ABSTRACT
<p><b>OBJECTIVE</b>To assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative).</p><p><b>METHODS</b>HES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay.</p><p><b>RESULTS</b>3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L.</p><p><b>CONCLUSION</b>HES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.</p>
Subject(s)
Humans , Binding Sites , Extracellular Space , Metabolism , Ouabain , Chemistry , Pharmacology , Peptides , Chemistry , Protein Binding , Sodium-Potassium-Exchanging ATPase , Chemistry , Genetics , MetabolismABSTRACT
In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.
Subject(s)
Animals , Rats , Amino Acid Sequence , Chromatography, High Pressure Liquid , Erythrocyte Membrane , Ouabain , Pharmacology , Peptide Fragments , Metabolism , Sodium-Potassium-Exchanging ATPase , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1.</p><p><b>METHODS</b>Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1.</p><p><b>RESULTS</b>After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells.</p><p><b>CONCLUSION</b>High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.</p>
Subject(s)
Animals , Cricetinae , Humans , Mice , Antibodies, Viral , Allergy and Immunology , Antibody Specificity , Allergy and Immunology , CHO Cells , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Chickens , Cricetulus , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins , Genetics , Allergy and Immunology , Metabolism , Immunization , Methods , Immunoglobulins , Allergy and Immunology , Immunohistochemistry , Mice, Inbred C57BL , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Metabolism , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , TransfectionABSTRACT
<p><b>AIM</b>To improve specificity and accuracy of endogenous ouabain measurement assay.</p><p><b>METHODS</b>Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared.</p><p><b>RESULTS</b>The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin.</p><p><b>CONCLUSION</b>The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.</p>
Subject(s)
Animals , Male , Rabbits , Antibody Specificity , Chickens , Allergy and Immunology , Cross Reactions , Egg Yolk , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , Immunoglobulin G , Allergy and Immunology , Immunoglobulins , Allergy and Immunology , OuabainABSTRACT
<p><b>OBJECTIVE</b>To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues.</p><p><b>METHODS</b>Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex.</p><p><b>CONCLUSION</b>Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.</p>
Subject(s)
Animals , Cattle , Rats , Calibration , Cell Line , Enzyme-Linked Immunosorbent Assay , Immunization , Methods , Immunoglobulins , Allergy and Immunology , Immunohistochemistry , Ouabain , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>Paclitaxel was used in a phase II trial in combination with cisplatin for esophageal cancer. The anti-tumor response, toxicity and survival of the treated patients were evaluated.</p><p><b>METHODS</b>Thirty patients with advanced, unresectable, or complicated with metastasis were allotted, twenty-seven patients had no prior chemotherapy while 3 patients had received adjuvant chemotherapy. Patients were given paclitaxel 175 mg/m(2) by 3-hour infusion on D1, and cisplatin 40 mg/m(2) daily on D2 and D3. Granulocyte colony-stimulating factor (G-CSF) was not routinely administered unless the patient had neutropenia. Treatment was recycled every 21 days.</p><p><b>RESULTS</b>Thirty patients (male/female, 28/2; median age 58) completed a median of 3 cycles and 27 patients were evaluable for response. Major objective responses were observed in 16 patients (59.3%; 95% confidence interval, 38.9% to 75.5%), including 5 complete responses (18.5%) and 11 partial responses (40.7%). The median time to tumor progression was 5.0 months (range, 1 to 23 months). The median actuarial survival was 9.7 months (range, 1 to 23 months). Twenty-eight patients were assessable for toxicity. The most common nonhematologic toxicity was alopecia. Grade 3 to 4 neutropenia was observed in 17.9% of the patients. Toxicity was manageable with dose attenuation and G-CSF support.</p><p><b>CONCLUSION</b>The combination of paclitaxel and cisplatin can be considered as a main regimen in the treatment of advanced esophageal cancer.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Alopecia , Antineoplastic Combined Chemotherapy Protocols , Therapeutic Uses , Carcinoma, Squamous Cell , Drug Therapy , Cisplatin , Drug Administration Schedule , Esophageal Neoplasms , Drug Therapy , Pathology , Liver Neoplasms , Drug Therapy , Lung Neoplasms , Drug Therapy , Neoplasm Staging , Neutropenia , Paclitaxel , Remission Induction , Survival RateABSTRACT
A percutaneous visceral tumor puncture director is introduced in this paper, which can be used for needle biopsy and brush biopsy in kinds of visceral tumor. The clinical informations show that the application of the director can increase the positive ratio of puncture, and it has important clinical value. The director is safe, practical and easy to operate.