Showing papers in "Cytotechnology in 2014"
TL;DR: It has been reported for the first time that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures.
Abstract: In this study, the cytotoxic, genotoxic/antigenotoxic and antioxidant/oxidant activity of copaene (COP), a plant-derived tricyclic sesquiterpene, on human lymphocyte cultures (n = 5) was investigated. COP was added into culture tubes at various concentrations (0, 10, 25, 50, 100, 200 and 400 mg/L). While the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used for viability and cytotoxic evaluations, the micronucleus (MN) and sister chromatid exchange (SCE) assays were used for genetic evaluations. Moreover, total antioxidant capacity (TAC) and total oxidative status analysis were used for biochemical evaluations. According to LDH and MTT assays COP significantly reduced cell proliferation at high concentrations (200 and 400 mg/L). In addition, there was no significant increase (P < 0.05) in both SCE and MN frequencies of cultures treated with COP as compared to controls. We have also concluded that concentrations of COP of 50 and 100 mg/L increased TAC level when compared to the controls. In conclusion, in this study it has been reported for the first time that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures.
56 citations
TL;DR: The applicability of this lentivirus-based vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines is demonstrated.
Abstract: Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at −80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 108 transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 105 TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.
54 citations
TL;DR: It can be concluded that the consumption of GSPE or FO might be useful for preventing nephrotoxicity caused by cisplatin treatment, particularly oxidative stress, endogenous antioxidant defense system and DNA damage indicating their curative effect.
Abstract: Cisplatin (CP) is a chemotherapeutic drug used in treatment of malignancies. However, its clinical utility is limited by nephrotoxicity. The purpose of the present study was to investigate the protective role of grape seed proanthocyanidin extract (GSPE) (100 mg/kg/day) or fish oil (FO) (5 ml/kg/day) against cisplatin induced nephrotoxicity in terms of biochemical parameters, oxidative stress and DNA damage. CP nephrotoxiciy is manifested by increased levels of serum creatinine, urea and uric acid, accompanied by their decrease in urine. Na, K and Ca levels were altered in both serum and urine. In addition, cisplatin caused a decrease in renal GSH, SH-group, SOD, GST, and Na–K–ATPase levels. However the levels of MDA, H2O2 and NO were increased. Also, we assessed the renal genotoxic potential of cisplatin as manifested by an increase in the tail length of DNA, tail intensity (DNA %) and tail moment. On the other hand, administration of GSPE or FO pre-cisplatin treatment ameliorated the current changes in most of the above tested parameters, particularly oxidative stress, endogenous antioxidant defense system and DNA damage indicating their curative effect. Thus, it can be concluded that the consumption of GSPE or FO might be useful for preventing nephrotoxicity caused by cisplatin treatment.
51 citations
TL;DR: In this paper, random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) was used for the molecular characterization of Dicentrarchuslabrax embryonic cells (DLEC) as a possible tool to detect DNA alterations in environmental genotoxic studies.
Abstract: Continuous cell lines could provide an important tool for studying epidemiology, toxicology, cellular physiology and the host–pathogen interactions. Random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) was used for the molecular characterization of Dicentrarchuslabrax embryonic cells (DLEC) as a possible tool to detect DNA alterations in environmental genotoxic studies. We studied the DNA pattern of the DLEC fish cell line, a fibroblast-like cell line derived from European sea bass. From a total of 15 primers only six showed good discriminatory power for the amplification process on DNA samples collected from cells by three different methods (organic extraction, salting-out method and chelating agent extraction). The results obtained show that the cell line chosen for this study could be used as a possible tool for the detection of potential genotoxicity of numerous chemical compounds.
51 citations
TL;DR: The present findings demonstrated that CVC could be a source of antioxidant and chemopreventive activities to be studied on cancer diseases.
Abstract: Carvacrol (CVC) is a phenolic monoterpene present in many essential oils of medicinal and aromatic plants and has attracted attention because of its beneficial biological activities. To date, although various biological activities of CVC have been demonstrated, its neurotoxicity on cultured primary rat neurons and N2a neuroblastoma cells has never been explored. Therefore, in this present study, we aimed to describe in vitro antiproliferative and/or cytotoxic properties (by 3-(4,5 dimetylthiazol -2-yl)-2,5 diphenlytetrazolium bromide (MTT) test), genotoxic damage potentials (by single cell gel electrophoresis (SCGE) or Comet assay) and antioxidant activities (by total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis) of CVC in vitro. Dose (0–400 mg/L) dependent effects of CVC were tested on both cultured primary rat neurons and N2a neuroblastoma cells. Statistical analysis of MTT assay results indicated significant (p 0.05). In addition, our results indicated that 10, 25 and 50 mg/L of CVC treatment caused increases of TAC levels in cultured primary rat neurons but not in the N2a cell line. However, CVC treatments led to increases of TOS levels in cultured primary rat neurons at only 400 mg/L while they led to increases of TOS levels in N2a neuroblastoma cells at 200 and 400 mg/L. The present findings demonstrated that CVC could be a source of antioxidant and chemopreventive activities to be studied on cancer diseases.
46 citations
TL;DR: YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.
Abstract: Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.
44 citations
TL;DR: It is suggested that both calendula and thyme extracts had anti-genotoxic effects due to their higher content of total phenolic compounds.
Abstract: The aims of the current work were to evaluate the hepatoprotective effect of calendula flowers and/or thyme leave extracts on aflatoxins (AFs)-induced oxidative stress, genotoxicity and alteration of p53 bax and bcl2 gene expressions. Eighty male Sprague–Dawley rats were divided into eight equal groups including: the control group, the group fed AFs-contaminated diet (2.5 mg/kg diet) for 5 weeks, the groups treated orally with thyme and/or calendula extract (0.5 g/kg b.w) for 6 weeks and the groups pretreated orally with thyme and/or calendula extract 1 week before and during AFs treatment for further 5 weeks. Blood, liver and bone marrow samples were collected for biochemical analysis, gene expression, DNA fragmentation and micronucleus assay. The results showed that AFs induced significant alterations in oxidative stress markers, increased serum AFP and inflammatory cytokine, percentage of DNA fragmentation, the expression of pro-apoptotic gene p53 and bax accompanied with a decrease in the expression of bcl2. Animals treated with the extracts 1 week before AFs treatment showed a significant decrease in oxidative damage markers, micronucleated cells, DNA fragmentation and modulation of the expression of pro-apoptotic genes. These results suggested that both calendula and thyme extracts had anti-genotoxic effects due to their higher content of total phenolic compounds.
44 citations
TL;DR: It was determined that carvacrol has at least a partially protective role on liver enzymes in comparison with diabetic control rats, and there were no significant differences in serum insulin levels, food-water intake values and body weight changes.
Abstract: Little is known about the protective effects of carvacrol on the symptoms of streptozotocin induced diabetes in rats. Hence, this present study was designed to evaluate the protective effect of the strong antioxidant, carvacrol, on the symptoms of streptozotocin induced diabetes in rats. Carvacrol at the doses of 25 and 50 mg/kg body weight were orally administered to diabetic rats for a period of 7 days after the onset of diabetes. Food-water intake and body weight changes were daily recorded. Biochemical parameters such as serum glucose, insulin, total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were measured. Although treatment of diabetic rats with oral administration of carvacrol resulted in a slight reduction in serum glucose level and significant reduction in serum total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in comparison with diabetic control rats, there were no significant differences in serum insulin levels, food-water intake values and body weight changes. Despite the inadequacy of carvacrol on diabetes treatments, it was determined to have at least a partially protective role on liver enzymes.
43 citations
TL;DR: Bee pollen appears more potent in exerting an ameliorative effect and this effect was more pronounced in testis, on the basis of the present assays.
Abstract: Bee pollen and propolis are popular, traditional health foods The objective of the current study was to investigate the anti-mutagenic, anti-histopathologic and antioxidant effects among water extracts of Egyptian bee pollen (WEBP) and brown powder of water-soluble derivative propolis (WSDP) on cisplatin (CDDP) induced hepatic, renal, testicular and genotoxicity in male albino mice (Musmuscullus), in addition to their effects on the oxidant/antioxidant status in the tested organs Hepatic, renal and testicular dysfunctions were evaluated histologically; while genotoxicity and cytotoxicity were evaluated by the bone marrow chromosomal aberration assay and mitotic index, respectively Moreover, oxidative stress was explored via determination of lipid peroxidation, catalase activity and the concentration of the reduced form of glutathione The treatment of mice with WEBP and WSDP at doses 140 and 84 mg/kg b wt/day, respectively for 14 days simultaneously with CDDP (28 mg/kg b wt) resulted in significant protection The positive control animals taken CDDP alone showed toxic histological and genetical manifestations (at P < 005) accompanied with an elevated content of peroxidized lipid and lowered catalase activity and glutathione concentration in the homogenate of liver, kidney and testis tissues (at P < 0001) These toxic side effects in all tested organs were greatly ablated with a significant reduction in lipid peroxidation level and elevation in catalase activity and glutathione concentration (P < 0001) when using both WEBP and WSDP On the basis of the present assays, Bee pollen appears more potent in exerting an ameliorative effect and this effect was more pronounced in testis
41 citations
TL;DR: For the first time this study proved the anticancer potential of SPME against human lung and breast cancer by inducing apoptosis through the modulation of Bcl-2 family proteins.
Abstract: Spondias pinnata, a commonly distributed tree in India, previously proven for various pharmacological properties and also reported for efficient anti-oxidant, free radical scavenging and iron chelating activity, continuing this, the present study is aimed to investigate the role of 70 % methanolic extract of S. pinnata bark (SPME) in promoting apoptosis in human lung adenocarcinoma cell line (A549) and human breast adenocarcinoma cell line (MCF-7). These two malignant cell lines and a normal cell line were treated with increasing concentrations of SPME and cell viability is calculated. SPME showed significant cytotoxicity to both A549 and MCF-7 cells with an IC50 value of 147.84 ± 3.74 and 149.34 ± 13.30 μg/ml, respectively, whereas, comparatively no cytotoxicity was found in normal human lung fibroblast cell line (WI-38): IC50 932.38 ± 84.44 μg/ml. Flow cytometric analysis and confocal microscopic studies confirmed that SPME is able to induce apoptosis in both malignant cell lines. Furthermore, immunoblot result proposed the pathway of apoptosis induction by increasing Bax/Bcl-2 ratio in both cell types, which results in the activation of the caspase-cascade and ultimately leads to the cleavage of Poly adeno ribose polymerase. For the first time this study proved the anticancer potential of SPME against human lung and breast cancer by inducing apoptosis through the modulation of Bcl-2 family proteins. This might take S. pinnata in light to investigate it for further development as therapeutic anticancer source.
39 citations
TL;DR: Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression, and proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.
Abstract: Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL−1 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These ‘nucleotide ratios’ changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.
TL;DR: It can be concluded that the tested extract exhibited a certain level of in vitro antioxidant, antimicrobial, genotoxic and anticancer activities.
Abstract: In this study, the antioxidant, antimicrobial, genotoxic and anticancer activities of Cetraria islandica methanol extract were determined by using free radical and superoxide anion scavenging activity, reducing power, determination of total phenolic compounds and flavonoid contents, broth microdilution minimal inhibitory concentration against five bacterial and five fungal species, cytokinesis block micronucleus (MN) assay on peripheral blood lymphocytes (PBLs) and the microculture tetrazolium test on FemX (human melanoma) and LS174 (human colon carcinoma) cell lines As a result of the study, we found that C islandica methanol extract exhibited moderate free-radical-scavenging activity with IC50 values 67838 μg/ml Moreover, the tested extract had effective reducing power and superoxide anion radical scavenging The minimal inhibitory concentration values against the tested microorganisms ranged from 0312 to 5 mg/ml The extract increased MN frequency in a dose dependent manner, but it was significant in higher tested concentrations (50, 100 and 200 μg/ml) No significant differences were observed between NDI values in all treatments and untreated PBLs In addition, the tested extract had strong anticancer activity towards both cell lines with IC50 values of 2268 and 3374 μg/ml It can be concluded that the tested extract exhibited a certain level of in vitro antioxidant, antimicrobial, genotoxic and anticancer activities
TL;DR: This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects.
Abstract: Cytotoxic and antimicrobial effects of Montivipera xanthina venom against LNCaP, MCF-7, HT-29, Saos-2, Hep3B, Vero cells and antimicrobial activity against selected bacterial and fungal species: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus epidermidis ATCC 12228, S. typhimirium CCM 5445, Proteus vulgaris ATCC 6957 and Candida albicans ATCC 10239 were studied for evaluating the potential medical benefit of this snake venom. Cytotoxicity of venom was determined using MTT assay. Snake venom cytotoxicity was expressed as the venom dose that killed 50 % of the cells (IC50). The antimicrobial activity of venom was studied by minimal inhibitory concentration (MIC) and disc diffusion assay. MIC was determined using broth dilution method. The estimated IC50 values of venom varied from 3.8 to 12.7 or from 1.9 to 7.2 μg/ml after treatment with crude venom for 24 or 48 h for LNCaP, MCF-7, HT-29 and Saos-2 cells. There was no observable cytotoxic effect on Hep3B and Vero cells. Venom exhibited the most potent activity against C. albicans (MIC, 7.8 μg/ml and minimal fungicidal concentration, 62.5 μg/ml) and S. aureus (MIC, 31.25 μg/ml). This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects.
TL;DR: It is found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments, which implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.
Abstract: The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.
TL;DR: This study demonstrated for the first time that G. glabra extracts provided increased resistance of DNA against CdCl2 induced genetic and oxidative damage in human lymphocytes so, the risk on target tissues of C dCl2 could be reduced and ensured early recovery from its toxicity.
Abstract: Cadmium is a modern environmental contaminant that is toxic and carcinogenic. Glycyrrhiza glabra is a traditional medicinal herb which grows in the various parts of the World. Recent studies demonstrated that G. glabra has antifungal, antimicrobial, antioxidant, and powerful antiinflammatory features. The purpose of this study was to investigate the genetic safety of extracts from G. glabra and its effects on cadmium (as CdCl2) induced genotoxicity. Therefore we evaluated the capability of G. glabra extract to inhibit the rate of micronucleus (MN), sister chromatid exchange (SCE) formations induced by CdCl2. Moreover, to assess the effects of G. glabra on cell viability and oxidative status, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and total antioxidant capacity (TAC) assays. Our results showed that there were significant increases (P < 0.05) in both SCE and MN frequencies of cultures treated with CdCl2 (5 ppm) as compared to controls. However, co-application of G. glabra extract (5, 10 and 20 ppm) and CdCl2 resulted in decreases of MN and SCE rates as compared to the group treated with CdCl2 alone. Again, the results of MTT and TAC assays clearly indicated dose dependent ameliorative effects of G. glabra extracts against CdCl2 toxicity. In conclusion, this study demonstrated for the first time that G. glabra extracts provided increased resistance of DNA against CdCl2 induced genetic and oxidative damage in human lymphocytes. So, the risk on target tissues of CdCl2 could be reduced and ensured early recovery from its toxicity.
TL;DR: Electroporation is proved to be the method of choice in transiently transfecting undifferentiated PC12 cells and it is demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.
Abstract: A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.
TL;DR: A bioprocess engineering strategy based on bioreactors and 3-D cultures is discussed in order to achieve the improved stem cell yield, function, and safety required for production under current good manufacturing practices.
Abstract: Stem cells, including mesenchymal stem cells and pluripotent stem cells, are becoming an indispensable tool for various biomedical applications including drug discovery, disease modeling, and tissue engineering. Bioprocess engineering, targeting large scale production, provides a platform to generate a controlled microenvironment that could potentially recreate the stem cell niche to promote stem cell proliferation or lineage-specific differentiation. This survey aims at defining the characteristics of stem cell populations currently in use and the present-day limits in their applications for therapeutic purposes. Furthermore, a bioprocess engineering strategy based on bioreactors and 3-D cultures is discussed in order to achieve the improved stem cell yield, function, and safety required for production under current good manufacturing practices.
TL;DR: In oxidative stress generated by hepatic ischemia–reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidants defence systems in the body.
Abstract: Little is known about the effective role of Hypericum perforatum on hepatic ischemia–reperfusion (I/R) injury in rats. Hence, albino rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. Hypericum perforatum extract (HPE) at the dose of 50 mg/kg body weight (HPE50) was intraperitonally injected as a single dose, 15 min prior to ischemia. Rats were sacrificed at the end of reperfusion period and then, biochemical investigations were made in serum and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) levels. At the same time alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were assayed in serum samples and compared statistically. While the ALT, AST, LDH activities and MDA levels were significantly increased, CAT and GPx activities significantly decreased in only I/R-induced control rats compared to normal control rats (p < 0.05). Treatment with HPE50 significantly decreased the ALT, AST, LDH activities and MDA levels, and markedly increased activities of CAT and GPx in tissue homogenates compared to I/R-induced rats without treatment–control group (p < 0.05). In oxidative stress generated by hepatic ischemia–reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidant defence systems in the body.
TL;DR: Investigation shows that O. cinnamomi has protective effects on ethanol-induced oxidative and mucosal damage and is effective in treating peptic ulcer disease and liver and kidney disorders induced by ethanol.
Abstract: Peptic ulcer disease is a gastrointestinal disorder defined by mucosal damage and free oxygen radicals associated with peptic ulcer and gastritis. Cinnamon is a traditional herb used for many diseases and it has also effects as an antioxidant, anti-inflammatory, antispasmodic and anti-ulcerative. Our research is based on oxidative stress and effects of Oleum cinnamomi on stomach, liver and kidney disorders induced by ethanol. In our experiment, 2–3 month old male Sprague–Dawley rats were used. One hour before the mucosal damage induced by 70 % ethanol, O. cinnamomi (2.5 ml/kg) was added into the groups. Gastric pH, analysis of gastric mucus and ulcer index were calculated from samples obtained from the stomach. Superoxide dismutase (SOD), malondialdehyde and catalase (CAT) levels were determined in stomach, liver and kidney homogenates and erythrocyte hemolysate. Histopathological examination of stomach, liver and kidney were determined with H&E staining. The non-treated ulcerative group showed higher scores than the control group which was treated with O. cinnamomi, when ulcer scores, gastric mucus and pH level of stomach are compared. Increased lipid peroxidation levels were observed in the liver, kidney and erythrocyte hemolysate. SOD activity was decreased in liver whereas increased in stomach of ethanol treated ulcerative groups. CAT levels were increased in stomach and liver of ethanol treated rats. Histopathological findings showed that ethanol treatment cause multiply organ damage such as stomach, liver and kidney injury. O. cinnamomi treatment protected these tissues from ethanol-induced damage. Consequently, the current investigation shows that O. cinnamomi has protective effects on ethanol-induced oxidative and mucosal damage.
TL;DR: It is suggested that bengkoang fiber extract has positive effects on the immune system in vitro and in vivo, and that BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells.
Abstract: Bengkoang (Pachyrhizus erosus (L.) Urban) is one of the most popular edible root vegetables in Indonesia. Bengkoang contains fairly large amounts of carbohydrates and crude fiber. The purpose of this research is to evaluate the immunomodulatory effect of the bengkoang fiber extract (BFE) in vitro and in vivo. BFE was prepared by heating the powder of bengkoang fiber suspended in distilled water at 121 °C for 20 min. BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells. In addition, production of IgM, IgG, and IgA by mouse primary splenocytes was facilitated by BFE in a dose-dependent manner. BFE also significantly facilitated production of both interleukin-5 and interleukin-10 by splenocytes. Immunoglobulin production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were significantly activated by oral administration of BFE to mice for 14 days. The serum immunoglobulin levels of IgG, IgM, and IgA were also significantly enhanced. Furthermore, cytokine production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were also facilitated by oral administration of BFE. These results suggest that BFE has positive effects on the immune system in vitro and in vivo.
TL;DR: The oldest to latest works published in major indices are reviewed to gather information within this article to review genotoxicologic and carcinogenic profile of the artificial sweetener aspartame.
Abstract: The objective of this article is to review genotoxicologic and carcinogenic profile of the artificial sweetener aspartame. Aspartame is a synthetic dipeptide, nearly 180–200 times sweeter than sucrose. It is the most widely used artificial sweetener especially in carbonated and powdered soft drinks, beverages, drugs and hygiene products. There is a discussion ongoing for many years whether aspartame posses genotoxic and carcinogenic risk for humans. This question led to many studies to specify the adverse effects of aspartame. Therefore, we aimed to review the oldest to latest works published in major indices to gather information within this article. With respect to published data, genotoxicity and carcinogenicity of aspartame is still confusing. So, consumers should be aware of the potential side effects of aspartame before they consume it.
TL;DR: It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.
Abstract: The purpose of the present study was to investigate the osteoprotective effects of soybean oil (SbO) and sesame oil (SO) in ovarictomized (OVX) rats. The results indicated that the OVX rats exhibited a significant decrease in Ca and P level in both serum and bone, the activities of the antioxidant enzymes SOD and CAT and the antioxidant biomarker GSH accompanied with a marked increase in the oxidative stress markers MDA and PC, the inflammatory indices (TNF-α, CRP levels, WBCs counts and ACP activity) in, both, bone and serum. Supplementating the diet of the OVX rats with SbO (15 % w/w) or SO (10 % w/w) for 2 months to resulted in modulation of the alterations in all tested parameters and succeeded to restore minerals, antioxidant enzymes, antioxidant biomarkers, oxidative stress markers, inflammatory indices, and WBCs counts. It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.
TL;DR: It is demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in B MSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.
Abstract: Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72 h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.
TL;DR: Results of this study showed that the PE fraction of A. lancea inhibited the growth of BGC-823 and SGC-7901 cells in a dose- and time-dependent manner.
Abstract: The purpose of this study was to investigate the pharmacological effect of fraction of Atractylodes lancea (Thunb.) DC. (A. lancea) extract. In this study, we isolated different polarity fractions, including petroleum ether (PE), ethyl acetate, n-butanol, and the remaining H2O fractions from the water extract of A. lancea. The antigastric cancer properties of the different fractions in BGC-823 and SGC-7901 cells were evaluated. Apoptotic cells were treated with PE fraction and stained with Hoechst 33342 and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide. The cell cycle was analyzed via flow cytometry. The main compounds of PE fraction were determined by HPLC–ESI–MS. Results of this study showed that the PE fraction of A. lancea inhibited the growth of BGC-823 and SGC-7901 cells in a dose- and time-dependent manner. The morphological and mitochondrial transmembrane potential changes suggested that the cells showed preliminary apoptosis characteristics after treatment with the three different polarities. The main compounds of PE fraction include two sesquiterpene compounds: eudesm-4(15),7-diene-9α,11-diol and eudesm-4(15)-ene-7a,11-diol; three sesquiterpene lactone compounds: atractylenolid I, atractylenolid III and 3-β-acetyl-atractylenolid III and one polyacetylenic compound: 4,6,12-tetradecatriene-8,10-diyne-1,3,14-triol.
TL;DR: Bisplatin + bilberry administration seems to reduce the cisplatin induced ovarian toxicity thus it alleviates free radical damage, but it dose not protect completely rat ovary tissues.
Abstract: Cisplatin is one of the most effective chemotherapeutic agents but injury may occur at higher doses. The aim of this study was to investigate the effect of bilberry on cisplatin induced toxic effects in rat ovary. Twenty-one female Wistar–Albino rats were utilized to form three groups: In group 1 (control group), each rat received intraperitoneal injection of 1 mL of 0.9 % NaCl saline solution during 10-days. In group 2 (cisplatin group), a single dose of 7.5 mg/kg b.w. cisplatin was given. In group 3 (cisplatin + bilberry group), a single dose of 7.5 mg/kg cisplatin and bilberry at 200 mg/kg b.w. were given for 10 days. Ovaries were surgically removed in all groups and prepared for biochemical and light microscopic investigations at the examination times. Malondialdehyde (MDA) levels and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) of tissue samples were measured. Histopathological damages in cisplatin administrated rats were seen such as severe edema, vascular congestion, hemorrhage and follicular degeneration in the ovary tissue. Moderate pathological alterations were observed in rats treated with bilberry plus cisplatin. Cisplatin administration significantly increased MDA production and decreased SOD, CAT, GPx and GST activities in the ovarian tissue when compared to the control group (p < 0.05). Cisplatin + bilberry administration increased antioxidant enzymes activities and reduced MDA levels. Bilberry administration seems to reduce the cisplatin induced ovarian toxicity thus it alleviates free radical damage. But it dose not protect completely rat ovary tissues.
TL;DR: To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCS.
Abstract: Spermatogonial stem cells (SSCs) are the only type of cells that transmit genes to the subsequent generations. The proliferation, cultivation and identification of SSCs in vitro are critical to understanding of male infertility, genetic resources and conservation of endangered species. To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCs. The testes of 6–8 d mouse were harvested and digested by two-step enzyme digestion method. The SSCs and Sertoli cells were separated by differential plating. Then the SSCs were identified by alkaline phosphatase staining, RT-PCR and indirect immunofluorescence cell analysis. The cellular proliferation capacity was measured by methyl thiazolyl tetrazolium assay. The results showed that addition of 20 and 40 ng/ml of GDNF could strongly promote growth of mouse SSCs (p 0.05). However, the combination of 20 ng/ml GDNF and 1,000 U/ml LIF could significantly enhance the invitro proliferation of mouse SSCs (p < 0.05), and the OD490 value was 0.696 at day 5 of culture when the density of SSCs was 5–10 × 104 cells/ml.
TL;DR: Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways.
Abstract: Venom from the sea anemone, Heteractis magnifica, has multiple biological effects including, cytotoxic, cytolytic and hemolytic activities. In this study, cytotoxicity induced by H. magnifica venom was investigated using the crystal violet assay on human breast cancer T47D and MCF7 cell lines and normal human breast 184B5 cell line. Apoptosis was also assayed via Annexin V-flourescein isothiocyanate and propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle progression and mitochondria membrane potential were studied via flow cytometry following PI and JC-1 staining respectively. H. magnifica venom induced significant reductions in viable cell numbers and increases in apoptosis in T47D and MCF7 in dose-dependent manners. A significant apoptosis-related increase in the sub G1 peak of the cell cycle in both breast cancer cell lines was also observed. Moreover, treatment by venom cleaved caspase-8, caspase-9, and activated caspase-3. Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways. Consequently, H. magnifica venom has potential for the development of a breast cancer therapeutic.
TL;DR: It is reported that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.
Abstract: Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.
TL;DR: Optimal culture conditions may promote early differentiation of tenocytes in adipose and umbilical cord blood stem cells, improving their ability to aid tendon regeneration and facilitating more efficient recovery from tendon injury.
Abstract: Recovery from tendon injury is based on long periods of rest, which results in sub-optimal repair, often replacing tendon with fibrocartilage scar tissue. Recently, the use of stem cells in equine tendon repair has been attempted with variable success. The objective of this work was to determine the expression of scleraxis (scx) and tenascin C (TnC), two markers of tenocytes, in adipose (AdMSC) and umbilical cord blood (UCB) stem cells during culture on various substrata and in response to fibroblast growth factor (FGF) treatment. Equine UCB and AdMSC were cultured on gelatin-coated plasticware, 30 % matrigel or collagen-coated Cytodex beads and treated with 10 ng/ml FGF2, FGF4 or FGF5 prior to measurement of proliferation, kinase activity and tenocyte gene expression. Supplementation with FGF2 or FGF5 activated the ERK1/2 signaling pathway in AdMSC and UCB; no effect of FGF4 was observed in UCB. FGF2 increased proliferation in AdMSC but not UCB. Conversely, FGF5 stimulated proliferation of UCB. Culture in matrigel increased scx expression in both cell populations and increased TnC in AdMSC. In AdMSC grown in matrigel, supplementation with FGF2 or FGF5 increased TnC expression. Thus, culture conditions (substrata and FGF supplementation) impact markers of tenocytes in AdMSC and UCB stem cells, indicating that careful consideration should be given to culture conditions prior to use of UCB or AdMSC as therapeutic aids. Optimal culture conditions may promote early differentiation of these cells, improving their ability to aid tendon regeneration and facilitating more efficient recovery from tendon injury.
TL;DR: Results indicate that direct adherence was a simple and convenient method for isolation and cultivation of bone marrow stromal cells, and BMSCs can be induced in vitro to differentiate into neuronal cells by using GDNF, which could achieve a more persistent and stable inducing effect than when using BDNF.
Abstract: The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). They can be obtained directly from patients and successfully induced to form various differentiated cell types. In addition, cell-based transplantation therapies have been proven to be promising strategies for curing disease of the nerve system. Therefore, it was particularly important to establish an easy and feasible method for the isolation, purification, and differentiation of bone marrow stromal cells (BMSCs). The aim of this study was to isolate and characterize putative bone marrow derived MSCs from Sprague–Dawley (SD) rats. Furthermore, differentiation effects were compared between the GDNF-induction group and the BDNF-induction group. Of these, BMSCs were isolated from the SD rats in a traditional manner, and identified based on plastic adherence, morphology, and surface phenotype assays. After induction with GDNF and BDNF, viability of BMSCs was detected by MTT assay and neuronal differentiation of BMSCs was confirmed by using immunofluorescence and Western blotting. Besides, the number of BMSCs that obviously exhibited neuronal morphology was counted and the results were compared between the GDNF-induction group and BDNF-induction groups. Our results indicate that direct adherence was a simple and convenient method for isolation and cultivation of BMSCs. Furthermore, BMSCs can be induced in vitro to differentiate into neuronal cells by using GDNF, which could achieve a more persistent and stable inducing effect than when using BDNF.