The 5-hydroxytryptamine 2A receptor (5-HT_2AR) is one of the key targets in the development of novel antidepressants. To develop new antidepressants targeting the 5-HT_2A receptor, this study established a high-throughput screening method for drugs targeting the 5-HT_2A receptor based on the principle of detecting calcium flux signals. The immunofluorescence assay and western blotting were employed to evaluate receptor expression levels in the 5-HT_2AR-CHO cell line. The reaction system parameters, including cell seeding density, DMSO concentration, and dye incubation time, were optimized with Z'-factor and signal window values as evaluation indicators. The specificity, precision, stability, and applicability of the method were assessed. Results indicated that the 5-HT_2AR-CHO cell line stably expressed high levels of the 5-HT_2A receptor. The optimized screening method involved a reaction system with 10 000 cells/well, 0.2% DMSO, and 2 h incubation with Calcium 6 dye. The method demonstrated excellent specificity, with inter-batch precision below 10% for the detection of 5-hydroxytryptamine (5-HT) at low, medium, and high concentrations. Testing four compounds that target the 5-HT_2A receptor- agonists 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and lysergic acid diethylamide (LSD), along with the antagonist MDL100907-yielded Z'-factors (at EC₈₀) greater than 0.85 and signal window values over 0.91. The EC₅₀ values of these compounds were in the nanomolar range, and their potency rank order aligned with previously reported data, confirming the reliability of the established method. When being applied to the detection of 38 known active compounds, the method efficiently identified 5-HT_2A receptor agonists and antagonists while showing no response to non-target compounds. In conclusion, this study successfully constructs a high-throughput screening approach for 5-HT_2A receptor-targeting drugs based on calcium flux signals. The method possesses strong specificity, high sensitivity, and robust stability, being suitable for screening antidepressants targeting the 5-HT_2A receptor.
High-Throughput Screening Assays/methods* ; Receptor, Serotonin, 5-HT2A/metabolism* ; Animals ; CHO Cells ; Cricetulus ; Calcium Signaling/drug effects* ; Antidepressive Agents/pharmacology* ; Humans ; Serotonin 5-HT2 Receptor Antagonists/pharmacology* ; Calcium/metabolism*

OBJECTIVETo investigate whether inhalable particulate matters can cause the damage of chromosome or mitotic apparatus to produce micronucleus, and to evaluate genetic toxicology of moxa smoke on chromosome.

METHODSBy MTT method, the 24 h half maximal inhibitory concentration (IC50) of moxa smoke condensation (MSC) on Chinese hamster ovary (CHO) cells was 0.087 mg/mL. CHO cells, which were cultured in vitro, were divided into a solvent control group, a positive control group (cyclophosphamide as solvent), a low concentration group, a moderate concentration group and a high concentration group. The low concentration group, moderate concentration group and high concentration group were set approximately 1/8, 1/4, 1/2 of IC50, respectively. Whether micronucleus had dose-effect response induced by the damage of chromosome or mitotic apparatus was observed after CHO cells were contaminated by MSC in the low concentration group, moderate concentration group and high concentration group.

RESULTSThe rate of micronucleus induced by MSC in the low concentration group, moderate concentration group and high concentration group was higher than that in the solvent control group (all P < 0.05), which presented dosage-effect response. The experiment was repeated 3 times, indicating it was repeatable with statistical significance.

CONCLUSIONHigh concentration of MSC shows toxicity to induce chromosome damage, which disappears at low concentration. The genetic toxicology is also dependent on concentration, and the concentration of moxa smoke is essential. In clinical treatment, it is noted to control the level of moxa smoke, while the clinical safety standard of moxa smoke concentration is in need of further study.

Air Pollutants ; adverse effects ; Animals ; CHO Cells ; Cell Nucleus ; drug effects ; genetics ; Cricetinae ; Cricetulus ; Inhalation Exposure ; adverse effects ; analysis ; Micronucleus Tests ; Moxibustion ; adverse effects ; Particulate Matter ; adverse effects ; Smoke ; adverse effects ; analysis

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. The relatively short half-life of FSH in vivo requires daily injections for more than 10 days that is inconvenient and possibly contribute to the stress perceived by the patients. The goal of the present study was to increase FSH glycosylation, in order to develop a long-acting recombinant FSH. The cDNA of native alpha and beta subunit of human FSH was linked by a sequence with two N-linked glycosylation sites, and the resulted DNA was inserted into pcDNA3.1 vector to generate a recombinant vector of pcDNA3.1-FSH. The pcDNA3.1-FSH was linearized and transfected into CHO-K1, positive transformants were selected by G418 and confirmed by PCR and Western blotting. A single chain recombinant FSH was expressed, with molecular weight of about 49 kDa. The recombinant FSH expression level in CHO-K1 cell strain in serum-free culture was 3 mg/L. Single injection of this recombinant FSH could induce folliculogenesis and ovulation in rats, the efficacy was similar with the commercially available FSH preparation (Folltropin-V) administrated 8 times consecutively. The results suggested a long-acting FSH was produced successfully.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Follicle Stimulating Hormone, Human ; biosynthesis ; Genetic Vectors ; Half-Life ; Humans ; Ovarian Follicle ; drug effects ; Ovulation ; drug effects ; Rats ; Recombinant Proteins ; biosynthesis ; Transfection

The present paper is aimed to explore the biological osteoinductive activity of recombinant human bone morphogenetic protein 9 (rhBMP-9) by various biological technologies. In this study, we firstly obtained hBMP-9 cDNA by PCR and inserted it into vector pcDNA4/His Max to reconstruct hBMP-9 eukaryotic expression vector pcDNA4/His Max-BMP-9. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level rhBMP-9 was reconstructed by co-transfecting the expression vectors pcDNA4/His* Max-hBMP-9 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification by the methotrexate. We finally obtained a monoclonal cell line expressing the highest level protein. We purified the medium after culturing the highest-producing monoclonal by Ni-NTA His-Bind Resin columns and concentrated to by a Centricon 50 at 4 degrees C and stored at 70 degrees C until it was used. Western blot and SDS-PAGE analyses showed a specific band of about 32kD in pro-region lane and a specific band of about 50kD in pro-region complex lane. Biological activities of rhBMP-9 were tested by colorimetric determination and histochemical staining of Alkaline Phosphatase (ALP) Activity, osteocalcin and oesteopontin for C3H10 T1/2 cells, which were stimulated culture by different concentration (20, 50, 100 microg/mL) of rhBMP-9. The results showed that the rhBMP-9 could induce osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro, and were proportional to the amount. This study can provide experimental data for further tests in vivo and clinical applications.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Growth Differentiation Factor 2 ; biosynthesis ; isolation & purification ; pharmacology ; Humans ; Osteogenesis ; drug effects ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Transfection

OBJECTIVETo investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector.

METHODSCCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.

RESULTSGene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks.

CONCLUSIONCCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

Animals ; CD40 Ligand ; genetics ; pharmacology ; CHO Cells ; Cell Line, Tumor ; Chemokine CCL21 ; genetics ; pharmacology ; Colonic Neoplasms ; therapy ; Cricetulus ; Dendritic Cells ; drug effects ; physiology ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology

Recent advances in hematopoietic stem cells (HSCs) expansion by growth factors including angiopoietin-like proteins (Angptls) have opened up the possibility to use HSCs in regenerative medicine. However, the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion. Here, we report the functional role of mouse Angptls 1, 2, 3, 4, 6 and 7 and growth factors SCF, TPO, IGF-2 and FGF-1 on purified mouse bone-marrow (BM) Lineage(-)Sca-1(+)(Lin-Sca-1(+)) HSCs. The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors (SCF, TPO, IGF-2 and FGF-1). None of the Angptls stimulated HSC proliferation, enhanced or inhibited HSCs colony formation, but they did support the survival of HSCs. By contrast, any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3- to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone. These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.
Angiopoietin-like 4 Protein ; Angiopoietin-like Proteins ; Angiopoietins ; genetics ; metabolism ; Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; cytology ; CHO Cells ; Cell Differentiation ; drug effects ; Cell Lineage ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cricetinae ; Cricetulus ; Culture Media, Conditioned ; pharmacology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Membrane Proteins ; metabolism ; Mice ; Transfection

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.
Acanthamoeba castellanii/drug effects/metabolism/*pathogenicity ; Amebiasis/*parasitology ; Animals ; CHO Cells ; Cell Adhesion/drug effects ; Cell Survival ; Cricetinae ; Cricetulus ; Escherichia coli K12/metabolism ; Female ; Mannose/*pharmacology ; Mannose-Binding Lectin/*metabolism ; Phagocytosis ; Protozoan Proteins/metabolism

To explore an efficient strategy for further development of anticancer fluoroquinolone candidates derived from ciprofloxacin, a heterocyclic ring as the bioisosteric replacement of C3 carboxyl group led to a key intermediate, oxadiazole thiol (5), which was further modified to the bis-oxadiazole methylsulfides (7a-7h) and the corresponding dimethylpiperazinium iodides (8a-8h), respectively. Structures were characterized by elemental analysis and spectra data, and their anticancer activities in vitro against CHO, HL60 and L1210 cancer cells were also evaluated by MTT assay. The preliminary results show that piperazinium compounds (8) possess more potent activity than that of corresponding free bases (7).
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; CHO Cells ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Ciprofloxacin ; chemistry ; Cricetinae ; Cricetulus ; Drug Design ; HL-60 Cells ; Humans ; Inhibitory Concentration 50 ; Leukemia L1210 ; Molecular Structure ; Oxadiazoles ; chemical synthesis ; chemistry ; pharmacology ; Piperazines ; chemical synthesis ; chemistry ; pharmacology

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.
Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Arabinonucleosides ; pharmacology ; CHO Cells ; Cricetinae ; Cricetulus ; Cytarabine ; analogs & derivatives ; pharmacology ; Cytosine ; analogs & derivatives ; pharmacology ; DNA Breaks, Double-Stranded ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; deficiency ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Homologous Recombination ; genetics ; Inhibitory Concentration 50 ; Prodrugs

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the intrahepatic bile duct epithelium, has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Thus, there is an urgent need to develop new effective therapeutic strategies for this disease. We previously found that L1 cell adhesion molecule (L1CAM) plays an important role in tumor progression of ICC, and we generated a murine mAb, A10-A3 (IgG1), that binds to the Ig1 domain of L1CAM. In the present study, we further characterized A10-A3, constructed a chimeric A10-A3 antibody (cA10-A3) containing the constant regions of human IgG1, and evaluated the therapeutic potential in a human ICC xenograft nude mice model. The affinities (K D) of A10-A3 and cA10-A3 for soluble L1CAM were 1.8 nM and 1.9 nM, respectively, as determined by competition ELISA. A10-A3 inhibited L1CAM homophilic binding and was slowly internalized into the tumor cells, but it did not significantly inhibit proliferation of ICC cells in vitro. cA10-A3 mediated antibody-dependent cell-mediated cytotoxicity in vitro and displayed anti-tumor activity in the ICC animal model. These results suggest that the humanized A10-A3 antibody may have potential as an anticancer agent for the treatment of ICC.
Animals ; Antibodies, Monoclonal/genetics/*immunology ; Antibody-Dependent Cell Cytotoxicity ; Bile Ducts, Intrahepatic/drug effects/immunology/pathology ; CHO Cells ; Cell Adhesion/drug effects ; Cell Proliferation/drug effects ; Cholangiocarcinoma/*drug therapy/immunology/pathology ; Cricetinae ; Disease Models, Animal ; Endocytosis/drug effects ; Humans ; Immunoglobulin G/genetics/*immunology ; Liver Neoplasms/*drug therapy/immunology/pathology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neural Cell Adhesion Molecule L1/genetics/*immunology/metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Fusion Proteins/immunology/metabolism/*therapeutic use