ABSTRACT
SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.
RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.
Subject(s)
Animals , Carnosine/pharmacology , Fibroblasts/drug effects , Kidney/cytology , Superoxide Dismutase/drug effects , Vimentin/drug effects , Biological Assay , Cell Survival/drug effects , Fluorescent Antibody Technique , Cricetinae , Cell Culture Techniques , Energy MetabolismABSTRACT
Objective: To explore the immunogenicity and influencing factors of hepatitis B vaccination based on different vaccination schedules among chronic kidney disease (CKD) patients. Methods: CKD patients who participated in randomized controlled trials in four hospitals in Shanxi province and completed three doses of 20 µg vaccination (at months 0, 1 and 6) and four doses of 20 µg or 60 µg vaccination (at months 0, 1, 2, and 6) were surveyed from May 2019 to July 2020.According to the ratio of 1∶1∶1, 273 CKD patients were divided into 3 groups randomly. Quantification of the anti-hepatitis B surface antigen-antibody (anti-HBs) in serum samples was performed using chemiluminescent microparticle immunoassay at months 1 and 6 after the entire course of the vaccinations. The positive rate, high-level positive rate, geometric mean concentration (GMC) of anti-HBs, and the influencing factors were analyzed by χ2 tests, analysis of variance, unconditional logistic regression analysis. Results: A total of 273 CKD patitents were participants.The positive rates in the CKD patients with four doses of 20 µg vaccination (92.96%,66/71) or 60 µg vaccination (93.15%, 68/73) were higher than that in the CKD patients with three doses of 20 µg vaccination (81.69%, 58/71) at month one after the full course of the vaccinations (P<0.05). The GMCs of anti-HBs showed similar results (2 091.11 mIU/ml and 2 441.50 mIU/ml vs. 1 675.21 mIU/ml) (P<0.05). The positive rate was higher in the CKD patients with four doses of 60 µg vaccination (94.83%,55/58) than in those with three doses of 20 µg vaccination (78.79%,52/66) (P<0.05) at month six after the full course of the vaccinations. And the GMC of anti-HBs in the patients with four doses of 60 µg vaccination (824.28 mIU/ml) was significantly higher than those in the patients with 3 or 4 doses of 20 µg vaccination (639.74 mIU/ml and 755.53 mIU/ml) (P<0.05). After controlling the confounding factors, the positive rate in the CKD patients with four doses of 60 µg vaccination were 3.19 (95%CI: 1.02-9.96) and 5.32 (95%CI: 1.27-22.19) times higher than those in the patients with three doses of 20 µg vaccination at months 1 and 6 after the full course of the vaccinations, respectively. The positive rate in CKD patients without immune suppression or hormone therapy was 3.33 (95%CI: 1.26-8.80) and 4.78 (95%CI: 1.47-15.57) times higher than those in the patients with such therapy, respectively. Conclusions: Four doses of 20 µg or 60 µg hepatitis B vaccination could improve the immunogenicity in patients with CKD. And four doses of 60 µg vaccination might play a positive role in maintaining anti-HBs in this population. The immunogenicity in the CKD patients with immune suppression or hormone therapy was poor.
Subject(s)
Animals , Cricetinae , Humans , CHO Cells , Cricetulus , Follow-Up Studies , Hepatitis B/prevention & control , Hepatitis B Antibodies , Hepatitis B Surface Antigens , Hepatitis B Vaccines , Immunization, Secondary , Renal Insufficiency, Chronic , VaccinationABSTRACT
SUMMARY: Adipose tissue morphology of different fat tissue depots can be described using the number of adipocytes and cell surface of adipocytes. This study deals with characteristics and morphometric analysis of white and brown adipose tissue depots in healthy adult laboratory mice, hamsters and rats of both sexes. The number of unilocular adipocytes in white adipose tissue differs from one adipose tissue depot to another, with the largest number of adipocytes in mice and a similar number in hamsters and rats. The smallest surface area and the largest percentage of small unilocular adipocytes were found in mice. White adipose tissue in hamsters and rats was predominantly made out of a larger percentage of medium-sized adipocytes and a smaller percentage of small and medium-sized adipocytes. Uncoupling protein 1 positive multilocular adipocytes were found in classic brown adipose tissue depots with larger percentages in mice (93.20 %) and hamsters (91.30 %), while rats had a smaller percentage (78.10 %). In white and brown adipose tissue, significant differences between species and both sexes within the same species were found, indicating the influence of sexual dimorphism. The presented morphometric results could serve as a basis for further studies concerning experimental animal models of metabolic disorders and obesity.
RESUMEN: La morfología del tejido adiposo de diferentes depósitos de tejido graso se puede describir utilizando el número de adipocitos y la superficie celular de los adipocitos. Este estudio analiza las características y el análisis morfométrico de los depósitos de tejido adiposo blanco y marrón en ratones, hamsters y ratas de laboratorio, adultos sanos de ambos sexos. El número de adipocitos uniloculares en el tejido adiposo blanco difiere de un depósito de tejido adiposo a otro, con el mayor número de adipocitos en ratones y un número similar en hámsteres y ratas. La superficie más pequeña y el mayor porcentaje de adipocitos uniloculares pequeños se encontraron en ratones. El tejido adiposo blanco en hámsteres y ratas estaba compuesto predominantemente por un mayor porcentaje de adipocitos de tamaño mediano y un porcentaje menor de adipocitos de tamaño pequeño y mediano. Los adipocitos multiloculares positivos para la proteína desacopladora 1 se encontraron en depósitos de tejido adiposo marrón clásico con mayores porcentajes en ratones (93,20 %) y hámsters (91,30 %), mientras que las ratas tenían un porcentaje menor (78,10 %). En el tejido adiposo blanco y pardo se encontraron diferencias significativas entre especies y entre ambos sexos dentro de una misma especie, lo que indica la influencia del dimorfismo sexual. Los resultados morfométricos presentados podrían servir como base para futuros estudios sobre modelos animales experimentales de trastornos metabólicos y obesidad.
Subject(s)
Animals , Male , Female , Mice , Rats , Adipose Tissue, Brown/anatomy & histology , Subcutaneous Fat/anatomy & histology , Adipose Tissue, White/anatomy & histology , Viscera/anatomy & histology , Cricetinae , Sex Characteristics , Models, AnimalABSTRACT
High fat diet induced hyperlipidemia hamster model was used to explore the anti-hyperlipidemia effect of water extract of Moringa oleifera leaves( WEMOL). On this basis,the possible action mechanism was predicted by network pharmacology. Golden hamsters were randomly divided into normal diet group( NFD),high-fat diet group( HFD),simvastatin group,high dose group of WEMOL( HIWEMOL) and low dose group of WEMOL( LOWEMOL). The model was administered simultaneously for 66 days,during which the body weight changes of hamsters were recorded. At the end of the experiment,serum lipid level and serum transaminase level of golden hamsters in each group were detected,and the pathological changes of liver were observed by hematoxylin-eosin( HE) staining. The results showed that WEMOL could significantly decrease the serum total cholesterol( TC),total triglyceride( TG),low density lipoprotein cholesterol( LDL-c) levels,and reduce the lipid deposition in liver tissue,thus improving the hyperlipidemia of golden hamsters. According to the prediction of network pharmacology,219 targets of potential active components of M.oleifera leaves and 185 targets of water-soluble potential active components of M. oleifera leaves for the treatment of hyperlipidemia were obtained separately. The MCODE analysis was performed on the PPI network of 219 targets and 185 targets obtained above and got five and four clusters respectively. The signaling pathway analysis of clusters showed that among the common pathways,nonalcoholic fatty liver,insulin resistance,MAPK signaling pathway,estrogen signaling pathway,cell apoptosis and HIF-1 signaling pathway were associated with hyperlipidemia. In addition,the potential active components of M. oleifera leaves could also inhibit the metabolic inflammation of hyperlipidemia by modulating complement and coagulation cascades signaling pathway,and GSK3 B,F2,AKT1,RELA,SERPINE1 might be the key targets. The water-soluble potential active components of M. oliefera leaves could modulate lipid metabolism by modulating AMPK signaling pathway and JAK-STAT signaling pathway,with PIK3 CB,PIK3 CA,CASP3,AKT1 and BCL2 as the key targets. These results suggested that WEMOL had anti hyperlipidemia effect,and its mechanism might be related to the protein expression regulation of lipid metabolism,nonalcoholic fatty liver disease and atherosclerosis related signaling pathways.
Subject(s)
Animals , Cricetinae , Diet, High-Fat , Glycogen Synthase Kinase 3 , Hyperlipidemias/drug therapy , Liver , Moringa oleifera , Plant LeavesABSTRACT
OBJECTIVE@#To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As@*METHODS@#Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique. Four groups of recipient rats (n=6 in each) were treated with normal saline (control), As@*RESULTS@#Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As@*CONCLUSION@#Combination treatment with As
Subject(s)
Animals , Cricetinae , Rats , Arsenic Trioxide , Heart Transplantation , Heme Oxygenase-1/metabolism , Heterografts , Leflunomide , NF-E2-Related Factor 2/metabolism , Rats, Inbred Lew , Signal TransductionABSTRACT
Objective@#To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers.@*Methods@#Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types.@*Results@#A mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types.@*Conclusions@#The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.
Subject(s)
Animals , Cricetinae , Humans , Blotting, Western , Brain , Brain Chemistry , Electrophoresis, Polyacrylamide Gel , Neoplasms/chemistry , Prion Proteins/analysisABSTRACT
To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.
Subject(s)
Animals , Cricetinae , CHO Cells , Cricetulus , Enzymes/metabolism , Recombinant Proteins/geneticsABSTRACT
Prolactin (PRL) plays critical roles in regulation of biological functions with the binding of specific prolactin receptor (PRLR). Revealing the expression patterns of PRLR at different developmental stages is beneficial to better understand the role of PRL and its mechanism of action in striped hamsters. In this study, the cDNA sequence of PRLR (2866-base-pairs) was harvested from the pituitary of mature female striped hamsters (Cricetulus barabensis) that contains an 834-base-pair 5′-untranslated region (1-834 bp), a 1848-base-pair open reading frame (835-2682 bp), and a 184-base-pair 3′-untranslated region (2683-2866). The 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids. In the mature PRLR, two prolactin-binding motifs, 12 cysteines, and five potential Asn-linked glycosylation sites were detected. Our results showed that the PRLR mRNA quantity in the hypothalamus, pituitary, ovaries, or testis was developmental-stage-dependent, with the highest level at sub-adult stage and the lowest level at old stage. We also found that PRLR mRNAs were highest in pituitary, medium level in hypothalamus, and lowest in ovaries or testis. PRLR mRNAs were significantly higher in males than in females, except in the hypothalamus and pituitary from 7-week-old striped hamsters. Moreover, the PRLR mRNAs in the hypothalamus, pituitary, and ovaries or testis were positively correlated with the expression levels of GnRH in the hypothalamus. These results indicated that the PRLR has conserved domain in striped hamster, but also possesses specific character. PRLR has multiple biological functions including positively regulating reproduction in the striped hamster.
Subject(s)
Animals , Male , Female , Prolactin/genetics , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , Pituitary Gland/metabolism , Cricetinae , Sequence Analysis , DNA, Complementary/geneticsABSTRACT
Abstract Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.
Resumo A mucosite oral é uma complicação inflamatória comum em pacientes com câncer. Este estudo avaliou os marcadores histopatológicos, estereológicos e antioxidantes de Eucalyptus 2% na mucosite oral induzida em hamsters dourados machos. Neste estudo em animais, a mucosite oral foi induzida em 30 hamsters golden masculinos por 5-FU (60 mg / kg) nos dias 0, 5 e 10 do estudo. A bolsa da bochecha foi arranhada com uma agulha estéril uma vez ao dia nos dias 3 e 4. Nos dias 14 a 17, os grupos de gel de eucalipto a 2% e curativos à base de gel foram tratados e comparados com um grupo controle. Foram avaliados os escores histopatológicos e estereológicos e o conteúdo de malondialdeído na bolsa, bem como as atividades de glutationa e mieloperoxidase no tecido da bolsa. Os escores histopatológicos de mucosite foram menores no grupo de gel de eucalipto a 2% do que os do gel e do grupo controle (p <0,05). Além disso, um nível mais baixo de malondialdeído e maiores atividades de mieloperoxidase e glutationa foram detectadas no grupo tratado com eucalipto em comparação aos grupos à base de gel e controle (p <0,001). A espessura da mucosa e submucosa aumentou no grupo Eucalyptus. A densidade numérica do fibroblasto e a densidade do volume do colágeno aumentaram significativamente nos grupos tratados com eucalipto. Em conclusão, o uso do extrato hidroalcoólico de Eucalyptus pode estar associado a menor intensidade de mucosite oral, diminuição da concentração de malondialdeído, aumento da atividade de mieloperoxidase e glutationa, aumento do volume de mucosa e submucosa, aumento de fibroblastos e colágeno na mucosite oral induzida em hamsters dourados em consumo de 5 UF.
Subject(s)
Animals , Male , Stomatitis , Mucositis , Eucalyptus , Plant Extracts , Cricetinae , Mesocricetus , Fluorouracil , Mouth MucosaABSTRACT
Abstract The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.
Resumo O papel dos roedores como reservatórios de helmintos de importância para a saúde pública não é bem conhecido. O potencial zoonótico de Syphacia spp. foi confirmado; portanto, o estudo teve como objetivo estimar a ocorrência de nematóides oxiurídeos em pequenos roedores de pet shops e clubes de reprodução na Eslováquia. Amostras fecais de 586 roedores mantidos em 133 gaiolas foram coletadas entre 2016 e 2018 e examinadas pelo método de flotação de Faust. Foram detectadas quatro espécies de nematódeos oxiurídeos, Syphacia muris, S. obvelata, Aspiculuris tetraptera e Paraspidodera uncinata, A. tetraptera foi encontrado nas amostras fecais de todas as espécies de roedores incluídas nesta pesquisa. O número de gaiolas positivas variou de 5,4% em hamsters a 70,0% em camundongos. A prevalência de Syphacia muris foi maior nos gerbilos da Mongólia, onde até 75,0% das gaiolas foram positivas; S. obvelata foi encontrada em 26,7% das gaiolas com camundongos, 25,0% das gaiolas com gerbilos da Mongólia e 3,2% das gaiolas com ratos. A alta prevalência de Syphacia spp. em todas as espécies animais aponta o risco de infecção para os seres humanos. Animais oferecidos para venda estão frequentemente em contato próximo com seres humanos; portanto, eles devem ser regularmente testados quanto a parasitas e, então, efetivamente desparasitados.
Subject(s)
Animals , Oxyuriasis/veterinary , Oxyuroidea/isolation & purification , Rodent Diseases/parasitology , Feces/parasitology , Pets/parasitology , Neglected Diseases/veterinary , Oxyuriasis/diagnosis , Oxyuriasis/epidemiology , Oxyuroidea/classification , Rats/parasitology , Rodent Diseases/diagnosis , Rodent Diseases/epidemiology , Prevalence , Gerbillinae/parasitology , Cricetinae/parasitology , Slovakia/epidemiology , Pets/classification , Neglected Diseases/diagnosis , Neglected Diseases/epidemiology , Guinea Pigs/parasitology , Mice/parasitologyABSTRACT
BACKGROUND Visceral leishmaniasis (VL) is a tropical neglected disease with high associated rates of mortality. Several studies have highlighted the importance of the intestinal tract (IT) and gut microbiota (GM) in the host immunological defense. Data in the literature on parasite life cycle and host immune defense against VL are scarce regarding the effects of infection on the IT and GM. OBJECTIVES This study aimed to investigate changes observed in the colon of Leishmania infantum-infected hamsters, including alterations in the enteric nervous system (ENS) and GM (specifically, levels of bifidobacteria and lactobacilli). METHODS Male hamsters were inoculated with L. infantum and euthanised at four or eight months post-infection. Intestines were processed for histological analysis and GM analysis. Quantitative polymerase chain reaction (qPCR) was performed to quantify each group of bacteria: Bifidobacterium spp. (Bf) and Lactobacillus spp (LacB). FINDINGS Infected hamsters showed histoarchitectural loss in the colon wall, with increased thickness in the submucosa and the mucosa layer, as well as greater numbers of intraepithelial lymphocytes. Forms suggestive of amastigotes were seen inside mononuclear cells. L. infantum infection induced changes in ENS, as evidenced by increases in the area of colonic enteric ganglia. Despite the absence of changes in the levels of Bf and LacB during the course of infection, the relative abundance of these bacteria was associated with parasite load and histological alterations. MAIN CONCLUSIONS Our results indicate that L. infantum infection leads to important changes in the colon and suggest that bacteria in the GM play a protective role.
Subject(s)
Animals , Bifidobacterium , Leishmania infantum , Gastrointestinal Microbiome , Lactobacillus , Leishmaniasis, Visceral , Cricetinae , Parasite Load , Intestines/parasitologyABSTRACT
Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.
Subject(s)
Animals , Cricetinae , Batch Cell Culture Techniques , Bioreactors , Reference Standards , CHO Cells , Cell Count , Computer Simulation , Cricetulus , Industrial Microbiology , MethodsABSTRACT
In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Subject(s)
Animals , Cricetinae , Humans , Mice , Antibodies, Viral , Genetics , Allergy and Immunology , Cytomegalovirus , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Gene Expression , Immunoglobulin M , Allergy and Immunology , Protein Sorting Signals , Recombinant Fusion Proteins , Allergy and ImmunologyABSTRACT
BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.
Subject(s)
Animals , Female , Bacterial Proteins/administration & dosage , Transcription Factors/administration & dosage , Bacterial Vaccines/administration & dosage , Vaccines, DNA/administration & dosage , Leptospirosis/prevention & control , Antigens, Bacterial/administration & dosage , Bacterial Proteins/immunology , Transcription Factors/immunology , Bacterial Vaccines/immunology , Cricetinae , Fluorescent Antibody Technique, Indirect , Vaccines, DNA/immunology , Disease Models, Animal , Nanoparticles , Leptospira interrogans/immunology , Leptospirosis/immunology , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunologyABSTRACT
BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
Subject(s)
Animals , Cell Survival , Apoptosis , Cell Proliferation , Caspase 7/deficiency , Cricetulus , Cricetinae , CHO Cells , Caspase 7/geneticsABSTRACT
OBJECTIVE@#To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function.@*METHODS@#293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested.@*RESULTS@#293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen.@*CONCLUSION@#To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Subject(s)
Animals , Cricetinae , Humans , Amino Acid Motifs , CHO Cells , Cell Adhesion , Cricetulus , HEK293 Cells , Integrin beta3 , Genetics , Metabolism , Platelet Glycoprotein GPIIb-IIIa Complex , Genetics , Metabolism , Signal TransductionABSTRACT
Objective To evaluate the chemopreventive effects of 8-allyl garcinol on oral squamous cell carcinoma(OSCC).Methods OSCC cell line CAL27 were cultured and treated with different concentrations of garcinol or 8-allyl garcinol. Their effects on the biological behaviors of OSCC cell line CAL27 were measured by MTT assay,clony formation assay,scratch migration assay,and flow cytometry with Annexin V-FITC/PI staining assay. We established DMBA-induced hamster cheek pouch models of dysplasia. While the negative control group was not treated,the positive group was treated with 0.5% DMBA solution tropically to the left cheek pouch three times per week for three consecutive weeks. The other four groups received 0.5 mmol/L or 1.0 mmol/L garcinol or 8-allyl garcinol respectively three times within the following two weeks after DMBA treatment. Hamsters were sacrificed at the fifth week to obtain tissue samples of the left cheek pouch. The samples were examined by histopathology and BrdU immunohistochemisty.Results MTT assay showed that both garcinol and 8-allyl garcinol inhibited the proliferation of CAL27 cells in a concentration-and time-dependent manner. The half maximal inhibitory concentration(IC)of 8-allyl garcinol[(13.13±2.55)μmol/L] was significantly lower than garcinol[(32.20±3.24)μmol/L;t=8.008,P=0.001]. Comparing the two grougs of medicine in the same concentration,the inhibiting proliferation effects 8-allyl garcinol had significantly stronger effect in inhibiting proliferation than garcinol when the same dose was applied,and the difference was largest at the concentrations of 10(24 h:t=8.012,P=0.001;48 h:t=5.939,P=0.001;72 h:t=12.551,P=0.001)and 20 μmol/L(24 h:t=8.887,P=0.001;48 h:t=9.324,P=0.002;72 h:t=5.361,P=0.002). The clone formation assay showed the clone formation rates after the treatment with 20 μmol/L garcinol and 20 μmol/L 8-allyl garcinol were(44.1±0.4)% and(23.6±0.6)%,respectively,which were significantly lower than those after treatment with 10 μmol/L garcinol[(55.6±2.8)%;t=6.894,P=0.019] and 10 μmol/L 8-allyl garcinol[(31.0±0.6)%;t=15.556,P=0.001]. The inhibiting effects of 8-allyl garcinol at the concentrations of 10 μmol/L(t=14.682,P=0.003)and 20 μmol/L(t=51.514,P=0.001)were significantly stronger than garcinol.Scratch test showed the relative cell migration rates after treatment with 10 and 20 μmol/L garcinol for 12 hours were(16.00±4.55)%(t=3.139,P=0.026)and(3.00±3.16)%(t=6.608,P=0.001),respectively,which were lower than negative control [(30.33±7.64)%]. The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 12 hours were(16.25±3.86)%(t=3.245,P=0.023)and(6.00±2.65)%(t=5.214,P=0.006),respectively,which were also lower than negative control[(30.33±7.64)%]. In addition,the relative cell migration rates after treatment with 10 and 20 μmol/L garcinol for 24 hours were(23.75±4.57)%(t=4.718,P=0.005)and(5.75±1.50)%(t=10.432,P=0.001),respectively,which were lower than negative control[(45.33±7.64)%]. The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 24 hours were(23.50±2.38)%(t=5.529,P=0.003)and(11.67±2.31)%(t=7.308,P=0.002),respectively,which were also lower than negative control[(45.33±7.64)%]. Furthermore,the relative cell migration rate after treatment with 20 μmol/L garcinol for 24 hours was significantly lower than after treatment with 8-allyl garcinol(t=4.151,P=0.009). The apoptosis experiments showed that the early apoptosis rate of CAL27 cells was(5.00±0.10)% after treatment with 10 μmol/L garcinol,which was significantly higher than negative control[(1.57±0.21)%;F=70.950,P=0.001]. The early and late apoptosis rates of CAL27 cells were(5.90±0.78)%(t=39.384,P=0.001)and(9.73±1.67)%(t=10.101,P=0.001),respectively,after treatment with 20 μmol/L garcinol,which were also significantly higher than negative control. The early apoptosis rate of CAL27 cells was(4.63±1.16)% after treatment with 8-allyl garcinol,which was significantly higher than negative control(t=4.511,P=0.041). The effects of 8-allyl garcinol in promoting cell apoptosis were weaker than garcinol(10 μmol/L:t=5.982,P=0.004;20 μmol/L:t=8.578,P=0.001). The histopathological test also showed that the hyperplastic areas of oral mucosal epithelium in hamsters after treatment with 0.5 mmol/L garcinol(t=2.546,P=0.031),0.5 mmol/L 8-allyl garcinol(t=3.485,P=0.008),1.0 mmol/L garcinol(t=4.556,P=0.001),and 1.0 mmol/L 8-allyl garcinol(t=5.393,P=0.001)were significantly smaller than positive control. The dysplasia areas of oral mucosal epithelium in hamsters after treatment with 0.5 mmol/L 8-allyl garcinol(t=2.130,P=0.046),1.0 mmol/L garcinol(t=3.434,P=0.010),and 1.0 mmol/L 8-allyl garcinol(t=4.518,P=0.004)were also smaller than positive control;1.0 mmol/L garcinol group(t=2.793,P=0.023)and 1.0 mmol/L 8-allyl garcinol group(t=4.997,P=0.001)were smaller than 0.5 mmol/L garcinol treatment group. Immunohistochemical staining of BrdU showed that the BrdU-labeled indicators were significantly lower in negative control group(t=7.563,P=0.001),0.5 mmol/L garcinol(t=2.862,P=0.029),0.5 mmol/L 8-allyl garcinol(t=4.693,P=0.002),1.0 mmol/L garcinol(t=5.071,P=0.002),and 1.0 mmol/L 8-allyl garcinol(t=5.133,P=0.001)when compared with the positive control. The BrdU-labeled indicators in 0.5 mmol/L 8-allyl garcinol(t=3.724,P=0.007),1.0 mmol/L garcinol(t=7.000,P=0.001),and 1.0 mmol/L 8-allyl garcinol(t=4.413,P=0.003)were also significantly lower than in 0.5 mmol/L garcinol group.Conclusions 8-allyl garcinol could inhibit the proliferation and migration of OSCC cell line CAL27 and promotes apoptosis. It also has prominent inhibitory effects on DMBA-induced hamster cheek pouch dysplasia. However,the specific effects are slightly different from garcinol.
Subject(s)
Animals , Cricetinae , Apoptosis , Carcinoma, Squamous Cell , Cell Proliferation , Chemoprevention , Mouth Neoplasms , TerpenesABSTRACT
We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighbor-joining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.
Subject(s)
Adult , Animals , Cricetinae , Female , Humans , Body Size , DNA, Mitochondrial , Electron Transport Complex IV , Korea , Metacercariae , Ovary , Phylogeny , Saline Waters , Smegmamorpha , Thailand , Trees , WrestlingABSTRACT
The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.
Subject(s)
Animals , Cricetinae , Humans , Animals, Genetically Modified , CHO Cells , Cricetulus , Gene Expression , Introns , TransfectionABSTRACT
Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.