Hybrid Cell Lines Research Articles

In vitro Generation of Cytotoxic T Cells With Potential for Adoptive Tumor Immunotherapy of Multiple Myeloma.

Multiple myeloma is a life-threatening hematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using tumor antigen-specific, cytotoxic T-lymphocytes, may represent an alternative or additional treatment for multiple myeloma. In this study, we used hybrid cell lines, generated by fusion of an EBV B-lymphoblastoid cell line (B-LCL) and myeloma cells, to stimulate in vitro peripheral blood lymphocytes (PBLs) from patients with multiple myeloma. We investigated induction of antigen-specific, cytotoxic T-lymphocytes to the well-defined tumor associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1, which have been shown to be expressed in a high proportion of cases of multiple myeloma. HLA-A2-peptide pentamer staining, interferon-γ and perforin ELISpot assays, as well as cytotoxicity assays were used. Following several rounds of in vitro stimulation, the hybrid cell lines induced antigen-specific, cytotoxic T-lymphocytes to four candidate TAAs in PBLs from HLA-A2+ multiple myeloma patients, using known HLA-A2 restricted peptide epitopes of the TAAs. In contrast, the HLA-A2+ myeloma cell line U266 failed to induce antigen-specific, cytotoxic T-lymphocytes in vitro. Our data indicate that B-LCL/myeloma hybrid cell lines induce antigen-specific, cytotoxic T-lymphocytes in PBLs isolated from multiple myeloma patients in vitro and may represent a novel strategy for use in adoptive immunotherapy of multiple myeloma.

Immunohistochemical analysis of adiponectin in atherosclerotic lesions of human aorta.

BACKGROUNDMetabolic syndrome, a cluster of interrelated disorders including abdominal obesity, insulin resistance, dyslipidemia, and hypertension (HTN) plays an important role in development of atherosclerotic lesions in arterial wall. Dysregulation of adipose tissue hormones (adipokines) production is a possible link between abdominal obesity and other manifestations of metabolic syndrome. Adiponectin is a well-known adipokine which affects metabolism and inflammatory response. However, data on its role in atherogenesis are still controversial. The aim of this study is to investigate whether adiponectin is present in atherosclerotic lesions of human aorta.METHODSThirty-five autopsy segments from abdominal, thoracic aortas, and aortic arch of four men (mean age: 57 years) were fixed and stained for lipids [Oil Red O (ORO)], cells [hematoxylin-eosin (H&E)], and adiponectin [indirect immunoperoxidase assay (IPA) method]. Samples of both stable and unstable plaques were selected for analysis. Human adipose tissue, THP-1 monocytes/macrophages, and human endothelial hybrid cell line (EA.hy926) were chosen for detection of adiponectin messenger ribonucleic acid (mRNA) using reverse transcription polymerase chain reaction (RT-PCR).RESULTSAdiponectin accumulations were found inside endothelial cells covering both stable and unstable atherosclerotic plaques. Focal depositions of adiponectin were also found in fibrous caps of stable lesions and atheromatous core of both stable and unstable plaques and also in adventitia. RT-PCR revealed mRNA expression of adiponectin gene in adipose tissue, but not in mononuclears and endothelial cells.CONCLUSIONAdiponectin is present in aortic plaques of humans, but is not synthesized in endothelial cells and mononuclears, at least in culture conditions. Detection of adiponectin in atherosclerotic lesions can serve as indirect evidence of possible participation of this adipokine in atherogenesis.

Cell models and drug discovery for mitochondrial diseases.

Mitochondrion is a semi-autonomous organelle, important for cell energy metabolism, apoptosis, the production of reactive oxygen species (ROS), and Ca2+ homeostasis. Mitochondrial DNA (mtDNA) mutation is one of the primary factors in mitochondrial disorders. Though much progress has been made, there remain many difficulties in constructing cell models for mitochondrial diseases. This seriously restricts studies related to targeted drug discovery and the mechanism and therapy for such diseases. Here we summarize the characteristics of patient-specific immortalized lymphoblastoid cells, fibroblastoid cells, cytoplasmic hybrid (cybrid) cell lines, and induced pluripotent stem cells (iPSCs)-derived differentiation cells in the study of mitochondrial disorders, as well as offering discussion of roles and advances of these cell models, particularly in the screening of drugs.

The Differentiation of Spinal Cord Motor Neurons is Associated with Changes of the Mitochondrial Phospholipid Cardiolipin

Motor neuron damage caused by diseases, traumatic insults or de-afferentation of the spinal cord is often incurable due to the poor intrinsic regenerative capacity. Moreover, regenerated peripheral nerves often do not reach normal functionality. Here, we investigated cardiolipin in the process of neuro-differentiation, since cardiolipin is closely linked to the mitochondrial energy supply in cells. The NSC-34 hybrid cell line, produced by fusing neuroblastoma cells with primary spinal cord motor neurons, was used, since it shares several morphological and physiological characteristics with mature primary motor neurons. Their neuro-differentiation was supported by switching from normal to differentiation medium or by fatty acid supplementation. Differentiation was evaluated by measuring neurite-sprouting parameters and PPARα expression. Cellular fatty acid distribution was analyzed to indicate changes in lipid metabolism during differentiation. Cardiolipin was characterized by acyl-chain composition and the distribution of molecular cardiolipin species. Both, the switch from normal to differentiation medium as well as the administration of palmitic and oleic acid promoted neuro-differentiation. Stimulated differentiation was accompanied by changes in cardiolipin content and composition. The positive correlation between neuro-differentiation and concentration of those molecular cardiolipin species containing palmitic and oleic acid implied a link between differentiation of NSC-34 cells and cardiolipin metabolism. We further demonstrated the impact of cellular lipid metabolism, and particularly cardiolipin metabolism, during and NSC-34 neuritogenesis. Thus, cardiolipin may represent a new therapeutic target for axon regeneration after peripheral nerve injuries or when axon sprouting is required to compensate for motor neuron loss in response to aging and/or disease.

CGRPergic Nerve TRPA1 Channels Contribute to Epigallocatechin Gallate-Induced Neurogenic Vasodilation.

Green tea polyphenol epigallocatechin gallate (EGCG), promotes vasodilation and reduces blood pressure, mechanisms of which are not fully resolved. Recent reports suggested that EGCG can activate heterologously expressed mouse and zebrafish TRPA1 channels. Activation of TRPA1 in sensory neurons triggers the release of calcitonin gene-related peptide (CGRP), a potent vasodilator. Whether CGRP-containing (CGRPergic) sensory nerves contribute to EGCG-induced reduction in vascular resistance remains unclear. In this study, we demonstrate that intravenous infusion of EGCG elevated the plasma level of CGRP in mice, an effect that was attenuated by TRPA1 channel blocker A-967079. EGCG-induced increase in mesenteric artery blood flow and reduction in mean arterial pressure were reversed by A-967079, CGRP receptor antagonist CGRP8-37, and CGRP depletion in perivascular nerves. Moreover, EGCG stimulated TRPA1-dependent intracellular Ca2+ elevation and CGRP release in a differentiated rat embryonic dorsal root ganglion/mouse neuroblastoma hybrid cell line. Together, these data suggest that EGCG-induced activation of TRPA1 channels in perivascular CGRPergic nerves decreases vascular resistance via Ca2+-dependent exocytosis of CGRP.

Evaluation of Phosphodiesterase-5 Inhibitory Potential of Biofield Energy Treated DMEM by Determining cGMP Level in Human Endothelial Cell Line

Erectile dysfunction is a common disorder found in men, which occurs due to multiple factors such as psychogenic, hormonal imbalance, and neurovascular disturbances. The present investigation was undertaken to examine the effect of a Consciousness Energy Healing based DMEM medium on the Human Endothelial Hybrid Cell Line (EA. hy926) to evaluate the level of cyclic guanosine monophosphate (cGMP). The test item (DMEM medium) was divided into three parts, first part received a one-time Consciousness Energy Healing Treatment by a renowned Biofield Energy Healer, Alice Branton and was labeled as the one-time Biofield Energy Treated (BT-I) DMEM, while second part received the two-times the Biofield Energy Treatment and is denoted as BT-II DMEM. The third part did not receive any treatment and defined as the untreated DMEM group. The level of cGMP for the inhibition of PDE-5 enzyme was assessed using cGMP ELISA assay kit (colorimetric). Sildenafil citrate, used as positive control, which showed a significant increase of cGMP levels in Ea. hy926 cells. The one-time Biofield Energy Healing Treated DMEM (BT-I) showed 29.92% alteration, while the two-times Biofield Energy Healing Treated DMEM (BT-II) showed a significantly (p≤0.001) increased level of intracellular cGMP by 307.09% in Ea. hy926 cells compared to the untreated DMEM group. Overall, experimental data suggested that the two-times Biofield Treated DMEM showed a significantly improved level of cGMP compared with the one-time Biofield Treated DMEM group. Therefore, data indicated that the Biofield Energy Healing Treatment can be used to treat erectile dysfunction along with other sexual disorders such as orgasmic disorders, female sexual arousal disorder, fetishistic disorder, frotteuristic disorder, hypoactive sexual desire disorder, sex addiction, sexual masochism and sadism, vaginismus, voyeuristic disorder, premature or delayed ejaculation.

Development of Immuno-Enzymatic Monoclonal Tests-Systems for the Detection of Glanders and Melioidosis Agents

Objective of the study was the development of immune-enzymatic monoclonal test-kit for detecting glanders and melioidosis agents. Materials and methods. We used microbial cultures and hybrid cell lines obtained from the collection of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation. Hybridoma cells were incubated in the peritoneal cavity of BALB/c mice. Preparations of glanders and melioidosis monoclonal antibodies were isolated from the ascetic fluids through precipitation with ammonium sulfate and purification by means of ion-exchange chromatography. Specific components of the test-kits were subjected to freeze drying in corresponding protective media. Study of diagnostic properties of the developed test systems was performed using ELISA. Results and conclusions. We have obtained preparations of monoclonal antibodies in vivo, as well as isolated and purified immunoglobulins from ascetic fluids. We also selected the pairs of monoclonal antibodies for manufacturing specific components. Experimental series of immune-enzymatic monoclonal test-systems allowing for specific detection of glanders and melioidosis causative agents in concentrations ranging from 0.5·106 CFU/ml and higher were made. The absence of cross-reactivity with closely related saprophytes and heterologous microorganisms in concentrations of 1,0·108 CFU/ml was shown. Demonstrated was the possibility in principle to differentiate between Burkholderia malleiand Burkholderia pseudomallei using ELISA. Test systems are promising for follow up state registration as medical products for in vitro diagnostics.

Development of Enzyme-Immunoassay for <i>Bacillus anthracis</i> Detection

Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.

Molecular mechanisms involved in the protective effect of pituitary adenylate cyclase-activating polypeptide in an in vitro model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Based on transcriptional profiles of motor cortex samples, in a previous work, we were able to classify two subgroups of sporadic ALS (SALS) patients, named SALS1 and SALS2. A further meta-analysis study has revealed sixteen drug targets commonly deregulated in SALS2 and superoxide dismutase 1 (SOD1) G93A mice. The identified candidate drug targets included pituitary adenylate cyclase-activating polypeptide (PACAP), epidermal growth factor receptor (EGFR) and matrix metallopeptidase-2 (MMP-2). By using a motor neuron-like hybrid cell line (NSC-34) expressing human SOD1 G93A as an in vitro model of ALS, here we investigated the functional correlation among these three genes. Our results have shown that PACAP increases cell viability following serum deprivation. This effect is induced through EGFR transactivation mediated by protein kinase A stimulation. Furthermore, EGFR phosphorylation activates mitogen-activated protein kinases/extracellular signal-regulated kinases 1 and 2 survival signaling pathway and increases MMP-2 expression, significantly reduced by serum starvation. These results suggest that a deeper characterization of mechanisms involved in PACAP/EGFR/MMP-2 axis activation in G93A SOD1 mutated neurons may allow identifying new targets for ALS therapy.

Isotyping antibodies produced from hybridoma A6G11C9

Hybridoma technology was discovered in 1975 to produce the monoclonal antibodies. By this way, we can produce a desired antibody in large amounts. From a single hybrid cell line, they grow and develop to produce a monoclonal antibody with large enough quantities to use for the research, treatment and diagnosis. The level of the antibody producing is depended on the cell density and the incubation period. The growth capacity of each hybrid cell line depends on the composition of the substances in the culture medium of animal cells. Among them, fetal bovine serum is the most commonly used serum-supplement for the in vitro cell culture. This is due to it having a very low level of antibodies and containing more growth factors. In the previous studies, the hybrid cell line (designed A6G11C9) was the best one secreting the highest anti-A monoclonal antibodies, whose specificity agglutinaned human red blood cells containing A antigen. This report showed the result of the study on the coditional culture and isotyping of the immunoglobulin. The hybrid cell line A6G11C9 was cultured in the DMEM medium with different level of fetal bovine serum. As the results, this hybridoma line grow in the DMEM medium containing 10% fetal bovine serum is five times faster than in the DMEM medium with 1% fetal bovine serum. The maximum of the cell density are 11.106 cells/ml after 50 hours innoculation. The maximum of the titer from culture supernatant are 1/1024 after 100 hours innoculation. The monoclonal antibodies derived from hybrid cell line A6G11C9 are IgM heavy chain and the kappa light chain.

Study on creating the hybridoma for producing a monoclonal antibody that causes agglutination of the red blood cell B group

ABO incompatibility is a potential lethal barrier in tranfusion therapy. ABO blood cell grouping for all of the donors and potential recipients is the unique way for ensuring the highest safety for potential recipients in blood transfusion. ABO blood cell grouping must be performed by both of the serum sample method and erythrocytes sample method. Nowadays, when applying the serum sample method, the monoclonal antibodies are commonly used to identify the antigens on the surface of the red blood cells. In this study, for the first time in Vietnam, the hybridoma technology was successefully developed and screened hybrid cells for producing monoclonal antibody specifically agglutinated with red blood cells B group. After being isolated from spleen and iliac lympho node of the human red blood cells B group immuned BALB/c mice, the lymphocytes B was fused with myeloma sp2/0. As the results, 10 single hybrid cell lines B4D6C6, B4D6E2, B4D10C9, B4D10B6, B4D10D5, B4D10D6, B4D10E4, B4H6C5, B8F6B6, B8F6D4 have been screened by a specific agglutination with sample red blood cells group B. Among them, the hybrid cell line B4D10C9 was the best secreting anti-B monoclonal antibody into culture, that presented by antibody titer reached 1/256, hence it was selected for further studies. The ELISA method helped to isotype the anti-B monoclonal antibody which was produced from B4D10C9 hybridoma. As a result, the anti-B monoclonal antibody contained IgM heavy chains and kappa light chains. The IgM antibody could be able to agglutinate red blood cells 25 times more than IgG antibody. The success of screening B4D10C9 hybridoma producing IgM monoclonal antibody is the most significant result of this study.

Prostaglandin E2 induces differentiation of NSC-34 cells with neuron-like features

Prostaglandin E2 (PGE2), one of the major lipid mediators produced by the arachidonic acid cascade, is involved in various physiological effects. We demonstrated previously that PGE2 suppressed cell proliferation and promoted neurite outgrowth, a morphological marker of neuronal cell differentiation, in NSC-34 cells, a hybrid cell line produced by fusion of motor neuron-enriched spinal cord cells with mouse neuroblastoma cells and that E-prostanoid receptor 2 subtype plays a role in the PGE2-induced differentiation of NSC-34 cells. However, electrophysiological characteristics of differentiated NSC-34 cells by PGE2 still remains to be elucidated. In the present study, we compared the electrophysiological properties of the NSC-34 cells differentiated by PGE2 to those by retinoic acid (RA) using whole-cell patch clamp techniques.

Production of a Human Cell Line with a Plant Chromosome.

It is a major challenge in biology to know whether chromosome functions of replication, segregation, gene expression, inheritance, etc. are conserved among evolutionary distant organisms where common structural features are maintained. Establishment of hybrid cell lines between evolutionary distant organisms, such as humans and plants, would be one of the promising synthetic approaches to study the evolutionary conservation of chromosome functions. In this chapter, we describe the protocol for successful establishment of human cell lines with a functional plant chromosome. Systematic analyses of hybrid cells will facilitate the evolutionary study of organisms with respect to chromosome functions. It will also provide a basic platform for genome writing and construction of chromosomal shuttle vectors .

Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells

Almost of the ABO blood grouping reagents is being trading derive from the monoclonal antibodies. There are two methods to produce the monoclonal antibodies from hybridoma lines, which were in vitro method (hybridoma cultured in the medium) and in vivo method (hybridoma cultured in the mice intra-abdominal). In Vietnam, Nguyen Thi Trung and co-authors was succesfully screened in hybridoma cell line A6G11C9 which generating of the anti A monoclonal antibody agglutinated A antigen on the surface of red blood cells. The fusion of mouse lymohocyte B generated anti-A antibody with mouse myeloma sp2/0 is formed that hybrid cell lines. The anti-A monoclonal antibody is produced from hybridoma cell line A6G11C9 have been highly intensive confirmed. It is capability of growth and anti B monoclonal antibody producing stability through the generations. In this study, the process to produce large amounts of monoclonal antibodies from B4D10C9 hybridoma by in vitro method are published. Firstly, hybridoma cells are stored in liquid nitrogen to wake by culture in medium. Then, First, hybrid cells are stored frozen in liquid nitrogen to wake cultured cells. Then, they were first inoculated to produce enough biomass to serve a larger scale. Cell biomass continues to be second inoculated into DMEM containing 10% fetal bovin serum for 10 days. The culture medium contained anti-A monoclonal antibodies were collected by centrifugation to remove cells. The anti-A monoclonal antibody levels in culture medium was concentrated and remove phenol red indicator by the precipitation with NH4SO4 50% saturated. The anti-A monoclonal antibody solution at 5 times concentrated have been better agglutinated with erythrocytes containing A antigen than monoclonal antibody solution non-concentration. 150 ml of concentrated antibodies were produced. Antibody titer of the anti-A monoclonal antibodies in the concentrated 5 times solution was 1/512. The intensity of the reaction anti-A monclonal antibody with red blood cell containing A antigen was 4+. Citation: Nguyen Thi Trung, Truong Nam Hai, 2018. Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells. Tap chi Sinh hoc, 40(1): x-xx. DOI: 10.15625/0866-7160/v40n1.9154. *Corresponding author: tnhai@ibt.ac.vn Received 12 January 2017, accepted 20 December 2017

Two approaches reveal a new paradigm of \u2018switchable or genetics-influenced allele-specific DNA methylation\u2019 with potential in human disease

Imprinted genes are vulnerable to environmental influences during early embryonic development, thereby contributing to the onset of disease in adulthood. Monoallelic methylation at several germline imprints has been reported as DNMT1-dependent. However, which of these two epigenetic attributes, DNMT1-dependence or allelic methylation, renders imprinted genes susceptible to environmental stressors has not been determined. Herein, we developed a new approach, referred to as NORED, to identify 2468 DNMT1-dependent DNA methylation patterns in the mouse genome. We further developed an algorithm based on a genetic variation-independent approach (referred to as MethylMosaic) to detect 2487 regions with bimodal methylation patterns. Two approaches identified 207 regions, including known imprinted germline allele-specific methylation patterns (ASMs), that were both NORED and MethylMosaic regions. Examination of methylation in four independent mouse embryonic stem cell lines shows that two regions identified by both NORED and MethylMosaic (Hcn2 and Park7) did not display parent-of-origin-dependent allelic methylation. In these four F1 hybrid cell lines, genetic variation in Cast allele at Hcn2 locus introduces a transcription factor binding site for MTF-1 that may predispose Cast allelic hypomethylation in a reciprocal cross with either C57 or 129 strains. In contrast, each allele of Hcn2 ASM in J1 inbred cell line and Park7 ASM in four F1 hybrid cell lines seems to exhibit similar propensity to be either hypo- or hypermethylated, suggesting a ‘random, switchable’ ASM. Together with published results, our data on ASMs prompted us to propose a hypothesis of regional ‘autosomal chromosome inactivation (ACI)’ that may control a subset of autosomal genes. Therefore, our results open a new avenue to understand monoallelic methylation and provide a rich resource of candidate genes to examine in environmental and nutritional exposure models.

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB.

Cellular transformation assays have been utilized for many years as powerful in vitro methods for examining neoplastic transformation potential/frequency and mechanisms of carcinogenesis for both chemical and radiological carcinogens. These mouse and human cell based assays are labor intensive but do provide quantitative information on the numbers of neoplastically transformed foci produced after carcinogenic exposure and potential molecular mechanisms involved. Several mouse and human cell systems have been generated to undertake these studies, and they vary in experimental length and endpoint assessment. The CGL1 human cell hybrid neoplastic model is a non-tumorigenic pre-neoplastic cell that was derived from the fusion of HeLa cervical cancer cells and a normal human skin fibroblast. It has been utilized for the several decades to study the carcinogenic/neoplastic transformation potential of a variety of ionizing radiation doses, dose rates and radiation types, including UV, X ray, gamma ray, neutrons, protons and alpha particles. It is unique in that the CGL1 assay has a relatively short assay time of 18-21 days, and rather than relying on morphological endpoints to detect neoplastic transformation utilizes a simple staining method that detects the tumorigenic marker alkaline phosphatase on the neoplastically transformed cells cell surface. In addition to being of human origin, the CGL1 assay is able to detect and quantify the carcinogenic potential of very low doses of ionizing radiation (in the mGy range), and utilizes a neoplastic endpoint (re-expression of alkaline phosphatase) that can be detected on both viable and paraformaldehyde fixed cells. In this article, we review the history of the CGL1 neoplastic transformation model system from its initial development through the wide variety of studies examining the effects of all types of ionizing radiation on neoplastic transformation. In addition, we discuss the potential of the CGL1 model system to investigate the effects of near zero background radiation levels available within the radiation biology lab we have established in SNOLAB.

A missense MT-ND5 mutation in differentiated Parkinson Disease cytoplasmic hybrid induces ROS-dependent DNA Damage Response amplified by DROSHA

Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally differentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson’s disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon differentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identified a rare and heteroplasmic missense mutation affecting a highly conserved residue of the ND5-subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the first evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells.

Extracts of Terminalia sericea Enhance Cell Migratory Activity of Endothelial Hybrid and Fibroblast Cells In Vitro.

Terminalia sericea is a plant that has been used amongst others medicinally to treat wounds. The aim of this study was to assess the in vitro wound healing ability of T. sericea. Hot water, methanol, ethyl acetate, and hexane extracts were prepared. Thin layer chromatography (TLC) and ultra-performance liquid chromatography time of flight mass spectrometry (UPLC-TOF-MS) were used to determine the phytochemical classes and genus specific compounds present in the plant. Cytotoxicity was assessed in the SC-1 fibroblast and EA.hy926 endothelial hybrid cell lines using the sulforhodamine B assay. The effect of the extracts on cellular migration in both cell lines was assessed using the scratch assay. The major phytochemical classes detected in the extracts using TLC were alkaloids, coumarins, flavonoids, glycosides, phenolics, saponins, sterols, and terpenoids. The genus-specific compounds punicalagin, sericoside, anolignan B, and arjunic acid were identified in the extracts by means of UPLC-QTOF-MS. Cytotoxicity was not observed after 24 h of exposure and a generally low cytotoxic trend was noted after 72 h. A significant (p < 0.05) enhancement of cell migration in both cell lines was noted in the scratch assay. The wound healing ability of T. sericea is mainly attributed to the migratory and proliferative activity of the extracts responsible for the acceleration of wound closure. Isolation and individualized testing of the active compounds is warranted.

A hypertension-associated mitochondrial DNA mutation alters the tertiary interaction and function of tRNALeu(UUR)

Several mitochondrial tRNA mutations have been associated with hypertension, but their pathophysiology remains poorly understood. In this report, we identified a novel homoplasmic 3253T→C mutation in the mitochondrial tRNALeu(UUR) gene in a Han Chinese family with maternally inherited hypertension. The m.3253T→C mutation affected a highly conserved uridine at position 22 at the D-stem of tRNALeu(UUR), introducing a G-C base pairing (G13-C22) at the D-stem and a tertiary base pairing (C22-G46) between the D-stem and the variable loop. We therefore hypothesized that the m.3253T→C mutation altered both the structure and function of tRNALeu(UUR) Using cytoplasmic hybrid (cybrid) cell lines derived from this Chinese family, we demonstrated that the m.3253T→C mutation perturbed the conformation and stability of tRNALeu(UUR), as suggested by faster electrophoretic mobility of mutated tRNA relative to the wild-type molecule. Northern blot analysis revealed an ∼45% decrease in the steady-state level of tRNALeu(UUR) in the mutant cell lines carrying the m.3253T→C mutation, as compared with control cell lines. Moreover, an ∼35% reduction in aminoacylation efficiency of tRNALeu(UUR) was observed in the m.3253T→C mutant cells. These alterations in tRNALeu(UUR) metabolism impaired mitochondrial translation, especially for those polypeptides with a high proportion of Leu(UUR) codons, such as ND6. Furthermore, we demonstrated that the m.3253T→C mutation decreased the activities of mitochondrial complexes I and V, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the cells. In conclusion, our findings may provide new insights into the pathophysiology of maternally inherited hypertension.

Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184\u201316193 poly-cytosine tract), which confer resistance to cisplatin

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 μg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184–16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.