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1.
Protein Expr Purif ; 225: 106596, 2025 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39218246

RESUMO

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.


Assuntos
Gonadotropina Coriônica , Cricetulus , Proteínas Recombinantes , Células CHO , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Humanos , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/biossíntese , Gonadotropina Coriônica/farmacologia , Cricetinae , Sinais Direcionadores de Proteínas/genética , Expressão Gênica , Transfecção
2.
J Ethnopharmacol ; 336: 118716, 2025 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-39179055

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng flowers, which are the buds of the traditional Chinese medicinal herb Sanqi, are widely used in China for their cough-ameliorating properties, with demonstrated therapeutic effects in the treatment of both acute and chronic coughs. However, both the antitussive mechanism and active compound basis of P. notoginseng flowers remain poorly understood. AIM OF THE STUDY: We investigated the antitussive effects of P. notoginseng flowers, identified the bioactive constituents responsible for alleviating cough symptoms, and elucidated the underlying pharmacological mechanisms. MATERIALS AND METHODS: We analyzed the major chemical constituents of aqueous extracts of P. notoginseng flowers using liquid chromatography-mass spectrometry and quantitatively analyzed the key component, 20S-ginsenoside Rh2, using high-performance liquid chromatography. Using a cough reflex model in healthy mice and an ovalbumin-induced, highly sensitive guinea pig cough model, we verified the suppressive effects of P. notoginseng flowers and their saponin constituents on coughing. Furthermore, we explored the mechanisms of action of the key ion channels, NaV1.7 and TRPV1, using whole-cell patch-clamp techniques and molecular docking. Finally, the therapeutic mechanisms of P. notoginseng flowers on pathological cough were revealed using hematoxylin and eosin staining, immunohistochemistry, and western blotting. RESULTS: The active components of P. notoginseng flowers were primarily protopanaxadiol-type saponins, among which 20S-ginsenoside Rh2 had the highest content (51.46 mg/g). In the mouse model, P. notoginseng flowers exhibited antitussive effects comparable to those of pentoxyverine citrate. Although its main saponin component, 20S-ginsenoside Rh2, showed slightly weaker effects, it still demonstrated concentration-dependent inhibition of channel activity. The whole-cell patch-clamp technique and virtual molecular docking showed that Rh2 might exert its effects by directly binding to the NaV1.7 and TRPV1 channels. In the guinea pig model, P. notoginseng flowers and their saponin components not only reduced cough frequency and prolonged the latency period before cough onset, but also significantly inhibited tracheal and pulmonary inflammation and the overexpression of TRPV1. CONCLUSIONS: 20S-Ginsenoside Rh2, the major bioactive saponin in P. notoginseng flowers, exhibits potent antitussive effects. The potential mechanism of action of 20S-Ginsenoside Rh2 in the treatment of cough may involve inhibiting NaV1.7 and TRPV1 channel currents through direct binding to core protein active sites and downregulating TRPV1 expression.


Assuntos
Antitussígenos , Tosse , Regulação para Baixo , Flores , Ginsenosídeos , Canal de Sódio Disparado por Voltagem NAV1.7 , Panax notoginseng , Canais de Cátion TRPV , Animais , Canais de Cátion TRPV/metabolismo , Cobaias , Flores/química , Tosse/tratamento farmacológico , Ginsenosídeos/farmacologia , Antitussígenos/farmacologia , Masculino , Camundongos , Panax notoginseng/química , Regulação para Baixo/efeitos dos fármacos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Células HEK293 , Simulação de Acoplamento Molecular , Cricetulus , Modelos Animais de Doenças , Células CHO , Saponinas/farmacologia , Ovalbumina
3.
Drug Dev Res ; 85(6): e22258, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39253992

RESUMO

The 2-nitroimidazole based 99mTc-radiopharmaceuticals are widely explored for imaging tumor hypoxia. Radiopharmaceuticals for targeting hypoxia are often lipophilic and therefore, show significant uptake in liver and other vital organs. In this context, lipophilic radiopharmaceuticals with design features enabling faster clearance from liver may be more desirable. A dipicolylamine-NCS bifunctional chelator that could generate a thiourea-bridge up on conjugation to primary amine bearing molecule was used to synthesize a 2-nitroimidazole-dipicolyl amine ligand for radiolabeling with 99mTc(CO)3 core. Corresponding Re(CO)3-analogue was prepared to establish the structure of 2-nitroimidazole-99mTc(CO)3 complex prepared in trace level. The 2-nitroimidazole-99mTc(CO)3 complex showed a hypoxic to normoxic ratio of ~2.5 in CHO cells at 3 h. In vivo, the complex showed accumulation and retention in tumor with high tumor to blood and tumor to muscle ratio. The study demonstrated the utility of metabolizable thiourea-bridge in 2-nitroimidazole-99mTc(CO)3 complex in inducing faster clearance of the radiotracer from liver. The dipicolylamine-NCS bifunctional chelator reported herein can also be used for radiolabeling other class of target specific molecules with 99mTc(CO)3 core.


Assuntos
Compostos Radiofarmacêuticos , Tioureia , Hipóxia Tumoral , Animais , Compostos Radiofarmacêuticos/farmacocinética , Células CHO , Tioureia/análogos & derivados , Tioureia/farmacocinética , Tioureia/química , Cricetulus , Camundongos , Nitroimidazóis/farmacocinética , Nitroimidazóis/química , Compostos de Organotecnécio/farmacocinética , Compostos de Organotecnécio/química , Compostos de Organotecnécio/administração & dosagem , Distribuição Tecidual , Ácidos Picolínicos/farmacocinética , Ácidos Picolínicos/química , Humanos , Tecnécio
4.
Biochemistry (Mosc) ; 89(8): 1392-1401, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39245452

RESUMO

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5'-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.


Assuntos
Channelrhodopsins , Clorófitas , Clorófitas/genética , Clorófitas/metabolismo , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Células CHO , Cricetulus , Optogenética/métodos , Luz
5.
Sci Rep ; 14(1): 20885, 2024 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-39242794

RESUMO

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.


Assuntos
Membrana Celular , Sobrevivência Celular , Micro-Ondas , Membrana Celular/metabolismo , Animais , Humanos , Transfecção , Células CHO , Cricetulus
6.
Sci Rep ; 14(1): 20856, 2024 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-39242806

RESUMO

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.


Assuntos
Cricetulus , Proteínas Recombinantes , Células CHO , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Cricetinae , Proteína Vermelha Fluorescente , Meios de Cultura/química
7.
PLoS One ; 19(9): e0305312, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39259753

RESUMO

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and ß-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.


Assuntos
Quimiocinas CC , Cricetulus , Receptores CCR8 , Animais , Receptores CCR8/metabolismo , Receptores CCR8/genética , Humanos , Células CHO , Camundongos , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Cricetinae , Quimiocina CCL1/metabolismo , Movimento Celular , Linhagem Celular , Quimiocina CCL11/metabolismo , AMP Cíclico/metabolismo
8.
Virol J ; 21(1): 215, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261951

RESUMO

BACKGROUND: Dengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear. RESULTS: We exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50-60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding. CONCLUSIONS: This study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.


Assuntos
Cricetulus , Vírus da Dengue , Pinças Ópticas , Receptores de Dopamina D2 , Receptores de Dopamina D2/metabolismo , Vírus da Dengue/fisiologia , Vírus da Dengue/efeitos dos fármacos , Animais , Células CHO , Dengue/virologia , Ligação Proteica , Humanos , Ligação Viral/efeitos dos fármacos , Cricetinae , Antagonistas de Dopamina/farmacologia
9.
Oncol Rep ; 52(5)2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39219278

RESUMO

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti­CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody­drug conjugates and chimeric antigen receptor­T cell therapy. Anti-pan­CD44 mAbs, C44Mab­5 and C44Mab­46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti­pan­CD44 mAbs (5­mG2a and C44Mab­46­mG2a) to evaluate the antitumor activities against CD44­positive cells. Both 5­mG2a and C44Mab­46­mG2a recognized CD44s­overexpressed CHO­K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5­mG2a and C44Mab­46­mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5­mG2a and C44Mab­46­mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5­mG2a and C44Mab­46­mG2a could exert antitumor activities against CD44­positive cancers and be a promising therapeutic regimen for tumors.


Assuntos
Anticorpos Monoclonais , Cricetulus , Neoplasias Esofágicas , Receptores de Hialuronatos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Camundongos , Humanos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/patologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Células CHO , Proliferação de Células/efeitos dos fármacos , Feminino , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico
10.
Int J Mol Sci ; 25(17)2024 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-39273634

RESUMO

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.


Assuntos
Anticorpos Monoclonais , Cricetulus , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/química , Células CHO , Animais , Trastuzumab/química , Trastuzumab/análise , Humanos
11.
MAbs ; 16(1): 2400414, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39245969

RESUMO

During the development process of therapeutic monoclonal antibodies (mAbs), it is crucial to control (critical) quality attributes such as N-glycosylation influencing pharmacokinetics (PK) and Fc effector functions. Previous reports have shown that mAbs containing high-mannose N-glycans are cleared faster from blood circulation, leading to reduced half-lives. The high-mannose N-glycan content of mAbs can be influenced during the cell culture process by factors such as cell lines, process conditions, and media. Furthermore, mAbs have either one high mannose N-glycan (asymmetrical high-mannose glyco-pair) or two high mannose N-glycans (symmetrical high-mannose glyco-pair). The hypothesis that the mannose receptor (MR, CD206) accelerates clearance by facilitating their internalization and subsequent lysosomal degradation is widespread. However, the interaction between MR and mAbs has not been explicitly demonstrated. This study aimed to investigate this interaction, providing the first systematic demonstration of MR binding to the Fc region of mAbs with high-mannose N-glycans. Two novel analytical methods, MR surface plasmon resonance and MR affinity chromatography, were developed and applied to investigate the MR-mAb interaction. The interaction is found to be dependent on high-mannose content, but is independent of the mAb format or sequence. However, different glyco-pairs exhibited varying binding affinities to the MR, with the symmetrical high-mannose glyco-pair showing the strongest binding properties. These findings strengthen the hypothesis for the MR-mediated mAb interaction and contribute to a deeper understanding of the MR-mAb interaction, which could affect the criticality of high-mannose containing mAbs development strategies of IgG-based molecules and improve their PK profiles.


Assuntos
Anticorpos Monoclonais , Lectinas Tipo C , Receptor de Manose , Lectinas de Ligação a Manose , Manose , Polissacarídeos , Receptores de Superfície Celular , Polissacarídeos/metabolismo , Polissacarídeos/química , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Lectinas Tipo C/metabolismo , Manose/metabolismo , Manose/química , Humanos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/imunologia , Animais , Glicosilação , Cricetulus , Células CHO , Ressonância de Plasmônio de Superfície , Ligação Proteica
12.
J Chromatogr A ; 1735: 465281, 2024 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-39243589

RESUMO

Therapeutic formats derived from the monoclonal antibody structure have been gaining significant traction in the biopharmaceutical market. Being structurally similar to mAbs, most Fc-containing therapeutics exhibit product-related impurities in the form of aggregates, charge variants, fragments, and glycoforms, which are inherently challenging to remove. In this work, we developed a workflow that employed rapid resin screening in conjunction with an in silico tool to identify and rank orthogonally selective processes for the removal of product-related impurities from a Fc-containing therapeutic product. Linear salt gradient screens were performed at various pH conditions on a set of ion-exchange, multimodal ion-exchange, and hydrophobic interaction resins. Select fractions from the screening experiments were analyzed by three different analytical techniques to characterize aggregates, charge variants, fragments, and glycoforms. The retention database generated by the resin screens and subsequent impurity characterization were then processed by an in silico tool that generated and ranked all possible two-step resin sequences for the removal of product-related impurities. A highly-ranked process was then evaluated and refined at the bench-scale to develop a completely flowthrough two-step polishing process which resulted in complete removal of the Man5 glycoform and aggregate impurities with a 73% overall yield. The successful implementation of the in silico mediated workflow suggests the possibility of a platformable workflow that could facilitate polishing process development for a wide variety of mAb-based therapeutics.


Assuntos
Anticorpos Monoclonais , Simulação por Computador , Contaminação de Medicamentos , Fragmentos Fc das Imunoglobulinas , Fluxo de Trabalho , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Cricetulus , Interações Hidrofóbicas e Hidrofílicas , Células CHO , Animais
13.
Cells ; 13(17)2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39273052

RESUMO

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)2 was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR's strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines.


Assuntos
Apoptose , Cricetulus , Dissulfetos , Dobramento de Proteína , Células CHO , Animais , Dissulfetos/metabolismo , Dissulfetos/química , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Formação de Anticorpos , Anticorpos Monoclonais , Cricetinae , Survivina/metabolismo , Humanos , Retículo Endoplasmático/metabolismo , Estresse Oxidativo
14.
Int J Mol Sci ; 25(17)2024 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-39273139

RESUMO

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3-10-overexpressed CHO-K1 (CHO/CD44v3-10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3-10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3-10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.


Assuntos
Anticorpos Monoclonais , Carcinoma de Células Escamosas , Receptores de Hialuronatos , Neoplasias Bucais , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/imunologia , Camundongos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Humanos , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Células CHO , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Cricetulus , Antineoplásicos Imunológicos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos BALB C
15.
Sci Rep ; 14(1): 20239, 2024 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-39215123

RESUMO

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.


Assuntos
Contaminação de Medicamentos , Eletroforese Capilar , Espectrometria de Massas , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Contaminação de Medicamentos/prevenção & controle , Animais , Células CHO , Cricetulus , Anticorpos Monoclonais/química , Anticorpos Monoclonais/análise , Concentração de Íons de Hidrogênio , Catepsinas/análise , Reatores Biológicos
16.
Environ Sci Technol ; 58(35): 15816-15826, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39166926

RESUMO

Recently, seven dihalohydroxybenzonitriles (diHHBNs) have been determined as concerning nitrogenous aromatic disinfection byproducts (DBPs) in drinking water. Herein, eight new monohalohydroxybenzonitriles (monoHHBNs), including 3-chloro-2-hydroxybenzonitrile, 5-chloro-2-hydroxybenzonitrile, 3-chloro-4-hydroxybenzonitrile, 3-bromo-2-hydroxybenzonitrile, 5-bromo-2-hydroxybenzonitrile, 3-bromo-4-hydroxybenzonitrile, 5-iodo-2-hydroxybenzonitrile, and 3-iodo-4-hydroxybenzonitrile, were detected and identified in drinking water for the first time. Thereafter, the relative concentration-cytotoxicity contribution of each HHBN was calculated based on the acquired occurrence level and cytotoxicity data in this study, the genome-scale cytotoxicity mechanism was explored, and a quantitative structure-activity relationship (QSAR) model was developed. Results indicated that new monoHHBNs were present in drinking water at concentrations of 0.04-1.83 ng/L and exhibited higher cytotoxicity than some other monohalogenated aromatic DBPs. Notably, monoHHBNs showed concentration-cytotoxicity contribution comparable to diHHBNs, which have been previously identified as potential toxicity drivers in drinking water. Transcriptomic analysis revealed immunotoxicity and genotoxicity as dominant cytotoxicity mechanisms for HHBNs in Chinese hamster ovary (CHO-K1) cells, with potential carcinogenic effects. The QSAR model suggested oxidative stress and cellular uptake efficiency as important factors for their cytotoxicity, highlighting the importance of potential iodinated HHBNs in drinking water, such as 3,5-diiodo-2-hydroxybenzonitrile, for future studies. These findings are meaningful for better understanding the health risk and toxicological significance of HHBNs in drinking water.


Assuntos
Desinfecção , Água Potável , Água Potável/química , Animais , Poluentes Químicos da Água/toxicidade , Cricetulus , Células CHO , Desinfetantes/toxicidade , Nitrilas/toxicidade , Relação Quantitativa Estrutura-Atividade , Purificação da Água
17.
Eur J Pharmacol ; 981: 176880, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39128804

RESUMO

Ghrelin modulates several biological functions via selective activation of the growth hormone secretagogue receptor (GHSR). GHSR agonists may be useful for the treatment of anorexia and cachexia, while antagonists and inverse agonists may represent new drugs for the treatment of metabolic and substance use disorders. Thus, the identification and pharmacodynamic characterization of new GHSR ligands is of high interest. In the present work the label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of a panel of GHSR ligands. This includes the endogenous peptides ghrelin, desacyl-ghrelin and LEAP2(1-14). Among synthetic compounds, the agonists anamorelin and HM01, the antagonists HM04 and YIL-781, and the inverse agonist PF-05190457 have been tested, together with HM03, R011, and H1498 from patent literature. The DMR results have been compared to those obtained in parallel experiments with the calcium mobilization assay. Ghrelin, anamorelin, HM01, and HM03 behaved as potent full GHSR agonists. YIL-781 behaved as a partial GHSR agonist and R011 as antagonist in both the assays. LEAP2(1-14) resulted a GHSR inverse agonist in DMR but not in calcium mobilization assay. PF-05190457, HM04, and H1498 behaved as GHSR inverse agonists in DMR experiments, while they acted as antagonists in calcium mobilization studies. In conclusion, this study provided a systematic pharmacodynamic characterization of several GHSR ligands in two different pharmacological assays. It demonstrated that the DMR assay can be successfully used particularly to discriminate between antagonists and inverse agonists. This study may be useful for the selection of the most appropriate compounds to be used in future studies.


Assuntos
Cálcio , Grelina , Receptores de Grelina , Receptores de Grelina/agonistas , Receptores de Grelina/metabolismo , Receptores de Grelina/antagonistas & inibidores , Ligantes , Grelina/farmacologia , Grelina/metabolismo , Cálcio/metabolismo , Humanos , Animais , Cricetulus , Células CHO , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Hidrazinas , Piperidinas , Quinazolinonas
18.
J Ethnopharmacol ; 335: 118687, 2024 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-39128798

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana officinalis L., commonly known as "valerian", is a traditional herbal medicine distributed in the north temperate zones of America, Europe and Asia. In traditional Chinese medicine, valerian and its roots were used for the treatment of restlessness of the heart and mind, palpitation and insomnia caused by internal depression of emotions and moods. However, safety evaluation of valerian remains deeply unclear. AIM OF THE STUDY: This study aimed to evaluate the genotoxicity, 14-days acute oral toxicity test, 90-day subchronic oral toxicity test and teratogenicity test of aqueous extract of valerian root (AEVR). MATERIALS AND METHODS: The genotoxicity of AEVR was evaluated with bacterial reverse mutation, mouse erythrocyte micronucleus test and in vitro mammalian cell chromosome aberration test. In the 14-days acute toxicity study, Kunming mice were administered at a dosage of 96 g/kg body weigh by gavage. In the 90-day subchronic toxicity study, Sprague-Dawley rats received oral doses of 0, 3.5, 7 and 14 g/kg body weight of AEVR. In the teratogenicity study, pregnant Sprague-Dawley rats received a dose of 0, 3.5, 7 and 14 g/kg body weight of AEVR. RESULTS: AEVR did not show any genotoxicity based on the bacterial reverse mutation, mouse erythrocyte micronucleus test and in vitro mammalian cell chromosome aberration test. In the acute toxicity study, AEVR at a dose of 96 g/kg body weight did not cause death or abnormal behavior in male or female mice. In the subchronic toxicity study, at the doses of 0, 3.5, 7, 14 g/kg body weight, no dose-related effects on clinical observation, body weight, organ weight, hematology, serum biochemistry and urinalysis of AEVR were detected in male or female rats. Teratogenicity test shown that there were no significant toxicologically changes in embryonic formation, body weight of pregnant rats, external, skeletal and visceral examination observed in pregnant and fetal rats at the dosage of 0, 3.5, 7, 14 g/kg body weight. CONCLUSION: In vivo or in vitro assays demonstrated that AEVR does not exhibit genotoxicity. The LD50 of AEVR was greater than 96 g/kg body weight in both sex of mice according to acute oral toxicity study. Subchronic toxicity and teratogenicity tests showed that the no observed adverse effect level (NOAEL) of AEVR was no less than 14 g/kg body weight. This study established a non-toxic dose of AEVR, providing a foundation for the use of valerian as a new resource food in some countries and regions.


Assuntos
Testes de Mutagenicidade , Extratos Vegetais , Raízes de Plantas , Ratos Sprague-Dawley , Testes de Toxicidade Aguda , Testes de Toxicidade Subcrônica , Valeriana , Animais , Masculino , Feminino , Extratos Vegetais/toxicidade , Extratos Vegetais/administração & dosagem , Valeriana/química , Camundongos , Aberrações Cromossômicas , Ratos , Testes para Micronúcleos , Relação Dose-Resposta a Droga , Cricetulus , Gravidez , Células CHO , Animais não Endogâmicos
19.
J Pharmacol Sci ; 156(2): 77-81, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39179337

RESUMO

Dopamine (DA) D2 receptors (D2Rs) have 2 isoforms, a long form (D2L) and a short form (D2S). D2L is predominantly postsynaptic in the striatal medium spiny neurons and cholinergic interneurons. D2S is principally presynaptic autoreceptors in the nigrostriatal DA neurons. Recently, we demonstrated that L-3,4-dihydroxyphenylalanine (L-DOPA) augments D2L function through the coupling between D2L and GPR143, a receptor of L-DOPA that was originally identified as the gene product of ocular albinism 1. Here we show that GPR143 modifies the functions of D2L and D2S in an opposite manner. Haloperidol-induced catalepsy was attenuated in DA neuron-specific Gpr143 gene-deficient (Dat-cre;Gpr143flox/y) mice, compared with wild-type (Wt) mice. Haloperidol increased in vivo DA release from the dorsolateral striatum, and this increase was augmented in Gpr143-/y mice compared with Wt mice. A D2R agonist quinpirole-induced increase in the phosphorylation of GSK3ß(pGSK3ß(S9)) was enhanced in Chinese hamster ovary (CHO) cells coexpressing D2L and GPR143 compared with cells expressing D2L alone, while it was suppressed in cells coexpressing D2S and GPR143 compared with D2S alone, suggesting that GPR143 differentially modifies D2R functions depending on its isoforms of D2L and D2S.


Assuntos
Cricetulus , Dopamina , Haloperidol , Receptores de Dopamina D2 , Animais , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Haloperidol/farmacologia , Células CHO , Dopamina/metabolismo , Corpo Estriado/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Camundongos , Levodopa/farmacologia , Catalepsia/induzido quimicamente , Catalepsia/genética , Catalepsia/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Quimpirol/farmacologia , Neurônios Dopaminérgicos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo
20.
Cells ; 13(16)2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39195235

RESUMO

BACKGROUND: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. METHODS: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. RESULTS: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. CONCLUSIONS: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.


Assuntos
Membrana Celular , Mutação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Humanos , Membrana Celular/metabolismo , Células CHO , Cricetulus , Mutação/genética , Fosforilação , Domínios Proteicos , Multimerização Proteica , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
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