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1.
Pharmacol Res Perspect ; 10(5): e01003, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36101495

RESUMO

α2-adrenoceptors, (α2A, α2B and α2C-subtypes), are Gi-coupled receptors. Central activation of brain α2A and α2C-adrenoceptors is the main site for α2-agonist mediated clinical responses in hypertension, ADHD, muscle spasm and ITU management of sedation, reduction in opiate requirements, nausea and delirium. However, despite having the same Gi-potency in functional assays, some α2-agonists also stimulate Gs-responses whilst others do not. This was investigated. Agonist responses to 49 different α-agonists were studied (CRE-gene transcription, cAMP, ERK1/2-phosphorylation and binding affinity) in CHO cells stably expressing the human α2A, α2B or α2C-adrenoceptor, enabling ligand intrinsic efficacy to be determined (binding KD /Gi-IC50 ). Ligands with high intrinsic efficacy (e.g., brimonidine and moxonidine at α2A) stimulated biphasic (Gi-Gs) concentration responses, however for ligands with low intrinsic efficacy (e.g., naphazoline), responses were monophasic (Gi-only). ERK1/2-phosphorylation responses appeared to be Gi-mediated. For Gs-mediated responses to be observed, both a system with high receptor reserve and high agonist intrinsic efficacy were required. From the Gi-mediated efficacy ratio, the degree of Gs-coupling could be predicted. The clinical relevance and precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy, remains to be determined. Comparison with α1 and ß1/ß2-adrenoceptors demonstrated subclass affinity selectivity for some compounds (e.g., α2:dexmedetomidine, α1:A61603) whilst e.g., oxymetazoline had high affinity for both α2A and α1A-subtypes, compared to all others. Some compounds had subclass selectivity due to selective intrinsic efficacy (e.g., α2:brimonidine, α1:methoxamine/etilefrine). A detailed knowledge of these agonist characteristics is vital for improving computer-based deep-learning and drug design.


Assuntos
Ligantes , Animais , Tartarato de Brimonidina , Células CHO , Cricetinae , Cricetulus , Humanos
2.
Phys Med Biol ; 67(18)2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36097336

RESUMO

Objective. To develop a new model (Mayo Clinic Florida microdosimetric kinetic model, MCF MKM) capable of accurately describing thein vitroclonogenic survival at low and high linear energy transfer (LET) using single-event microdosimetric spectra in a single target.Methodology. The MCF MKM is based on the 'post-processing average' implementation of the non-Poisson microdosimetric kinetic model and includes a novel expression to compute the particle-specific quadratic-dependence of the cell survival with respect to dose (ßof the linear-quadratic model). A new methodology toa prioricalculate the mean radius of the MCF MKM subnuclear domains is also introduced. Lineal energy spectra were simulated with the Particle and Heavy Ion Transport code System (PHITS) for1H,4He,12C,20Ne,40Ar,56Fe, and132Xe ions and used in combination with the MCF MKM to calculate the ion-specific LET-dependence of the relative biological effectiveness (RBE) for Chinese hamster lung fibroblasts (V79 cell line) and human salivary gland tumor cells (HSG cell line). The results were compared within vitrodata from the Particle Irradiation Data Ensemble (PIDE) andin silicoresults of different models. The possibility of performing experiment-specific predictions to explain the scatter in thein vitroRBE data was also investigated. Finally, a sensitivity analysis on the model parameters is also included.Main results. The RBE values predicted with the MCF MKM were found to be in good agreement with thein vitrodata for all tested conditions. Though all MCF MKM model parameters were determineda priori, the accuracy of the MCF MKM was found to be comparable or superior to that of other models. The model parameters determineda prioriwere in good agreement with the ones obtained by fitting all availablein vitrodata.Significance. The MCF MKM will be considered for implementation in cancer radiotherapy treatment planning with accelerated ions.


Assuntos
Benchmarking , Transferência Linear de Energia , Animais , Cricetinae , Cricetulus , Florida , Humanos , Cinética , Eficiência Biológica Relativa
3.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077460

RESUMO

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, -4, and -5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, -4, and -5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and -4 or TRPC1 and -5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, -4, and -5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.


Assuntos
Moléculas de Adesão de Célula Nervosa , Canais de Cátion TRPC , Animais , Células CHO , Cricetinae , Cricetulus , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPC/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1864(11): 184034, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35981654

RESUMO

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-µs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-µs pulse decreased to 57 ± 8 V/cm for a 100-µs burst of 100-ns, 0.25-MHz pulses (p < 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.


Assuntos
Eletroporação , Neurônios , Animais , Células CHO , Permeabilidade da Membrana Celular/fisiologia , Cricetinae , Cricetulus
5.
ACS Infect Dis ; 8(9): 1883-1893, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-35980012

RESUMO

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Antivirais , Células CHO , Cricetinae , Cricetulus , Fucose , Humanos , Imunoglobulina G , Ácido N-Acetilneuramínico , Glicoproteína da Espícula de Coronavírus
6.
Appl Microbiol Biotechnol ; 106(18): 6157-6167, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36038753

RESUMO

The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival-pathways such as the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT), mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and RAP1 pathways were observed, offering novel insights into how cell surface receptors and ligand-mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/survival receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior. KEY POINTS: • PI3K/AKT, MAPK, JAK-STAT, and RAP1 pathway receptors are enriched on the CHO cell surface and downstream pathways present on mRNA level. • Detected pathways indicate strong CHO survival and growth phenotypes. • Potential self-induction of surface receptors and respective ligands.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Animais , Células CHO , Cricetinae , Cricetulus , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética
7.
J Chromatogr A ; 1679: 463363, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35964464

RESUMO

α-1 antitrypsin (AAT) deficiency, a major risk factor for chronic obstructive pulmonary disease, is one of the most prevalent and fatal hereditary diseases. The rising demand of AAT poses a defined need for new processes of AAT manufacturing from recombinant sources. Commercial affinity adsorbents for AAT purification present the intrinsic limitations of protein ligands - chiefly, the high cost and the lability towards the proteases in the feedstocks and the cleaning-in-place utilized in biomanufacturing - which limit their application despite their high capacity and selectivity. This work presents the development of small peptide affinity ligands for the purification of AAT from Chinese hamster ovary (CHO) cell culture harvests. An ensemble of ligand candidates identified via library screening were conjugated on Toyopearl resin and evaluated via experimental and in silico AAT-binding studies. Initial ranking based on equilibrium binding capacity indicated WHAKKSKFG- (12.9 mg of AAT per mL of resin), WHAKKSHFG- (16.3 mg/mL), and KWKHSHKWG- (15.8 mg/mL) Toyopearl resins as top performing adsorbents. Notably, the fitting of adsorption data to Langmuir isotherms concurred with molecular docking and dynamics in returning values of dissociation constant (KD) between 1 - 10 µM. These peptide-based adsorbents were thus selected for AAT purification from CHO fluids, affording values of AAT binding capacity up to 13 gram per liter of resin, and product yield and purity up to 77% and 97%. WHAKKSHFG-Toyopearl resin maintained its purification activity upon 20 consecutive uses, demonstrating its potential for AAT manufacturing from recombinant sources.


Assuntos
Peptídeos , alfa 1-Antitripsina , Animais , Células CHO , Técnicas de Cultura de Células , Cromatografia de Afinidade , Cricetinae , Cricetulus , Ligantes , Simulação de Acoplamento Molecular
8.
Int J Mol Sci ; 23(16)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-36012438

RESUMO

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.


Assuntos
Proteínas Interatuantes com Canais de Kv , Canais de Potássio Shal , Animais , Cricetinae , Cricetulus , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo
9.
MAbs ; 14(1): 2111748, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36018829

RESUMO

Although several antibody fragments and antibody fragment-fusion proteins produced in Escherichia coli (E. coli) are approved as therapeutics for various human diseases, a full-length monoclonal or a bispecific antibody produced in E. coli has not yet been approved. The past decade witnessed substantial progress in expression of full-length antibodies in the E. coli cytoplasm and periplasm, as well as in cell-free expression systems. The equivalency of E. coli-produced aglycosylated antibodies and their mammalian cell-produced counterparts, with respect to biochemical and biophysical properties, including antigen binding, in vitro and in vivo serum stability, pharmacokinetics, and in vivo serum half-life, has been demonstrated. Extensive engineering of the Fc domain of aglycosylated antibodies enables recruitment of various effector functions, despite the lack of N-linked glycans. This review summarizes recent research, preclinical advancements, and clinical development of E. coli-produced aglycosylated therapeutic antibodies as monoclonal, bispecific, and antibody-drug conjugates for use in autoimmune, oncology, and immuno-oncology areas.Abbreviations: ADA Anti-drug antibody; ADCC Antibody-dependent cellular cytotoxicity; ADCP Antibody-dependent cellular phagocytosis; ADC Antibody-drug conjugate; aFc Aglycosylated Fc; AMD Age-related macular degeneration aTTP Acquired thrombotic thrombocytopenic purpura; BCMA B-cell maturation antigen; BLA Biologics license application; BsAb Bispecific antibody; C1q Complement protein C1q; CDC Complement-dependent cytotoxicity; CDCC Complement-dependent cellular cytotoxicity; CDCP Complement-dependent cellular phagocytosis; CEX Cation exchange chromatography; CFPS Cell-free protein expression; CHO Chinese Hamster Ovary; CH1-3 Constant heavy chain 1-3; CL Constant light chain; DLBCL Diffuse large B-cell lymphoma; DAR Drug antibody ratio; DC Dendritic cell; dsFv Disulfide-stabilized Fv; EU European Union; EGFR Epidermal growth factor receptor; E. coli Escherichia coli; EpCAM Epithelial cell adhesion molecule; Fab Fragment antigen binding; FACS Fluorescence activated cell sorting; Fc Fragment crystallizable; FcRn Neonatal Fc receptor; FcÉ£Rs Fc gamma receptors; FDA Food and Drug Administration; FL-IgG Full-length immunoglobulin; Fv Fragment variable; FolRαa Folate receptor alpha; gFc Glycosylated Fc; GM-CSF Granulocyte macrophage-colony stimulating factor; GPx7 Human peroxidase 7; HCL Hairy cell leukemia; HIV Human immunodeficiency virusl; HER2 Human epidermal growth factor receptor 2; HGF Hepatocyte growth factor; HIC Hydrophobic interaction chromatography; HLA Human leukocyte antigen; IBs Inclusion bodies; IgG1-4 Immunoglobulin 1-4; IP Intraperitoneal; ITC Isothermal titration calorimetry; ITP Immune thrombocytopenia; IV Intravenous; kDa Kilodalton; KiH Knob-into-Hole; mAb Monoclonal antibody; MAC Membrane-attack complex; mCRC Metastatic colorectal cancer; MM Multipl myeloma; MOA Mechanism of action; MS Mass spectrometry; MUC1 Mucin 1; MG Myasthenia gravis; NB Nanobody; NK Natural killer; nsAA Nonstandard amino acid; NSCLC Non-small cell lung cancer; P. aeruginosa Pseudomonas aeruginosa; PD-1 Programmed cell death 1; PD-L1 Programmed cell death-ligand 1; PDI Protein disulfide isomerase; PECS Periplasmic expression cytometric screening; PK Pharmacokinetics; P. pastoris Pichia pastoris; PTM Post-translational modification; Rg Radius of gyration; RA Rheumatoid arthritis; RT-PCR Reverse transcription polymerase chain reaction; SAXS Small angle X-ray scattering; scF Single chain Fv; SCLC Small cell lung cancer; SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC Size exclusion chromatography; SEED Strand-exchange engineered domain; sRNA Small regulatory RNA; SRP Signal recognition particle; T1/2 Half-life; Tagg Aggregation temperature; TCR T cell receptor; TDB T cell-dependent bispecific; TF Tissue factor; TIR Translation initiation region; Tm Melting temperature; TNBC Triple-negative breast cancer; TNF Tumor necrosis factor; TPO Thrombopoietin; VEGF Vascular endothelial growth factor; vH Variable heavy chain; vL Variable light chain; vWF von Willebrand factor; WT Wild type.


Assuntos
Anticorpos Biespecíficos , Carcinoma Pulmonar de Células não Pequenas , Proteínas de Escherichia coli , Neoplasias Pulmonares , Animais , Anticorpos Monoclonais , Células CHO , Complemento C1q , Cricetinae , Cricetulus , Enzimas de Restrição do DNA , Escherichia coli , Humanos , Fragmentos Fc das Imunoglobulinas , Recém-Nascido , Espalhamento a Baixo Ângulo , Fator A de Crescimento do Endotélio Vascular , Difração de Raios X
10.
Monoclon Antib Immunodiagn Immunother ; 41(4): 221-227, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917553

RESUMO

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.


Assuntos
Anticorpos Monoclonais , Células Endoteliais , Alanina , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos , Glicoproteínas de Membrana , Fatores de Transcrição
11.
Monoclon Antib Immunodiagn Immunother ; 41(4): 194-201, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917562

RESUMO

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.


Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Células Endoteliais , Glicoproteínas de Membrana , Fatores de Transcrição
12.
Monoclon Antib Immunodiagn Immunother ; 41(4): 214-220, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917564

RESUMO

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.


Assuntos
Anticorpos Monoclonais , Quimiocina CCL17 , Animais , Células CHO , Quimiocina CCL22 , Cricetinae , Cricetulus , Epitopos , Receptores CCR4
13.
J Therm Biol ; 108: 103303, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36031224

RESUMO

The thermoneutral zone (TNZ), one of the most well-recognized concepts of thermal physiology of homeothermic organisms, is observed to differ between animal species, and may be associated with energy expenditure. However, the characteristics of the TNZ of lactating females, the stage of life history with typically the highest energy demands, remain unclear. In this study, we examined body mass, metabolic rate, TNZ and body composition, and milk energy output, in striped hamsters (Cricetulus barabensis, mean body mass: 29.1 ± 4.4g ranging from 20.0 to 36.6g) at peak lactation, and in hamsters raising small, medium, and large litter sizes throughout lactation. There was a significant downward shift in the lower critical temperature (LCT) of the TNZ in lactating hamsters (TNZ = 22.5-35 °C), resulting in a wider TNZ compared to non-reproductive females (TNZ = 27.5-32.5 °C). At peak lactation, hamsters raising large litter sizes had a considerably lower LCT and a wider TNZ compared to hamsters raising medium and small sized litters, whose upper critical temperature of the TNZ remain fixed. Compared to virgin hamsters, hamsters at peak lactation consumed 2.5 times more food, and had significantly higher energy expenditure corresponding to a significantly higher resting metabolic rate and milk output to meet the requirements of their offspring, which increased with litter size. The organs with the highest oxygen consumption rates, such as the liver, kidneys, and digestive tracts, were considerably heavier in lactating hamsters, particular in those raising large litter sizes, compared to virgin hamsters. The data show that the increased energy expenditure during lactation induces a substantial downward shift of the LCT, consequently resulting in a wider TNZ. The morphological plasticity of organs with high energy requirements is likely involved in this TNZ shift.


Assuntos
Metabolismo Energético , Lactação , Animais , Metabolismo Basal , Cricetinae , Cricetulus , Feminino , Tamanho da Ninhada de Vivíparos , Gravidez
14.
Radiol Oncol ; 56(3): 326-335, 2022 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-35962956

RESUMO

BACKGROUND: Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 µs pulses and to investigate the effects of electric pulses on cisplatin structure. MATERIALS AND METHODS: Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. RESULTS: Nanosecond pulses equivalent to 8 × 100 µs pulses were established in vitro based on membrane permeabilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and accumulating cisplatin intracellularly as 8 × 100 µs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. CONCLUSIONS: Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 µs pulses.


Assuntos
Eletroquimioterapia , Animais , Células CHO , Sobrevivência Celular , Cisplatino/farmacologia , Cricetinae , Cricetulus , Eletroquimioterapia/métodos
15.
Biotechnol Lett ; 44(9): 1063-1072, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35918621

RESUMO

AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells. RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4-/--CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4-/--CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation. CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Células CHO , Sistemas CRISPR-Cas/genética , Cricetinae , Cricetulus , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética
16.
Chem Biol Interact ; 365: 110078, 2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-35940282

RESUMO

The problem of the efficient treatment of acute organophosphorus (OP) poisoning needs more efforts in the development of a versatile antidote, applicable for treatment of the injuries of both peripheral and central nervous systems. A series of N-H, N-methyl, N-butyl, and N-phenyl derivatives of benzhydroxamic (1a-1d), 3-methoxybenzhydroxamic (2a-2d), 4-methoxybenzhydroxamic (3a-3d) acids, and corresponding salycilhydroxamates (4a-4d) was prepared. Their predicted hydrophobicity (log P) was evaluated as regards to ВВВ score by the open access cheminformatics tools; prediction of the passive transport across the BBB was found by means on the parallel artificial membrane permeability assay (PAMPA). The data on reactivation capacity of human acetylcholinesterase (HssAChE) inhibited by GB, VX, and paraoxon was supported by molecular docking study on binding to the active site of the AChE, viability study against mammalian cells (Chinese hamster ovary CHO-K1), and biodegradability (Closed Bottle test OECD 301D). Among the studied compounds, N-butyl derivatives have better balanced combination of properties; among them, N-butylsalicylhydroxamic acid is most promising. The studied compounds demonstrate modest reactivation capacity; change of N-H by N-Me ensures the reactivation capacity in studied concentrations on all studied OP substrates; among N-butyl derivatives, the N-butylsalicylhydroxamic acid demonstrates most promising results within the series. The found regularities may lead to selection of perspective structures to complement current formulations for medical countermeasures against poisoning by organophosphorus toxicants.


Assuntos
Reativadores da Colinesterase , Intoxicação por Organofosfatos , Acetilcolinesterase/metabolismo , Animais , Antídotos/farmacologia , Células CHO , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Reativadores da Colinesterase/química , Reativadores da Colinesterase/farmacologia , Cricetinae , Cricetulus , Humanos , Simulação de Acoplamento Molecular , Oximas/química , Relação Estrutura-Atividade
17.
Artigo em Inglês | MEDLINE | ID: mdl-35964489

RESUMO

Short peptide biomimetic chromatography technology as a developing protein separation technology has huge potential for antibody purification. In this study, four tetrapeptide ligands (Ac-FYKH, Ac-YEHF, Ac-YFLH and Ac-FYHI) with high potential binding ability to antibody were selected for the optimal ligand to antibody purification. The results showed that Ac-YEHF-4FF resin had higher binding capacity and selectivity for hIgG among the four resins. And at pH 7.0 and 0.3 ml/min, the highest Q10%-hIgG of Ac-YEHF-4FF resin was 26.2 mg/ml resin while its Q10%-BSA was just 2.2 mg/ml resin. Further, Ac-YEHF-4FF resin was used to purify protein mixtures. By binding at pH 7.0 and being eluted at pH 5.0 and pH 4.0, Ac-YEHF-4FF resin was well used to separate hIgG from BSA containing feedstock, HSA containing feedstock and human serum with the purity and yield both more than 95 %. And the screened resin could also separate mAb from CHO cell culture supernatant with purity 94.3 % and yield 97.5 %. The adsorption and separation results of Ac-YEHF-4FF resin indicated that the goal of getting the efficacy of critical residues from protein A to biomimetic its structure and function could be achieved, which had great significance to the establishment and improvement of tetrapeptide biomimetic chromatography, and also provided a new method for the field of antibody separation and purification.


Assuntos
Biomimética , Imunoglobulina G , Adsorção , Animais , Células CHO , Cromatografia , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/metabolismo , Ligantes
18.
Drug Discov Ther ; 16(4): 148-153, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-36002309

RESUMO

Phenochalasin A, a unique phenol-containing cytochalasin produced by the marine-derived fungus Phomopsis sp. FT-0211, was originally discovered in a cell morphological assay of observing the inhibition of lipid droplet formation in mouse peritoneal macrophages. To investigate the mode of action and binding proteins, phenochalasin A was radio-labeled by 125I. Iodinated phenochalasin A exhibited the same biological activity as phenochalasin A. [125I]Phenochalasin A was found to be associated with an approximately 40 kDa protein, which was identified as G-actin. Furthermore, detail analyses of F-actin formation in Chinese hamster ovary cells (CHO-K1 cells) indicated that phenochalasin A (2 µM) caused elimination of F-actin formation on the apical site of the cells, suggesting that actin-oriented specific function(s) in cytoskeletal processes are affected by phenochalasin A.


Assuntos
Actinas , Gotículas Lipídicas , Actinas/análise , Actinas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Citocalasinas/metabolismo , Citocalasinas/farmacologia , Indóis , Radioisótopos do Iodo , Lactonas , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Macrófagos Peritoneais/química , Macrófagos Peritoneais/metabolismo , Camundongos , Fenóis
19.
Biomater Adv ; 137: 212819, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35929256

RESUMO

Nanotechnology has immensely advanced the field of cancer diagnostics and treatment by introducing potential delivery vehicles as carriers for drugs or therapeutic agents. In due course, mesoporous silica nanoparticles (MSNs) have emerged as excellent vehicles for delivering drugs, biomolecules, and biomaterials, attributed to their solid framework and porosity providing a higher surface area for decorating with various functional ligands. Recently, the metal tin (Sn) has gained huge importance in cancer research owing to its excellent cytotoxicity and ability to kill cancer cells. In the present work, we synthesized MSNs, conjugated them with organotin compounds, and characterized them using various physicochemical techniques. Subsequently, the biological evaluation of MSN (S1), MSN-MP (S2) and tin-conjugated MSNs (S3: MSN-MP-SnPh3) (MP = 3-mercaptopropyltriethoxysilane) revealed that these nanoconjugates induced cytotoxicity, necrosis, and apoptosis in MCF-7 cells. Moreover, these nanoconjugates exhibited anti-angiogenic properties as demonstrated in the chick embryo model. The increase of reactive oxygen species (ROS) was found as a one of the plausible mechanisms underlying cancer cell cytotoxicity induced by these nanoconjugates, encouraging their application for the treatment of cancer. The tin-conjugated MSNs demonstrated less toxicity to normal cells compared to cancer cells. Furthermore, the genotoxicity studies revealed the clastogenic and aneugenic effects of these nanoconjugates in CHO cells mostly at high concentrations. These interesting observations are behind the idea of developing tin-conjugated MSNs as prospective candidates for anticancer therapy.


Assuntos
Antineoplásicos , Dióxido de Silício , Animais , Antineoplásicos/farmacologia , Sobrevivência Celular , Embrião de Galinha , Cricetinae , Cricetulus , Portadores de Fármacos/química , Humanos , Nanoconjugados , Dióxido de Silício/química , Estanho/farmacologia
20.
J Chromatogr A ; 1679: 463385, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35933770

RESUMO

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.


Assuntos
Anticorpos Monoclonais , Proteína Estafilocócica A , Animais , Arginina , Células CHO , Cromatografia de Afinidade , Cricetinae , Cricetulus , Proteínas Recombinantes
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