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1.
Curr Protoc ; 3(2): e679, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36809687

ABSTRACT

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).


Subject(s)
DNA, Mitochondrial , Mitochondria , Mice , Rats , Animals , Humans , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Polymerase Chain Reaction , DNA Replication , Zalcitabine/metabolism , Zalcitabine/pharmacology , Ethidium/metabolism , Mammals/genetics , Mammals/metabolism
2.
Cells ; 11(14)2022 07 11.
Article in English | MEDLINE | ID: mdl-35883613

ABSTRACT

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.


Subject(s)
DNA-Binding Proteins , Transcription Factors , DNA, Mitochondrial/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Reverse Genetics , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Circ Res ; 129(12): e215-e233, 2021 12 03.
Article in English | MEDLINE | ID: mdl-34702049

ABSTRACT

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.


Subject(s)
Cell Dedifferentiation , Contractile Proteins/genetics , DNA Methylation , Focal Adhesion Kinase 1/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Animals , Cells, Cultured , Contractile Proteins/metabolism , DNA Methyltransferase 3A/genetics , DNA Methyltransferase 3A/metabolism , Focal Adhesion Kinase 1/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , Proteolysis , Ubiquitination , Up-Regulation
4.
Vet Microbiol ; 252: 108928, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33248402

ABSTRACT

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.


Subject(s)
Aziridines/pharmacology , Enterovirus B, Human/drug effects , Foot-and-Mouth Disease Virus/drug effects , Foot-and-Mouth Disease/prevention & control , Swine Diseases/prevention & control , Vesicular Stomatitis/prevention & control , Vesicular stomatitis Indiana virus/drug effects , Animals , Containment of Biohazards/veterinary , Swine , Swine Diseases/virology , Vesicular Stomatitis/virology , Virus Inactivation/drug effects
5.
Exp Mol Med ; 52(6): 877-886, 2020 06.
Article in English | MEDLINE | ID: mdl-32514188

ABSTRACT

Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that is frequently overexpressed in advanced human cancers. Recent studies have demonstrated that aside from FAK's catalytic activity in cancer cells, its cellular localization is also critical for regulating the transcription of chemokines that promote a favorable tumor microenvironment (TME) by suppressing destructive host immunity. In addition to the protumor roles of FAK in cancer cells, FAK activity within cells of the TME may also support tumor growth and metastasis through various mechanisms, including increased angiogenesis and vascular permeability and effects related to fibrosis in the stroma. Small molecule FAK inhibitors have demonstrated efficacy in alleviating tumor growth and metastasis, and some are currently in clinical development phases. However, several preclinical trials have shown increased benefits from dual therapies using FAK inhibitors in combination with other chemotherapies or with immune cell activators. This review will discuss the role of nuclear FAK as a driver for tumor cell survival as well as potential therapeutic strategies to target FAK in both tumors and the TME.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Tumor Microenvironment/physiology , Animals , Focal Adhesion Protein-Tyrosine Kinases/genetics , Humans , Tumor Microenvironment/genetics
6.
Transbound Emerg Dis ; 67(5): 1942-1953, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32145150

ABSTRACT

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF. The immunoperoxidase test (IPT) is a sensitive test for detecting ASF virus (ASFV) antibodies. However, due to the complexity of the procedure, the IPT is only suitable to be used as a confirmatory test. The ASFV p30 protein-based enzyme-linked immunosorbent assay (ELISA) is widely used for ASFV antibody screening, but the sensitivity is not comparable to the IPT. It is essential to have a better understanding of the antigenic properties of ASFV p30 to improve p30-based serologic tests. In this study, we developed a panel of 21 monoclonal antibodies (mAbs) against ASFV p30. With 14 out of the 21 mAbs, we defined 4 antigenic regions that contain at least 4 linear epitopes. Nine of the 14 mAbs mapped to antigenic regions 3 and 4 reacted with p30 in all serologic methods tested in this study, such as indirect immunofluorescence assay (IFA), ELISA and Western blot. The antigenic regions 3 and 4 are highly conserved and immunodominant in host antibody response. These mAbs and the defined p30 antigenic regions 3 and 4 provide valuable tools for the development and improvement of ASF serologic assays.

7.
Oxid Med Cell Longev ; 2019: 1754593, 2019.
Article in English | MEDLINE | ID: mdl-31285782

ABSTRACT

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring low concentrations of ROS in nonphagocytic cells. We found that dissolving luminol in NaOH, rather than DMSO, increased the H2O2-induced CL signal and that the addition of 4-iodophenylboronic acid (4IPBA) further increased CL intensity. Our new system also increased the rate and intensity of the CL signal in phorbol 12-myristate 13-acetate- (PMA-) treated HT-29 colon cancer cells compared to those in luminol only. We were able to quantify ROS levels from both cells and media in parallel using an H2O2 standard. A significant benefit to our system is that we can easily measure stimulus-induced ROS formation in a real-time manner and also investigate intracellular signaling pathways from a single sample simultaneously. We found that PMA induced tyrosine phosphorylation of protein tyrosine kinases (PTKs), such as focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), and Src, and increased actin stress fiber formation in a ROS-dependent manner. Interestingly, treatment with either N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) reduced the PMA-stimulated phosphorylation of these PTKs, implicating a potential role in cellular ROS signaling. Thus, our newly optimized CL assay using 4IPBA and a chemiluminescent imaging method provides a simple, real-time, and low-cost method for the quantification of low levels of ROS.


Subject(s)
Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Boron Compounds/pharmacology , Focal Adhesion Kinase 1/metabolism , HT29 Cells , Humans , Immunoblotting , Iodobenzenes/pharmacology , Onium Compounds/pharmacology , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects
8.
Sci Rep ; 9(1): 7617, 2019 05 20.
Article in English | MEDLINE | ID: mdl-31110200

ABSTRACT

Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1ß (IL-1ß) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1ß stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1ß signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE-/- mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.


Subject(s)
Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 2/genetics , Gene Expression/genetics , Inflammation/genetics , Interleukin-1beta/genetics , Tumor Necrosis Factor-alpha/genetics , Animals , Cell Adhesion/genetics , Cell Adhesion Molecules/genetics , Cells, Cultured , Cytokines/genetics , Endothelial Cells/metabolism , Humans , MAP Kinase Signaling System/genetics , Male , Mice , Mice, Inbred C57BL , Signal Transduction/genetics , Vascular Cell Adhesion Molecule-1/genetics
9.
Circ Res ; 125(2): 152-166, 2019 07 05.
Article in English | MEDLINE | ID: mdl-31096851

ABSTRACT

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.


Subject(s)
Cell Proliferation , Cyclin D1/metabolism , Focal Adhesion Kinase 1/metabolism , GATA4 Transcription Factor/metabolism , Myocytes, Smooth Muscle/metabolism , Tunica Intima/metabolism , Active Transport, Cell Nucleus , Animals , Cell Nucleus/metabolism , Cells, Cultured , Cyclin D1/genetics , Focal Adhesion Kinase 1/antagonists & inhibitors , Hyperplasia/metabolism , Mice , Mice, Inbred C57BL , Myocytes, Smooth Muscle/physiology , Tunica Intima/pathology
10.
Vaccine ; 37(26): 3435-3442, 2019 06 06.
Article in English | MEDLINE | ID: mdl-31085001

ABSTRACT

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMDxeno, or PBS, followed by intradermal challenge with FDMV strain O1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMDxeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMDxeno, when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMDxeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity.


Subject(s)
Capsid Proteins/immunology , Epitopes/immunology , Foot-and-Mouth Disease Virus/immunology , Foot-and-Mouth Disease/immunology , Swine/immunology , Adenoviridae/immunology , Adenoviridae Infections/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cell Line , Genetic Vectors/immunology , HEK293 Cells , Humans , Immunization/methods , Vaccination/methods , Viral Vaccines/immunology
11.
Biochem Biophys Res Commun ; 509(4): 1034-1040, 2019 02 19.
Article in English | MEDLINE | ID: mdl-30660359

ABSTRACT

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1). As VCAM-1 is a major receptor for α4 integrin and plays a key role in leukocyte recruitment, we reasoned that inhibition of FAK activity may reduce VCAM-1 expression within LNs and thus reduce metastasis of α4 integrin-expressing melanoma to LNs. First, we found that a pharmacological FAK inhibitor, PF-271, blocked tumor necrosis factor-α (TNF-α)-mediated VCAM-1 expression on human dermal lymphatic endothelial cells (HDLECs). In vitro, PF-271 significantly decreased B16F10 melanoma adhesion to and transmigration through HDLECs compared to TNF-α treated cells. Furthermore, in vivo FAK inhibition by oral PF-271 administration reduced VCAM-1 expression in inguinal, cervical, and popliteal LNs compared to vehicle treated mice. Finally, in a footpad metastasis model, B16F10 melanoma cells were injected into the right footpad of C57BL/6 mice, and PF-271 (50 mg/kg, twice daily for 6 days) was orally administrated after 1 week of tumor transplantation. While untreated mice exhibited significant metastatic melanoma lesions in popliteal LNs, PF-271 treated mice showed only marginal melanoma metastasis. These results support the possibility that FAK inhibitors may be a novel preventative option in melanoma metastasis by blocking VCAM-1 expression in LNs.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Integrin alpha4/metabolism , Lymph Nodes/pathology , Melanoma/pathology , Neoplasm Metastasis/prevention & control , Vascular Cell Adhesion Molecule-1/antagonists & inhibitors , Animals , Cell Line , Humans , Melanoma/chemistry , Melanoma, Experimental , Mice , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Vascular Cell Adhesion Molecule-1/metabolism
12.
Curr Protoc Cell Biol ; 78(1): 20.11.1-20.11.14, 2018 03.
Article in English | MEDLINE | ID: mdl-30040188

ABSTRACT

To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.


Subject(s)
Cells/metabolism , Cytological Techniques/methods , DNA, Mitochondrial/isolation & purification , Mammals/metabolism , Animals , Calibration , Cell Line , Ethidium/metabolism , Gene Dosage , Humans , Mice , Polymerase Chain Reaction , Uracil-DNA Glycosidase/metabolism , Zalcitabine/metabolism
13.
Virol J ; 13: 88, 2016 Jun 04.
Article in English | MEDLINE | ID: mdl-27260412

ABSTRACT

BACKGROUND: Diagnostics and research of high-consequence animal disease agents is often limited to laboratories with a high level of biosecurity that restrict the transport of biological material. Often, sharing of DNA with external partners is needed to support diagnostics, forensics, or research. Even in the absence of virus, RNA from positive-sense single stranded RNA (+ssRNA) viruses that may contaminate otherwise purified DNA preparations continues to pose a threat due to its potential to be infectious via direct translation to yield viral proteins. While the risk of animal infection or accidental reconstitution and release of a virus from RNA is very low, the high impact of an animal disease event associated with the accidental release of some + ssRNA viruses, such as classical swine fever or foot-and-mouth disease viruses, necessitates the precaution of having procedures to ensure the complete inactivation of viruses and + ssRNA viral genomes. RNA and DNA are differentially susceptible to enzymatic degradations; however, such procedures are susceptible to unintended DNA damage and/or failure due to enzyme or cofactor instabilities. Therefore, we describe the development and verification of a robust and simple chemical and physical method to selectively degrade RNA from purified DNA preparations. The procedure employs incubation of DNA in 0.25 N sodium hydroxide at 65 °C for 1 h followed by neutralization and boiling for 10 min to hydrolyze contaminating RNA and inactivate animal disease viruses from DNA preparations. Additional critical quality control elements include use of a synthetic control RNA (SCR) and an SCR-specific real-time RT-PCR to track effectiveness of the procedure in a parallel treated control sample, and a pH check of reagents to ensure proper neutralization of alkaline conditions. RESULTS: The new procedure reduced intact RNA beyond the limit of detection by realtime RT-PCR and inactivated viruses by in vitro culture infectivity assays. CONCLUSIONS: Treated DNA, while denatured, remains suitable for most common molecular biology procedures including PCR, transformation of E. coli, and molecular sequencing. The procedure ensures not only the inactivation of a variety of viruses but also the degradation through hydrolysis of potentially contaminating infectious + ssRNA viral genomes.


Subject(s)
DNA/isolation & purification , Disinfectants/pharmacology , Hydrolysis , RNA, Viral/drug effects , Sodium Hydroxide/pharmacology , Virus Inactivation , Animals , Hot Temperature , Hydrogen-Ion Concentration , Time Factors
14.
Rev. Fac. Med. (Caracas) ; 31(1): 19-28, jun. 2008. ilus, tab
Article in Spanish | LILACS | ID: lil-631536

ABSTRACT

La insuficiencia mitral de etiología isquémica, a diferencia de aquellas en las que existe compromiso “primario” de las valvas o del aparato subvalvular, tiene características propias que obligan a su análisis y abordaje con una visión diferente y muy particular. Se realizó una revisión de la literatura médica desde enero de 1995 hasta febrero 2007 bajo las palabras clave “ischemic mitral regurgitation” y se seleccionaron un total de 49 artículos relacionados con epidemiología de la insuficiencia mitral isquémica, la evaluación de sus mecanismos y las diferentes técnicas terapéuticas. Varios mecanismos han sido descritos entre los cuales están: desplazamiento, elongación o ruptura del músculo papilar, así como dilatación y deformación del anillo. Se han desarrollado diferentes técnicas terapéuticas en la insuficiencia mitral isquémica dirigidas a corregir cada uno de ellos: Prevención del remodelado postinfarto, reposicionamiento o reparación del músculo papilar, liberación o transposición de cuerdas tendinosas, plastia mitral y reemplazo valvular mitral, entre otras. La evaluación adecuada y la identificación individualizada, por parte del cardiólogo, de los mecanismos involucrados en la patogenia de la insuficiencia mitral isquémica, es determinante para lograr una correcta escogencia del tratamiento. Seguimiento a largo plazo de algunas técnicas terapéuticas empleadas es necesario para conocer su efectividad


Ischemic Mitral regurgitation, unlike those with a primary commitment of the valves and subvalvular apparatus, has particular characteristics that need an analysis and boarding with a different and special vision by the cardiologist. Was made an overhaul of medical publication from January 1995 to February 2007 with the key words “ischemic mitral regurgitation” and were selected a total of 49 articles related to ischemic mitral regurgitation epidemiology, evaluation of its mechanisms and the corresponding therapeutic techniques. Several mechanisms have been described: displacement, elongation or rupture of papillary muscle, as well as the expansion and deformation of the mitral ring. Different techniques directed to correct each one of these mechanisms have been developed: Post myocardial infarction remodeling prevention, papillary muscle relocation or repair, mitral chordae tendineae liberation or translocation, valve repair and valve replacement, among others. The suitable evaluation and individualized identification of the pathological mechanisms involved in the genesis of ischemic mitral regurgitation by de cardiologist is determinant for to choose the best therapy. Long term following of some therapeutic techniques is necessary for to know its effectiveness


Subject(s)
Humans , Mitral Valve Insufficiency/physiopathology , Mitral Valve Insufficiency/therapy , Myocardial Ischemia
15.
J Biol Chem ; 282(1): 700-9, 2007 Jan 05.
Article in English | MEDLINE | ID: mdl-17090525

ABSTRACT

The Th2 cytokine IL-13 is a major effector molecule in human allergic inflammation. Notably, IL-13 expression at birth correlates with subsequent susceptibility to atopic disease. In order to characterize the chromatin-based mechanisms that regulate IL-13 expression in human neonatal CD4(+) T cells, we analyzed patterns of DNase I hypersensitivity and epigenetic modifications within the IL-13 locus in cord blood CD4(+) T cells, naive or differentiated in vitro under Th1- or Th2-polarizing conditions. In naive CD4(+) T cells, hypersensitivity associated with DNA hypomethylation was limited to the distal promoter. Unexpectedly, during both Th1 and Th2 differentiation, the locus was extensively remodeled, as revealed by the formation of numerous HS sites and decreased DNA methylation. Obvious differences in chromatin architecture were limited to the proximal promoter, where strong hypersensitivity, hypomethylation, and permissive histone modifications were found selectively in Th2 cells. In addition to revealing the locations of putative cis-regulatory elements that may be required to control IL-13 expression in neonatal CD4(+) T cells, our results suggest that differential IL-13 expression may depend on the acquisition of a permissive chromatin architecture at the proximal promoter in Th2 cells rather than the formation of locus-wide repressive chromatin in Th1 cells.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chromatin/chemistry , Gene Expression Regulation , Interleukin-13/genetics , Chromatin/metabolism , DNA Methylation , Epigenesis, Genetic , Fetal Blood/metabolism , Flow Cytometry , Histones/metabolism , Humans , Infant, Newborn , Interleukin-13/metabolism , Models, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Th1 Cells/metabolism , Th2 Cells/metabolism
16.
Environ Health Perspect ; 113(6): 775-81, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15929903

ABSTRACT

We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7-11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18-79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials.


Subject(s)
Arsenic/metabolism , Arsenicals/urine , Glutathione Transferase/genetics , Methyltransferases/genetics , Polymorphism, Genetic , Purine-Nucleoside Phosphorylase/genetics , Water Pollutants, Chemical/metabolism , Adolescent , Adult , Aged , Arsenic/urine , Child , Environmental Monitoring , Female , Genotype , Humans , Male , Methylation , Mexico , Middle Aged , Molecular Sequence Data , Water Pollutants, Chemical/urine , Water Supply
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