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1.
Biol Reprod ; 105(1): 258-266, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33783478

RESUMO

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.


Assuntos
Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Embrião de Mamíferos/efeitos dos fármacos , Temperatura Alta , Vitrificação , Animais , Crioprotetores/farmacologia , Etilenoglicol/farmacologia , Ficoll/farmacologia , Ratos , Análise de Célula Única , Sacarose/farmacologia
2.
Biochem Biophys Res Commun ; 529(4): 984-990, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32819609

RESUMO

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.


Assuntos
Dextranos/farmacologia , Ficoll/farmacologia , Mucina-5AC/metabolismo , Polietilenoglicóis/farmacologia , Processamento de Proteína Pós-Traducional , Soroalbumina Bovina/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Dextranos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ficoll/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Mucina-5AC/química , Peptídeos/síntese química , Peptídeos/metabolismo , Polietilenoglicóis/química , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soroalbumina Bovina/química , Uridina Difosfato N-Acetilgalactosamina/análogos & derivados , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/metabolismo
3.
Biochemistry ; 58(28): 3087-3096, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31251033

RESUMO

Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.


Assuntos
Ficoll/metabolismo , Mononucleotídeo de Flavina/metabolismo , Heme/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/fisiologia , Ficoll/farmacologia , Mononucleotídeo de Flavina/química , Humanos , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/farmacologia
4.
Eur Biophys J ; 48(8): 789-801, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31654117

RESUMO

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.


Assuntos
Peixes-Gato , Glutationa Transferase/química , Desnaturação Proteica/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ficoll/farmacologia , Fígado/enzimologia , Peso Molecular , Polietilenoglicóis/farmacologia , Redobramento de Proteína/efeitos dos fármacos , Soluções
5.
Cryobiology ; 81: 132-137, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29428531

RESUMO

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-µl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.


Assuntos
Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Vitrificação , Animais , Sobrevivência Celular/efeitos dos fármacos , Etilenoglicol/farmacologia , Feminino , Ficoll/farmacologia , Camundongos , Sacarose/farmacologia
6.
Biophys J ; 111(1): 178-84, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27410745

RESUMO

Actomyosin kinetics is usually studied in dilute solutions, which do not reflect conditions in the cytoplasm. In cells, myosin and actin work in a dense macromolecular environment. High concentrations of macromolecules dramatically reduce the amount of free space available for all solutes, which results in an effective increase of the solutes' chemical potential and protein stabilization. Moreover, in a crowded solution, the chemical potential depends on the size of the solute, with larger molecules experiencing a larger excluded volume than smaller ones. Therefore, since myosin interacts with two ligands of different sizes (actin and ATP), macromolecular crowding can modulate the kinetics of individual steps of the actomyosin ATPase cycle. To emulate the effect of crowding in cells, we studied actomyosin cycle reactions in the presence of a high-molecular-weight polymer, Ficoll70. We observed an increase in the maximum velocity of the actomyosin ATPase cycle, and our transient-kinetics experiments showed that virtually all individual steps of the actomyosin cycle were affected by the addition of Ficoll70. The observed effects of macromolecular crowding on the myosin-ligand interaction cannot be explained by the increase of a solute's chemical potential. A time-resolved Förster resonance energy transfer experiment confirmed that the myosin head assumes a more compact conformation in the presence of Ficoll70 than in a dilute solution. We conclude that the crowding-induced myosin conformational change plays a major role in the changed kinetics of actomyosin ATPase.


Assuntos
Actomiosina/metabolismo , Ficoll/farmacologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise/efeitos dos fármacos , Cinética
7.
Biochem Biophys Res Commun ; 459(1): 75-80, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25712523

RESUMO

Guanine rich sequences present in the promoter region of oncogenes could fold into G-quadruplexes and modulate transcription. Equilibrium between folding and unfolding of the quadruplexes in these regions play important role in disease processes. We have studied the effect of a putative anticancer agent chelerythrine on G-rich NHE III1 present in the promoter region of c-myc oncogene. We have demonstrated the ability of chelerythrine, a telomerase inhibitor, to block the hybridization of Pu27 with its complementary strand via folding it into a quadruplex structure. Calorimetry shows that the association of Pu27 with chelerythrine is primarily enthalpy driven with high binding affinity (∼10(5) M(-1)). The association does not lead to any major structural perturbation of Pu27. The resulting 2:1 complex has enhanced stability as compared to free Pu27. Another notable feature is that the presence of molecular crowding agent like ficoll 70 does not change the mode of recognition though the binding affinity decreases. We suggest that the anticancer activity of chelerythrine could be ascribed to its ability to stabilize the quadruplex structure in the c-myc promoter region thereby downregulating its transcription.


Assuntos
Benzofenantridinas/farmacologia , Genes myc , Regiões Promotoras Genéticas/efeitos dos fármacos , Benzofenantridinas/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Entropia , Ficoll/farmacologia , Quadruplex G , Terapia de Alvo Molecular
8.
Zygote ; 23(5): 785-94, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25255836

RESUMO

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Fertilidade/efeitos dos fármacos , Ficoll/farmacologia , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Acrossomo/efeitos dos fármacos , Animais , Membrana Celular/química , Criopreservação/métodos , Feminino , Masculino , Gravidez , Taxa de Gravidez , Coelhos , Reprodução/efeitos dos fármacos , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Sacarose/farmacologia , Edulcorantes/farmacologia
9.
Blood ; 119(17): 3966-74, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22411869

RESUMO

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.


Assuntos
Linfócitos B/citologia , Linfócitos B/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/fisiologia , Animais , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiotaxia , Ficoll/análogos & derivados , Ficoll/farmacologia , Citometria de Fluxo , Hematopoese/fisiologia , Imunização , Técnicas Imunoenzimáticas , Integrases/metabolismo , Camundongos , Camundongos Knockout , Trinitrobenzenos/farmacologia
10.
Cryobiology ; 68(1): 152-4, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24463092

RESUMO

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.


Assuntos
Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Fertilização/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Dimetil Sulfóxido/farmacologia , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Etilenoglicol/farmacologia , Feminino , Fertilização/fisiologia , Fertilização in vitro , Ficoll/farmacologia , Masculino , Oócitos/citologia , Oócitos/fisiologia , Concentração Osmolar , Sacarose/farmacologia , Vitrificação
11.
J Immunol ; 187(10): 5183-95, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22003198

RESUMO

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.


Assuntos
Antígenos T-Independentes/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Regulação para Baixo/imunologia , Inibidores do Crescimento/fisiologia , Imunoglobulina G/biossíntese , Receptor de Morte Celular Programada 1/fisiologia , Animais , Células Cultivadas , Ficoll/análogos & derivados , Ficoll/farmacologia , Haptenos/fisiologia , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Tempo , Trinitrobenzenos/farmacologia
12.
Eur J Immunol ; 41(12): 3506-12, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21932446

RESUMO

Rapid production of neutralizing antibody can be critical for limiting the spread of infection. Such early antibody results when B-cell blasts mature directly to plasmablasts without forming germinal centers. These extrafollicular responses can involve Ig class switch recombination (CSR), producing antibody that can readily disseminate through infected tissues. The present study identifies the differentiation stage where CSR occurs in an extrafollicular response induced by 4-hydroxy-3-nitrophenyl acetyl (NP) conjugated to Ficoll (NP-Ficoll). To do this, we took advantage of the antigen dose dependency of CSR in this response. Thus, while both 30 and 1 µg NP-Ficoll induce plasmablasts, only the higher antigen dose induces CSR. Activation-induce cytidine deaminase (AID) is critical for CSR and in keeping with this a proportion of NP-specific B-cell blasts induced by 30 µg NP-Ficoll express AID. None of the B blasts responding to the non-CSR-inducing 1 µg dose of NP-Ficoll express AID. We confirmed that CSR occurs in B blasts by demonstrating the presence of rearranged heavy-chain transcripts in B blasts in the 30 µg response. CSR in this extrafollicular response is confined to B blasts, because NP-specific plasmablasts, identified by expressing CD138 and Blimp-1, no longer express AID and cannot undergo CSR.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Plasmócitos/imunologia , Animais , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Ficoll/farmacologia , Imunoconjugados/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nitrofenóis/farmacologia , Fenilacetatos/farmacologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Sindecana-1/genética , Sindecana-1/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
13.
Cytotherapy ; 14(7): 791-801, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22494074

RESUMO

BACKGROUND AIMS: Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations. METHODS: Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining. RESULTS: Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium. CONCLUSIONS: These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.


Assuntos
Diferenciação Celular , Hemólise , Células-Tronco Mesenquimais , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/análise , Animais , Medula Óssea/metabolismo , Transplante de Medula Óssea , Técnicas de Cultura de Células , Ficoll/farmacologia , Citometria de Fluxo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos
14.
Blood ; 114(11): 2254-62, 2009 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-19605846

RESUMO

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NF kappaB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI(-/-) background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.


Assuntos
Linfócitos B/imunologia , Imunodeficiência de Variável Comum/imunologia , Mutação de Sentido Incorreto , Transdução de Sinais/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Substituição de Aminoácidos , Animais , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/metabolismo , Ficoll/análogos & derivados , Ficoll/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Trinitrobenzenos/farmacologia
15.
Cryobiology ; 63(3): 292-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22020192

RESUMO

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2 °C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1° C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze-thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.


Assuntos
Cartilagem/citologia , Condrócitos/citologia , Criopreservação/métodos , Crioprotetores/farmacologia , Bancos de Tecidos , Vitrificação , Animais , Automação Laboratorial , Cartilagem/efeitos dos fármacos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Espécies em Perigo de Extinção , Etilenoglicol/farmacologia , Feminino , Feto , Ficoll/farmacologia , Congelamento , Técnicas de Transferência Nuclear , Gravidez , Cultura Primária de Células , Sacarose/farmacologia , Sobrevivência de Tecidos/efeitos dos fármacos
16.
J Sci Food Agric ; 91(14): 2518-28, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21445855

RESUMO

BACKGROUND: Disaccharides are, in general, the first choice as formulation compounds when freeze-drying microorganisms. Although polysaccharides and other biopolymers are considered too large to stabilise and interact with cell components in the same beneficial way as disaccharides, polymers have been reported to support cell survival. In the present study we compare the efficiency of sucrose and the polymers Ficoll, hydroxyethylcellulose, hydroxypropylmethylcellulose and polyvinylalcohol to support the survival of three bacterial strains during freeze drying. The initial osmotic conditions were adjusted to be similar for all formulations. Formulation characterisation was used to interpret the impact that different compound properties had on cell survival. RESULTS: Despite differences in molecular size, both sucrose and the sucrose-based polymer Ficoll supported cell survival after freeze drying equally well. All formulations became amorphous upon dehydration. Scanning electron microscopy and X-ray diffraction data showed that the discerned differences in structure of the dry formulations had little impact on the survival rates. The capability of the polymers to support cell survival correlated with the surface activity of the polymers in a similar way for all investigated bacterial strains. CONCLUSION: Polymer-based formulations can support cell survival as effectively as disaccharides if formulation properties of importance for maintaining cell viability are identified and controlled.


Assuntos
Arthrobacter/efeitos dos fármacos , Crioprotetores/química , Crioprotetores/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Pseudomonas putida/efeitos dos fármacos , Sphingomonas/efeitos dos fármacos , Arthrobacter/citologia , Arthrobacter/isolamento & purificação , Varredura Diferencial de Calorimetria , Celulose/análogos & derivados , Celulose/química , Celulose/farmacologia , Celulose/ultraestrutura , Contagem de Colônia Microbiana , Ficoll/química , Ficoll/farmacologia , Ficoll/ultraestrutura , Liofilização , Interações Hidrofóbicas e Hidrofílicas , Derivados da Hipromelose , Metilcelulose/análogos & derivados , Metilcelulose/química , Metilcelulose/farmacologia , Microscopia Eletrônica de Varredura , Álcool de Polivinil/química , Álcool de Polivinil/farmacologia , Pseudomonas putida/citologia , Pseudomonas putida/isolamento & purificação , Sphingomonas/citologia , Sphingomonas/isolamento & purificação , Relação Estrutura-Atividade , Sacarose/química , Sacarose/farmacologia , Tensão Superficial , Tensoativos/química , Tensoativos/farmacologia , Temperatura de Transição , Difração de Raios X
17.
Biochemistry ; 49(31): 6519-30, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20593812

RESUMO

Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.


Assuntos
Citocromos c/química , Substâncias Macromoleculares/farmacologia , Estabilidade Proteica , Dicroísmo Circular , Simulação por Computador , Dextranos/farmacologia , Ficoll/farmacologia , Guanidina/farmacologia , Temperatura Alta , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos
18.
J Appl Microbiol ; 108(3): 1032-1040, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19735322

RESUMO

AIMS: To investigate the effect of freeze-dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose-based formulations and to examine the relationship between amorphous matrix stability and cell viability. METHODS AND RESULTS: The resistance to moisture-induced sucrose crystallization and effects on the glass transition temperature (Tg) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. CONCLUSIONS: Polymers can be used to enhance the stability of freeze-dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50 degrees below its Tg for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid in the rational formulation design and proper determination of storage conditions for freeze-dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.


Assuntos
Ficoll/farmacologia , Lactobacillus/fisiologia , Viabilidade Microbiana , Povidona/farmacologia , Varredura Diferencial de Calorimetria , Cristalização , Liofilização , Lactobacillus/efeitos dos fármacos , Sacarose/química , Temperatura de Transição , Água/química
19.
Proc Natl Acad Sci U S A ; 104(48): 18976-81, 2007 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-18024596

RESUMO

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue single-domain alpha/beta protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (DeltaT(m) of 20 degrees C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20 degrees C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hard-sphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central beta-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.


Assuntos
Apoproteínas/química , Flavodoxina/química , Dobramento de Proteína , Apoproteínas/efeitos dos fármacos , Soluções Tampão , Dicroísmo Circular , Simulação por Computador , Desulfovibrio desulfuricans/química , Ficoll/farmacologia , Flavodoxina/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Termodinâmica
20.
Theriogenology ; 148: 180-185, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31757484

RESUMO

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.


Assuntos
Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Equidae/embriologia , Ficoll/farmacologia , Preservação de Tecido/veterinária , Vitrificação/efeitos dos fármacos , Animais , Criopreservação/métodos , Crioprotetores/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Preservação de Tecido/métodos
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