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The cytotoxic effects of red clover, blue false indigo, and red raspberry plant extracts were evaluated on CHO-K1 cells. The hormone-dependent CHO-K1 cells are ovarian cells derived from Chinese hamsters.Trifolium pratense(red clover) extracts were obtained from the blossoms and leaves of the red clover plant.Baptisia australis(blue false indigo) extracts were obtained from the roots, stems, and leaves of the blue false indigo plant.Rubus idaeus(red raspberry) extracts were prepared from the fruits of the red raspberry plant. Two methods, soxhlet and microwave assisted extractions, were utilized and evaluated for their effectiveness in producing phyto extracts. Methanol was the solvent used in both methods. In all experiments, the CHO-K1 cell line was exposed to the different extracts for a period of 48 hours. An MTS assay was performed to evaluate the effectiveness of the cytotoxic capabilities of each extract at different concentrations on the CHO-K1 cell line. After the collection of all the data, the 1:1 combination of red clover and red raspberry extracts, obtained via soxhlet extraction, yielded the most potent cytotoxic effects on the CHO-K1 cell line.&nbsp

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol–AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol–AmB structures at the cell surface, preventing AmB cytotoxicity. Electronic supplementary material The online version of this article (10.1007/s00018-019-03154-w) contains supplementary material, which is available to authorized users.

Made available in DSpace on 2019-10-06T15:35:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Made available in DSpace on 2019-10-06T15:35:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Most commercially available fluorescent dyes used for cellular images are toxic and their luminescence is often outshined by the intrinsic autofluorescence of the biological medium, the use of long emission lifetime inherent of the lanthanide compounds is a viable alternative. This paper reports the development of a luminescent hybrid based on silica nanoparticles decorated with Tb3+ complexes suitable for use in exploratory tests as biomarkers in the visualization of CHO-k1 cells by microscopy techniques. To achieve this aim, spherical/spheroidal (∼125 nm) functionalized silica particles were prepared by the sol-gel method and had their surface functionalized covalently with Schiff base ligands. Tb3+ Complexes were bonded onto the particles surface by a step-by-step methodology approach. By zeta potential, it was possible to investigate the particles surface charge after each synthesis step, reaching a negative value for the final hybrid, which showed intense luminescence in the green region, where the Tb3+ 5D4 → 7FJ transitions were observed in the emission spectrum. After the benzoic acid ligand (BzAc) addition, there was an intensification of Tb3+ emission, as well as an increase in the emission lifetime. Concerning the biological application of the material, the CHO-k1 cell viability was evaluated by the MTT method in the presence of the SiO2-[Tb(SB)(BzAc)y] hybrid, and high biocompatibility was observed. The absorption process of particles by the cells was investigated by TEM, suggesting an endocytosis mechanism as a probable uptake process. The confocal images indicated a green emission of the biomarker due to the Tb3+, distributed through the cytoplasm, mainly closely to the cell nuclei. Therefore, the silica particles decorated with Tb3+ complexes proved to be suitable to act as biomarkers in biological assays and cellular imaging. São Paulo State University (Unesp) School of Technology and Sciences São Paulo State University (Unesp) Institute of Biosciences Humanities and Exact Sciences São Paulo State University (Unesp) School of Technology and Sciences São Paulo State University (Unesp) Institute of Biosciences Humanities and Exact Sciences

Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

Ebolavirus causes severe hemorrhagic fever in humans and non-human primates. Entry of ebolavirus is mediated by the viral glycoprotein, GP; however, the required host factors have not been fully elucidated. A screen utilizing a recombinant Vesicular Stomatitis Virus (VSV) encoding Zaire ebolavirus GP identified four Chinese Hamster Ovary (CHO) cell lines resistant to GP-mediated viral entry. Susceptibility to vectors carrying SARS coronavirus S or VSV-G glycoproteins suggests that endocytic and processing pathways utilized by other viruses are intact in these cells. A cathepsin-activated form of the ebolaviral glycoprotein did not overcome the entry restriction, nor did expression of several host factors previously described as important for ebolavirus entry. Conversely, expression of the recently described ebolavirus host entry factor Niemann–Pick Type C1 (NPC1) restored infection. Resistant cells encode distinct mutations in the NPC1 gene, resulting in loss of protein expression. These studies reinforce the importance of NPC1 for ebolavirus entry.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:05:03Z No. of bitstreams: 1 S1415-47572010000100029.pdf: 82574 bytes, checksum: 878228800accf04126e289d24710e696 (MD5) Made available in DSpace on 2013-08-22T19:05:03Z (GMT). No. of bitstreams: 1 S1415-47572010000100029.pdf: 82574 bytes, checksum: 878228800accf04126e289d24710e696 (MD5) Previous issue date: 2010-01-01 Made available in DSpace on 2013-09-30T20:08:43Z (GMT). No. of bitstreams: 2 S1415-47572010000100029.pdf: 82574 bytes, checksum: 878228800accf04126e289d24710e696 (MD5) S1415-47572010000100029.pdf.txt: 27353 bytes, checksum: c37952b2887a8619a0df6baecde6cc46 (MD5) Previous issue date: 2010-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:54:50Z No. of bitstreams: 2 S1415-47572010000100029.pdf: 82574 bytes, checksum: 878228800accf04126e289d24710e696 (MD5) S1415-47572010000100029.pdf.txt: 27353 bytes, checksum: c37952b2887a8619a0df6baecde6cc46 (MD5) Made available in DSpace on 2014-05-20T13:54:50Z (GMT). No. of bitstreams: 2 S1415-47572010000100029.pdf: 82574 bytes, checksum: 878228800accf04126e289d24710e696 (MD5) S1415-47572010000100029.pdf.txt: 27353 bytes, checksum: c37952b2887a8619a0df6baecde6cc46 (MD5) Previous issue date: 2010-01-01 An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves. Universidade Federal do Espírito Santo (UFES) Departamento de Ciências Biológicas Universidade Estadual Paulista Júlio de Mesquita Filho Departamento de Biologia Universidade Estadual de Londrina (UEL) Departamento de Genética Universidade Federal do Espírito Santo (UFES) Departamento de Ciências Farmacêuticas Universidade Estadual Paulista Júlio de Mesquita Filho Departamento de Biologia

O Agaricus blazei Murrill ss. Heinemann (ABM), cogumelo comestível nativo do Brasil, tem sido utilizado na medicina popular no tratamento de inúmeras doenças, incluindo o câncer. O objetivo do presente estudo foi avaliar os efeitos do extrato ABM (0,4%) na clastogenicidade induzida pela exposição à radiação ultravioleta (UV), em culturas de células CHO-k1, pelo teste de aberração cromossômica. As células foram tratadas em diferentes condições (tratamento contínuo, pré-tratamento e pós-tratamento), associadas à indução de danos no DNA pela UV. A análise dos dados demonstrou que a UV e o ABM apresentaram atividade clastogênica. Nos protocolos de pré e pós-tratamento não foram evidenciados efeitos anticlastogênicos. No entanto, o protocolo de tratamento contínuo demonstrou efeito protetor com redução de danos de 86,1%. Os resultados não permitem inferir com clareza o tipo de mecanismo de ação do extrato de ABM, o qual poderia agir tanto por desmutagênese, quanto por bioantimutagênese. No entanto, é evidente o seu efeito na diminuição de danos causados por radiação não-ionizante, apesar de, em concentração muito elevada, apresentar atividade clastogênica.

The induction of the stress protein Grp75 in response to amino acid deprivation of Chinese Hamster Ovary cells was characterised using a specific monoclonal antibody. A 2-fold increase in the Grp75 protein content occurred over a period of 5–10 h after incubation of the cells in amino acid-free medium. A partial induction was obtained when either all non-essential amino acids or all essential amino acids were omitted from the medium indicating a broad-specificity response. Deletion of the single amino acids tryptophan, histidine or phenylalanine from otherwise complete medium also produced a partial induction of the protein. The increase in the level of Grp75 was completely blocked by cycloheximide, but only partially blocked by the inhibitors of mRNA synthesis actinomycin D and α-amanitin. A specific cDNA probe for Grp75 was generated by PCR and used to quantify mRNA levels. No increase in Grp75 mRNA was observed during the induction of the protein indicating that the primary regulation of Grp75 expression was not at the transcriptional level. These results contrast with the large increase in asparagine synthetase mRNA which has been shown to occur during amino acid deprivation, and indicate that cells respond to this form of stress by more than one mechanism.

The effect of culinary-medicinal Royal Sun Agaricus (Agaricus brasiliensis) hot water extract on methyl methane sulfonate (MMS) induced mutagenicity/genotoxity in Drosophila melanogaster was studied using a quick and broadly applicable in vivo assay, i.e., the wing somatic mutation and recombination test. We used 2(nd) instar larvae, trans-heterozygous for the third chromosome recessive markers, i.e., multiple wing hairs (mvh) and flare-3 [flr (3)], and fed them for 24 h with the aqueous extract of A. brasiliensis. For antigenotoxicity studies a 24-h pretreatment with the extract was done, followed by a 48-h treatment of the then 3(rd) instar larvae with MMS. The frequency of mutations of the wing blade changes (i.e., of the number of wing spots of different sizes) induced in somatic cells was determined as a parameter of genetic changes of the wing imaginal discs. The results showed that A. brasiliensis extract did not cause any genotoxic or mutagenic effects. No antigenotoxic and/or protective effect against the induction of mutations by MMS was observed. Instead, a possible enhanced mitotic recombination frequency by MMS was seen after pretreatment of the larvae with A. brasiliensis extract. Possible mechanisms of action are discussed. Ministry of Education and Science of the Republic of Serbia [173012, 173032]

To the Editor, Much attention has been focused on the attempt to undertake global analysis of protein expression and in particular to analyze the changes in protein expression associated with several processes such as differentiation and cell cycle progression. In general, cell differentiation— or proliferation—requires de novo protein synthesis and/or post-translational modification, in particular phosphorylation of proteins such as transcription factors. Consequently, there is a need to identify the proteins that are being modified, as well as to know when and where in the cell modifications occur. We focused our studies on the Chinese hamster ovary (CHO) mammalian tumor cell line, a good model to study the cell differentiation. It is known that cyclic 30,50monophosphate adenosine (cAMP) transforms CHO cells, in vitro: they lose characteristics associated with malignancy and adopt characteristics of normal fibroblasts (‘‘reverse transformation’’) [3, 7, 9]. Puck et al. [8–10] found that cAMP regulates the structure of the cell cytoskeleton, which in turn strongly influences many differentiation properties including the assumption of normal or cancerous behavior in mammalian cells. In a previous study, we analyzed how CHO-K1 cells display different nuclear protein contents when undergoing differentiation by cAMP [14]; our Mass Spectrometry (MS) investigation revealed a change in the composition of nuclear proteins associated to an inhibition of the protein expression with possible implications for the control of cell reverse transformation [14]. The mechanism of the transformation, however, is as yet obscure and need further exploration in all compartments of cell proteome which we have carried out with this study. Fundamental changes in properties of the cell membrane have indeed been reported but the molecular mechanisms generating these structural changes are largely unknown. The changes in the concentration of the different metabolites and the different protein composition associated with morphological changes observed are also largely unknown [8]. This study wants to explore more deeply the effect of cAMP differentiation on the proteome of CHO cells, considering that the changes in the levels of nuclear envelopeassociated proteins and chromatin-condensing proteins [14] constitutes only the first step toward developing an understanding of cellular mechanisms for controlling nuclear shape. Due to the complexity of eukaryotic proteomes, and the divergence of protein properties, it is beneficial to prepare standardized partial proteomes in order to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis [1]. The strategies used to prepare such a complex proteomic samples for MS analysis involve many steps. For protein extraction we chose to perform a subcellular fractionation to separate the whole cellular proteome in four fractions. In literature, there are several works in which this approach to proteome analysis is adopted [1, 6, 12, 13], but always to analyze the behavior of a restrict number of proteins, for example to investigate changes in subcellular localization of regulatory proteins impacted by experimental or disease parameters. R. Spera C. Nicolini Nanoworld Institute, Fondazione El.B.A. Nicolini, Largo Redaelli 7, Pradalunga, Bergamo, Italy e-mail: rspera@ibf.unige.it

The aim of this work was to evaluate the potential of hemolymph utilization as a culture medium supplement to cultivate the animal cell CHO-K1. For this purpose 1% v/v of hemolymph was added to DMEM medium containing 10% v/v of FBS and 1 or 4.5 g/L of glucose. The culture was grown in spinner flasks incubated in a 10% v/v CO2 environment, at 37ºC, with the Cytodex 1 microcarrier. Comparing the results obtained from the culture with hemolymph against those without hemolymph, a positive influence of the hemolymph was observed, as the experiment with hemolymph presented a 52% higher cell concentration and a higher productivity of up to 40%.Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB) é uma das grandes prioridades de pesquisa em desenvolvimento de processos com célula animal. O objetivo do presente trabalho foi realizar uma análise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da célula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37ºC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influência positiva da hemolinfa no cultivo, já que o ensaio com hemolinfa apresentou uma concentração máxima de células 52% maior e uma produtividade máxima de até 40% maior.

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIV-AIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional N-linked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 N-linked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation. Key words: CHO-K1, erythropoietin, glycosylation, MTT assay and TF-1 cell line.