK 11195) as the precursor with (11)C-CH(3)I and purified by semi-preparative reversed phase high performance liquid chromatography (HPLC). The radiochemical purity, chemical purity and stability of the product were evaluated by HPLC, and the toxicity was assessed in normal mice. The factors that affected (11)C-PK11195 synthesis were also studied.

RESULTS(11)C-PK11195 was successfully synthesized using the TracerLab FX(F-N) synthesizer. The synthesis time was about 35 min from the end of (11)C-carbon dioxide production by cyclotron to the end of (11)C-PK11195 synthesis (EOS), with a (11)C-methylation reaction time of 3-4 min. The uncorrected radiochemical yield for (11)C-methylation was (33-/+5)%. Analysis with radio-analytical HPLC showed a radiochemical purity and chemical purity of the product both exceeding 99%, with a specific radioactivity of 30-65 GBq/micromol at EOS (from the end of radionuclide production). The (11)C-PK11195 synthesized was radiochemically stable at room temperature and showed low toxicity in normal mice.

CONCLUSIONThe (11)C-PK11195 injection can be conveniently prepared using an automated synthesizer for clinical use in positron emission tomography.

Animals ; Carbon Radioisotopes ; Contrast Media ; chemical synthesis ; Isoquinolines ; adverse effects ; chemical synthesis ; Mice ; Positron-Emission Tomography ; Radioligand Assay ; Radiopharmaceuticals ; adverse effects ; chemical synthesis ; Receptors, GABA-A ; metabolism

Background: Graves disease is an autoimmune disease caused by TSH receptor antibodies. Thyrotropin binding inhibitor immunoglobulins(TBII) are detected in most Graves patients, but some patients have no TBII activities in their sera. It is unknown whether the clinical features of TBII-positive patients are different from those of TBII-negative patients. Methods: To evaluate the prevalence of TBII-negative Graves' patients and its clinical differences from TBII-positive patients, we examined TBII by radioreceptor assay in 686 consecutive untreated Graves patients. We found 84 TBII-negative patients(15 men and 69 women, mean age ±EM: 40.9±.4 years) and compared their clinical characteristics with 87 TBII-positive patients (22 men and 65 women, mean age±EM: 39.9±.5 years) who were selected randomly from the same patients group. Results: In this study, TBII was undetectable in 12.2% of patients with Graves' disease(84 of 686). TBII-negative group had a less weight loss than TBII-positive group. However, there was no significant differences in age, sex ratio, prevalence of ophthalmopathy, duration of illness and positive rate of family history for thyroid diseases between TBII-negative and
Antibodies ; Autoantibodies ; Autoimmune Diseases ; Female ; Graves Disease ; Humans ; Male ; Prevalence ; Radioligand Assay ; Receptors, Thyrotropin ; Sex Ratio ; Sodium Pertechnetate Tc 99m ; Thyroid Diseases ; Thyroid Gland ; Thyrotropin ; Weight Loss

OBJECTIVETo study the association between decreased ligand binding activity of glucocoid receptor (GR) and heat shock protein 90 (Hsp90), the molecular chaperone of GR, after acute lung injury (ALI) in mice.

METHODSIn mouse models of oleic acid-induced ALI, the levels of GR, Hsp90 and Hsp70 were dynamically observed using Western blotting, and the binding capacity and binding affinity of GR assessed with radioligand binding assay.

RESULTSAfter ALI, pulmonary edema was significantly aggravated in the mice with significantly increased lung body index and lung water ratio. GR increased within 1 h after the injury, but then decreased significantly to the lowest level at 12 h after the injury, and the levels of Hsp90 and Hsp70 was increased obviously and reached the highest at 12 h. Radioligand binding assay showed that the Bmax decreased gradually and Kd value increased, and these changes were consistent with the changes of Hsp90/GR.

CONCLUSIONThe ligand binding activity of GR is related to the changes of Hsp90 after ALI.

Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Binding Sites ; Female ; HSP70 Heat-Shock Proteins ; metabolism ; HSP90 Heat-Shock Proteins ; metabolism ; Male ; Mice ; Oleic Acid ; Radioligand Assay ; Random Allocation ; Receptors, Glucocorticoid ; metabolism

OBJECTIVETo observe the platelet activating factor (PAF) antagonistic effect of kaempferol.

METHODThe specific binding of [3H] PAF to rabbit platelet receptor was investigatedwith radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was determined with Fura-2 fluorescent technique.

RESULTThe 1, 2 or 4 nmol x L(-1) [3H]PAF specific binding to rabbit platelet receptor was inhibited by Kae dosage dependently and the IC50 were 30.8, 74.6 and 92.0 micro mol x L(-1), respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration elevation were inhibited by Kae in a dose-dependent manner. The IC50 of Kae to inhibit platelet adhesion was 65 micromol x L(-1).

CONCLUSIONKae is effective in inhibiting the action of PAF and it is a new PAF receptor antagonist.

Animals ; Blood Platelets ; drug effects ; physiology ; Calcium ; metabolism ; Kaempferols ; pharmacology ; Male ; Neutrophils ; metabolism ; Platelet Activating Factor ; metabolism ; Platelet Adhesiveness ; drug effects ; Platelet Membrane Glycoproteins ; antagonists & inhibitors ; metabolism ; Rabbits ; Radioligand Assay ; Receptors, G-Protein-Coupled ; antagonists & inhibitors ; metabolism