scholarly journals Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle

2005 ◽  
Vol 17 (8) ◽  
pp. 791 ◽  
Author(s):  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Christina T. Skou ◽  
R. Tayfur Tecirlioglu ◽  
Teija T. Peura ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Fluorescence Microscope ◽  
Time Dependent ◽  
Dependent Manner ◽  
Reconstructed Embryo ◽  
Ultraviolet Illumination ◽  
Oocyte Selection ◽  
Handmade Cloning

The purpose of the present study was to find an efficient and reliable chemically assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 μg mL−1 demecolcine for 2 h. Subsequently, zonae pellucidae were digested with pronase, and one-third of the cytoplasm connected to an extrusion cone was removed by hand using a microblade. The remaining two-thirds were used as recipients for HMC, and reconstructed and activated embryos were cultured for 7 days. The time-dependent manner of the development of extrusion cones, the efficiency (oriented bisection per oocyte; 94%), reliability (success per attempted enucleation; 98%), and the blastocyst per reconstructed embryo rates (48%) were measured. Ultrastructural analyses demonstrated that demecolcine treatment resulted in disoriented and haphazardly orientated microtubules. The general ultrastructure of the oocyte organelles, however, appeared to be unaltered by the treatments. Considering that no oocyte selection based on polar body presence was performed, this system seems to be more efficient and reliable than any other enucleation method. Moreover, expensive equipment (inverted fluorescence microscope) and a potentially harmful step (staining and ultraviolet illumination) can be eliminated from the HMC procedure without compromising the high in vitro efficiency.

scholarly journals 75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD FOR HANDMADE CLONING IN CATTLE AND SWINE

2004 ◽  
Vol 16 (2) ◽  
pp. 159 ◽  
Author(s):  
G. Vajta ◽  
T.T. Peura ◽  
L. Lai ◽  
C.N. Murphy ◽  
R.S. Prather ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Cumulus Cells ◽  
Dependent Manner ◽  
Uv Illumination ◽  
Reconstructed Embryo ◽  
Fetal Fibroblasts ◽  
Membrane Protrusions ◽  
Handmade Cloning

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P<0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.

scholarly journals Synthesis and Evaluation of the Antitumor Activity of Novel 1-(4-Substituted phenyl)-2-ethyl Imidazole Apoptosis Inducers In Vitro

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4293
Author(s):  
Zhen-Wang Li ◽  
Chun-Yan Zhong ◽  
Xiao-Ran Wang ◽  
Shi-Nian Li ◽  
Chun-Yuan Pan ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Cells ◽  
Antiproliferative Activity ◽  
Antitumor Agent ◽  
Positive Control ◽  
Selectivity Index ◽  
Time Dependent ◽  
Potential Candidate ◽  
Dependent Manner

Novel imidazole derivatives were designed, prepared, and evaluated in vitro for antitumor activity. The majority of the tested derivatives showed improved antiproliferative activity compared to the positive control drugs 5-FU and MTX. Among them, compound 4f exhibited outstanding antiproliferative activity against three cancer cell lines and was considerably more potent than both 5-FU and MTX. In particular, the selectivity index indicated that the tolerance of normal L-02 cells to 4f was 23–46-fold higher than that of tumor cells. This selectivity was significantly higher than that exhibited by the positive control drugs. Furthermore, compound 4f induced cell apoptosis by increasing the protein expression levels of Bax and decreasing those of Bcl-2 in a time-dependent manner. Therefore, 4f could be a potential candidate for the development of a novel antitumor agent.

scholarly journals Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

2020 ◽  
Vol 15 (1) ◽  
pp. 619-628
Author(s):  
Chen Yuan ◽  
Ya Mo ◽  
Jie Yang ◽  
Mei Zhang ◽  
Xuejun Xie
Keyword(s):  
Electrical Resistance ◽  
Cell Model ◽  
Polyclonal Antibodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Blood Retinal Barrier ◽  
Advanced Glycosylation End Products ◽  
End Products ◽  
Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

scholarly journals Metformin Inhibited Growth, Invasion and Metastasis of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

2018 ◽  
Vol 51 (3) ◽  
pp. 1276-1286 ◽  
Author(s):  
Feng Liang ◽  
Yu-Gang Wang ◽  
Changcheng Wang
Keyword(s):  
Squamous Cell Carcinoma ◽  
Plasma Glucose Level ◽  
Caspase 3 ◽  
Time Dependent ◽  
Dependent Manner ◽  
Metformin Treatment ◽  
Invasion And Migration ◽  
And Migration

Background/Aims: This study aimed at investigating the effects of metformin on the growth and metastasis of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. Methods: Two human ESCC cell lines EC9706 and Eca109 were selected and challenged with metformin in this study. Western blot assay was performed to detect th level of Bcl-2, Bax and Caspase-3. Scratch wound assay, transwell assay and Millicell invasion assay were used to assay the invasion and migration of EC9706 and Eca109 cells. Nude mice tumor models were used to assay the growth and lung metastasis of ESCC cells after metformin treatment. The plasma glucose level was also assayed. Results: We found that metformin significantly inhibited proliferation and induced apoptosis of both ESCC cell lines in a dose- and time-dependent manner, and the expression of Bcl-2 was down-regulated and Bax and Caspase-3 were up-regulated. Metformin significantly inhibited the invasion and migration of EC9706 and Eca109 cells (p < 0.05). mRNA and protein levels of MMP-2 and MMP-9 decreased significantly upon treatment with metformin of 10mM for 12, 24 and 48h in a time-dependent manner (p < 0.05). In line with in vitro results, in vivo experiments demonstrated that metformin inhibited tumorigenicity, inhibited lung metastasis and down-regulated the expression of MMP-2 and MMP-9. Moreover, we showed that metformin treatment did not cause significant alteration in liver and renal functions and plasma glucose level. Conclusion: Our study for the first time demonstrated the anti-invasive and anti-metastatic effects of metformin on human ESCC cells both in vitro and in vivo, which might be associated with the down-regulation of MMP-2 and MMP-9. As a whole, our results indicate the potential of metformin to be developed as a chemotherapeutic agent for patients with ESCC and might stimulate future studies on this area.

Synthesis of 70K stress protein by human leukocytes: effect of exercise in the heat

1991 ◽  
Vol 70 (1) ◽  
pp. 466-471 ◽  
Author(s):  
A. J. Ryan ◽  
C. V. Gisolfi ◽  
P. L. Moseley
Keyword(s):  
Protein Synthesis ◽  
Relative Humidity ◽  
Treadmill Exercise ◽  
Stress Protein ◽  
Exercise Bout ◽  
Time Dependent ◽  
Dependent Manner ◽  
Human Leukocytes ◽  
Before And After

To determine whether reinduction of 70,000-Da (70K) stress protein synthesis could be used as an assay for thermal history and/or cellular levels of 70K stress protein in hyperthermic humans, leukocytes were obtained before and after 2 h of exercise and then incubated at 37 or 41 degrees C. Five healthy males completed 2 h of treadmill exercise consisting of running at 4–6 km/h for 30–45 min followed by 75–90 min of walking up a 2–10% grade. This exercise bout was performed by two subjects in hot (46 degrees C, 15% relative humidity) and by five subjects in cooler (30 degrees C, 40% relative humidity) environmental conditions. Exercise resulting in rectal temperature (Tre) less than 40 degrees C did not alter the amount of 70K stress protein synthesized by leukocytes incubated at 41 degrees C. In contrast, exercise resulting in Tre greater than 40 degrees C reduced the amount of 70K stress protein synthesized by leukocytes incubated at 41 degrees C. A protein immunoblot, probed with an antibody specific for the inducible 72K stress protein, showed that the reduction of 35S-labeled 70K stress protein in these postexercise leukocyte samples occurred without marked elevations of this protein. In vitro incubation of human leukocytes at 40 degrees C for 15–120 min reduced, in a time-dependent manner, the amount of 70K stress protein synthesized during a subsequent 41 degrees C heat stress. This reduction of 70K stress protein synthesis in 41 degrees C-treated leukocytes was abolished when cycloheximide was present during the 40 degrees C preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 787
Author(s):  
Enrique García-Pérez ◽  
Dojin Ryu ◽  
Hwa-Young Kim ◽  
Hae Dun Kim ◽  
Hyun Jung Lee
Keyword(s):  
Oxidative Stress ◽  
Ochratoxin A ◽  
Cell Lines ◽  
Time Dependent ◽  
Mrna Levels ◽  
Dependent Manner ◽  
In Vitro System ◽  
Glutathione Peroxidase 1 ◽  
Glucose 6 Phosphate Dehydrogenase

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

SEB is cytotoxic and alters EC barrier function through protein tyrosine phosphorylation in vitro

1997 ◽  
Vol 273 (1) ◽  
pp. L31-L39 ◽  
Author(s):  
W. N. Campbell ◽  
M. Fitzpatrick ◽  
X. Ding ◽  
M. Jett ◽  
P. Gemski ◽  
...  
Keyword(s):  
Barrier Function ◽  
Tyrosine Kinases ◽  
Polymyxin B ◽  
Time Dependent ◽  
Dependent Manner ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Effector Cells ◽  
Protein Tyrosine

We studied whether Staphylococcal enterotoxin B (SEB) has direct effects on endothelial cells (EC) in the absence of effector cells or their products. Bovine or human pulmonary artery EC were grown to confluence on filters mounted in chemotaxis chambers. Barrier function was assessed by placing [14C]bovine serum albumin in the chamber and sampling the lower well for 14C activity. SEB exposures induced a significant (P < 0.001) dose- and time-dependent increase in albumin flux across both bovine and human EC monolayers. Albumin flux was temperature dependent, and cycloheximide pretreatment of the monolayers did not block the SEB-induced increase in permeability. Preincubation of SEB with trypsin or anti-SEB antibody significantly (P < 0.0001) reduced the effect, whereas pretreatment with polymyxin B did not. SEB at > or = 10 micrograms/ml significantly (P < 0.03) increased EC injury as measured by 51Cr release in a dose- and time-dependent manner. Herbimycin and genistein, inhibitors of protein tyrosine kinases, each protected against SEB-induced cytotoxicity, barrier dysfunction, and intercellular gap formation. We conclude that SEB perturbs endothelial barrier function and viability in the absence of effector cells or their mediators.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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