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1.
JTCVS Open ; 11: 398-411, 2022 Sep.
Article in English | MEDLINE | ID: mdl-36172446

ABSTRACT

Objective: To determine the incidence, outcomes, and evaluate diagnostic modalities for postoperative vocal cord dysfunction (VCD) following cardiothoracic surgery in children. Methods: A prospective mixed-methods study using principles of implementation science was completed. All patients undergoing surgery involving the aortic arch, ductus, or ligamentum arteriosum and vascular rings from September 2019 to December 2020 were enrolled. Patients underwent speech pathology assessment, laryngeal ultrasound, and flexible direct laryngoscopy. Results: Ninety-five patients were eligible for inclusion. The incidence of VCD ranged from 18% to 56% and varied according to procedure group. VCD occurred in 42% of neonates. Repair of hypoplastic aortic arch was associated with increased risk of VCD (57%; P = .002). There was no significant difference in duration of intubation, pediatric intensive care unit stay, or hospital stay. Forty percent children were able to achieve full oral feeding. Children with VCD were more likely to require nasogastric supplementary feeding at discharge (60% vs 36%; P = .044). Sixty-eight percent of patients demonstrated complete resolution of VCD at a median of 97 days postoperatively. Laryngeal ultrasound and speech pathology assessment combined had a sensitivity of 91% in comparison to flexible direct laryngoscopy. Conclusions: VCD occurred in one-third and resolved in two-thirds of patients at a median of 3 months following cardiac surgery. Aortic arch repair carried the highest risk of VCD. VCD adversely influenced feeding. Forty percent of patients achieved full oral feeding before discharge. VCD did not delay intensive care unit or hospital discharge. Speech pathology assessment and laryngeal ultrasound combined was reliable for diagnosis in most patients and was more patient friendly than flexible direct laryngoscopy.

2.
Cardiol Young ; 32(5): 775-781, 2022 May.
Article in English | MEDLINE | ID: mdl-34348806

ABSTRACT

Every year in Australia over a thousand children who are born with congenital heart disease require surgical intervention. Vocal cord dysfunction (VCD) can be an unavoidable and potentially devastating complication of surgery for congenital heart disease. Structured, multidisciplinary care pathways help to guide clinical care and reduce mortality and morbidity. An implementation study was conducted to embed a novel, multidisciplinary management pathway into practice using the consolidated framework for implementation research (CFIR). The goal of the pathway was to prepare children with postoperative vocal cord dysfunction to safely commence and transition to oral feeding. Education sessions to support pathway rollout were completed with clinical stakeholders. Other implementation strategies used included adaptation of the pre-procedural pathway to obtain consent, improving the process of identifying patients on the VCD pathway, and nominating a small team who were responsible for the ongoing monitoring of patients following recruitment. Implementation success was evaluated according to compliance with pathway defined management. Our study found that while there were several barriers to pathway adoption, implementation of the pathway was feasible despite pathway adaptations that were required in response to COVID-19.


Subject(s)
COVID-19 , Vocal Cord Dysfunction , Australia/epidemiology , Child , Critical Pathways , Humans
3.
J Biol Chem ; 297(5): 101172, 2021 11.
Article in English | MEDLINE | ID: mdl-34624316

ABSTRACT

The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.


Subject(s)
Glioblastoma/metabolism , Glycoproteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Neoplasm Proteins/metabolism , Signal Transduction , T-Lymphoma Invasion and Metastasis-inducing Protein 1/metabolism , Cell Line, Tumor , Gene Knockout Techniques , Glioblastoma/genetics , Glycoproteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Humans , Neoplasm Proteins/genetics , Phosphorylation/genetics , T-Lymphoma Invasion and Metastasis-inducing Protein 1/genetics
4.
J Clin Invest ; 128(10): 4654-4668, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30198904

ABSTRACT

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.


Subject(s)
B7-H1 Antigen/immunology , Immunotherapy , Killer Cells, Natural/immunology , Neoplasms, Experimental/therapy , Programmed Cell Death 1 Receptor/immunology , Animals , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/genetics , Humans , K562 Cells , Killer Cells, Natural/pathology , Mice , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/genetics
5.
Exp Cell Res ; 371(1): 83-91, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30059665

ABSTRACT

SIRT1 is a protein deacetylase with a broad range of biological functions, many of which are known to be important in carcinogenesis, however much of the literature regarding the role of SIRT1 in cancer remains conflicting. In this study we assessed the effect of SIRT1 on the initiation and progression of thymic T cell lymphomas. We employed mouse strains in which SIRT1 activity was absent or could be reversibly modulated in conjunction with thymic lymphoma induction using either the N-nitroso-N-methylurea (NMU) carcinogenesis or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) transgene. Decreased SIRT1 activity reduced the development of thymic lymphomas in the NMU-treated mice but was permissive for the formation of lung adenomas. Conversely, in the NPM-ALK transgenic mice, decreased SIRT1 activity had a modest promoting effect in the development of thymic lymphomas. The results of the work presented here add to the growing body of evidence that sirt1 is neither an outright oncogene nor a tumor suppressor. These opposing results in two models of the same disease suggest that the influence of sirt1 on carcinogenesis may lie in a role in tumor surveillance.


Subject(s)
Adenocarcinoma of Lung/genetics , Gene Expression Regulation, Neoplastic , Lymphoma, T-Cell/genetics , Oncogene Proteins, Fusion/genetics , Protein-Tyrosine Kinases/genetics , Sirtuin 1/genetics , Thymus Neoplasms/genetics , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/etiology , Adenocarcinoma of Lung/mortality , Administration, Oral , Animals , Antineoplastic Agents, Hormonal/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinogenesis/pathology , Lymphoma, T-Cell/drug therapy , Lymphoma, T-Cell/etiology , Lymphoma, T-Cell/mortality , Male , Methylnitrosourea/administration & dosage , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oncogene Proteins, Fusion/metabolism , Organ Specificity , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Sirtuin 1/metabolism , Survival Analysis , Tamoxifen/pharmacology , Thymus Gland/drug effects , Thymus Gland/metabolism , Thymus Gland/pathology , Thymus Neoplasms/drug therapy , Thymus Neoplasms/etiology , Thymus Neoplasms/mortality , Transfection
6.
PLoS One ; 12(3): e0173002, 2017.
Article in English | MEDLINE | ID: mdl-28273169

ABSTRACT

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4-5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2-4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.


Subject(s)
Sirtuin 1/metabolism , Alleles , Animals , Blood Glucose/drug effects , Catalysis , Cell Line , Enzyme Activation , Female , Gene Expression , Genotype , Male , Mice , Mice, Knockout , Mice, Transgenic , Phenotype , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sirtuin 1/genetics , Tamoxifen/pharmacology
7.
PLoS One ; 9(11): e112406, 2014.
Article in English | MEDLINE | ID: mdl-25380034

ABSTRACT

The protein deacetylase SIRT1 is involved in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. Both SIRT1 activity and tumorigenesis can be influenced by dietary fat and polyphenolics. We set out to determine whether dietary modulations of tumorigenesis are mediated by SIRT1 catalytic functions. We introduced a mammary gland tumor-inducing transgene, MMTV-PyMT, into stocks of mice bearing a H355Y point mutation in the Sirt1 gene that abolishes SIRT1 catalytic activity. Tumor latency was reduced in animals fed a high fat diet but this effect was not dependent on SIRT1 activity. Resveratrol had little effect on tumor formation except in animals heterozygous for the mutant Sirt1 gene. We conclude that the effects of these dietary interventions on tumorigenesis are not mediated by modulation of SIRT1 catalytic activity.


Subject(s)
Cell Transformation, Neoplastic/genetics , Diet, High-Fat , Point Mutation , Sirtuin 1/genetics , Analysis of Variance , Animals , Antigens, Polyomavirus Transforming/genetics , Antineoplastic Agents, Phytogenic/pharmacology , Biocatalysis , Cell Transformation, Neoplastic/drug effects , Heterozygote , Male , Mammary Neoplasms, Experimental/diet therapy , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mammary Tumor Virus, Mouse/genetics , Mice, Transgenic , Resveratrol , Sirtuin 1/metabolism , Stilbenes/pharmacology , Tumor Burden/drug effects , Tumor Burden/genetics
8.
FASEB J ; 28(3): 1306-16, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24297700

ABSTRACT

Type 2 diabetes, hepatic steatosis, and gut dysbiosis are pathophysiological consequences of obesity. Sirtuin (SIRT)-1 is a protein deacetylase implicated in the regulation of metabolic activity. We set out to determine whether the catalytic activity of SIRT1 plays a role in the development of metabolic syndrome, hepatic steatosis, and the distribution of gut microbiota. We challenged with a high-fat diet (HFD) a strain of mice homozygous for a Sirt1 allele carrying a point mutation that ablates the deacetylase activity of SIRT1. When compared to wild-type animals, mice lacking SIRT1 catalytic activity rapidly accumulated excessive hepatic lipid while fed the HFD, an effect evident within 2 wk of HFD feeding. Both white and brown adipose depots became hypertrophic, and the animals developed insulin resistance. The ratio of the major phyla of gut microbiota (Firmicutes and Bacteroidetes) increased rapidly in the SIRT1-deficient mice after HFD challenge. We conclude that the deacetylase activity of SIRT1 plays an important role in regulating glucose and hepatic lipid homeostasis. In addition, the composition of gut microbiota is influenced by both the animals' Sirt1 genotype and diet composition.


Subject(s)
Metabolic Syndrome/metabolism , Sirtuin 1/metabolism , Adipose Tissue/metabolism , Animals , Base Sequence , DNA Primers , Energy Metabolism , Glucose/metabolism , Homeostasis , Intestines/microbiology , Liver/pathology , Magnetic Resonance Imaging , Mice
9.
PLoS One ; 8(11): e82106, 2013.
Article in English | MEDLINE | ID: mdl-24278473

ABSTRACT

The protein deacetylase SIRT1 has been implicated in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. There are conflicting data that make it unclear whether Sirt1 functions as an oncogene or tumor suppressor. To assess the effect of SIRT1 on the emergence and progression of mammary tumors, we crossed mice that harbor a point mutation that abolishes SIRT1 catalytic activity with mice carrying the polyoma middle T transgene driven by the murine mammary tumor virus promoter (MMTV-PyMT). The absence of SIRT1 catalytic activity neither accelerated nor blocked the formation of tumors and metastases in this model. There was a lag in tumor latency that modestly extended survival in Sirt1 mutant mice that we attribute to a delay in mammary gland development and not to a direct effect of SIRT1 on carcinogenesis. These results are consistent with previous evidence suggesting that Sirt1 is not a tumor promoter or a tumor suppressor.


Subject(s)
Breast Neoplasms/metabolism , Disease Models, Animal , Sirtuin 1/metabolism , Animals , Breast Neoplasms/pathology , Catalysis , Female , Lung Neoplasms/secondary , Male , Mice
10.
Biochim Biophys Acta ; 1832(10): 1624-33, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23685312

ABSTRACT

Muscle insulin resistance is linked to oxidative stress and decreased mitochondrial function. However, the exact cause of muscle insulin resistance is still unknown. Since offspring of patients with type 2 diabetes mellitus (T2DM) are susceptible to developing insulin resistance, they are ideal for studying the early development of insulin resistance. By using primary muscle cells derived from obese non-diabetic subjects with (FH+) or without (FH-) a family history of T2DM, we aimed to better understand the link between mitochondrial function, oxidative stress, and muscle insulin resistance. Insulin-stimulated glucose uptake and glycogen synthesis were normal in FH+ myotubes. Resting oxygen consumption rate was not different between groups. However, proton leak was higher in FH+ myotubes. This was associated with lower ATP content and decreased mitochondrial membrane potential in FH+ myotubes. Surprisingly, mtDNA content was higher in FH+ myotubes. Oxidative stress level was not different between FH+ and FH- groups. Reactive oxygen species content was lower in FH+ myotubes when differentiated in high glucose/insulin (25mM/150pM), which could be due to higher oxidative stress defenses (SOD2 expression and uncoupled respiration). The increased antioxidant defenses and mtDNA content in FH+ myotubes suggest the existence of compensatory mechanisms, which may provisionally prevent the development of insulin resistance.


Subject(s)
Muscle Fibers, Skeletal/enzymology , Obesity/metabolism , Protons , Superoxide Dismutase/metabolism , Case-Control Studies , DNA, Mitochondrial/metabolism , Diabetes Mellitus, Type 2/genetics , Female , Genetic Predisposition to Disease , Humans , Insulin Resistance , Male , Membrane Potential, Mitochondrial , Middle Aged , Obesity/enzymology
11.
FASEB J ; 26(2): 555-66, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22006156

ABSTRACT

The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.


Subject(s)
Fertility/physiology , Sirtuin 1/metabolism , Amino Acid Substitution , Animals , Base Sequence , Caloric Restriction , Conserved Sequence , DNA Primers/genetics , Female , Fertility/genetics , Histidine/chemistry , Homeostasis , Infertility, Male/genetics , Infertility, Male/metabolism , Male , Mice , Mice, 129 Strain , Mice, Knockout , Mice, Mutant Strains , Motor Activity/genetics , Motor Activity/physiology , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Point Mutation , Pregnancy , Sirtuin 1/chemistry , Sirtuin 1/deficiency , Sirtuin 1/genetics , Spermatogenesis/genetics , Spermatogenesis/physiology
12.
PLoS One ; 6(9): e24643, 2011.
Article in English | MEDLINE | ID: mdl-21931792

ABSTRACT

BACKGROUND: Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. CONCLUSION/SIGNIFICANCE: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.


Subject(s)
Genetic Markers/genetics , Poxviridae/genetics , Recombination, Genetic/genetics , Animals , Cattle , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Genetic Vectors/genetics , Humans
13.
Exp Cell Res ; 316(13): 2189-93, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20412791

ABSTRACT

Sirtuin 1 (SIRT1) is an NAD-dependent histone deacetylase (HDAC) whose activity is thought to forestall the onset of a variety of age-related diseases. Mice carrying null mutations of the Sirt1 gene suffer high rates of neonatal lethality and those that survive are sterile, growth retarded, lean and their livers express high levels of insulin-like growth factor binding protein-1 (IGFBP1). IGFBP1 binds and regulates the bioavailability of Igfs. Interestingly, Igfbp1 transgenic mice largely phenocopy Sirt1-/- mice, suggesting the possibility that the over-expression of IGFBP1 in Sirt1-/- mice might be responsible for many of their phenotypes. We interbred Sirt1 heterozygote mice to Igfbp1-deficient mice to test the hypothesis that the disruption of one or both alleles of Igfbp1 would rescue the phenotype of Sirt1-/- mice. We report that mono- or bi-allelic disruption of the Igfbp1 gene had no effect on the embryonic and neonatal lethality of Sirt1-/- mice. However, we show that mice lacking at least one allele of both Sirt1 and Igfbp1 genes have a much higher incidence of malocclusion.


Subject(s)
Insulin-Like Growth Factor Binding Protein 1/physiology , Malocclusion/etiology , Malocclusion/pathology , Sirtuin 1/physiology , Animals , Crosses, Genetic , Genotype , Humans , Mice , Mice, Knockout , Mice, Transgenic , Phenotype
14.
Exp Cell Res ; 314(16): 3069-74, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18687325

ABSTRACT

The sirt1 gene encodes a protein deacetylase with a broad spectrum of reported substrates. Mice carrying null alleles for sirt1 are viable on outbred genetic backgrounds so we have examined them in detail to identify the biological processes that are dependent on SIRT1. Sera from adult sirt1-null mice contain antibodies that react with nuclear antigens and immune complexes become deposited in the livers and kidneys of these animals. Some of the sirt1-null animals develop a disease resembling diabetes insipidus when they approach 2 years of age although the relationship to the autoimmunity remains unclear. We interpret these observations as consistent with a role for SIRT1 in sustaining normal immune function and in this way delaying the onset of autoimmune disease.


Subject(s)
Autoimmune Diseases/immunology , Sirtuins/metabolism , Animals , Autoimmune Diseases/genetics , Cells, Cultured , Diabetes Insipidus/genetics , Diabetes Insipidus/immunology , Disease Models, Animal , Endotoxins/immunology , Female , Humans , Immunity, Innate , Immunoglobulins/immunology , Kidney/cytology , Kidney/metabolism , Liver/cytology , Liver/immunology , Mice , Mice, Knockout , Sirtuin 1 , Sirtuins/genetics , Urine
15.
PLoS One ; 3(3): e1759, 2008 Mar 12.
Article in English | MEDLINE | ID: mdl-18335035

ABSTRACT

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.


Subject(s)
Energy Intake , Energy Metabolism/physiology , Sirtuins/physiology , Animals , Calorimetry , Hormones/blood , Mice , Mice, Knockout , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Sirtuin 1
16.
Aging Cell ; 5(1): 81-8, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16441846

ABSTRACT

The SirT1 gene encodes a protein deacetylase that acts on a number of nuclear substrates. p53 was identified as a SirT1 substrate whose transcriptional activity was reported to be negatively regulated by SirT1-dependent deacetylation. We set out to determine whether developmental defects and perinatal lethality observed in SirT1-null mice were caused by p53 hyperactivity by creating mice deficient for both SirT1 and p53. Animals null for both proteins were smaller than normal at birth, had eyelid opening defects and died during the late prenatal and early postnatal periods, a phenotype indistinguishable from mice deficient for SirT1 alone. Upon re-examination of the role of SirT1 in modulating p53 activity, we found that while SirT1 interacts with p53, the SirT1 protein had little effect on p53-dependent transcription of transfected or endogenous genes and did not affect the sensitivity of thymocytes and splenocytes to radiation-induced apoptosis. These findings suggest that SirT1 does not affect many p53-mediated biological activities despite the fact that acetylated p53 has been shown to be a substrate for SirT1.


Subject(s)
Sirtuins/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis , Cells, Cultured , Crosses, Genetic , Dose-Response Relationship, Radiation , Eyelids/anatomy & histology , Mice , Mice, Transgenic , Phenotype , Protein Binding , Sirtuin 1 , Sirtuins/deficiency , Spleen/cytology , Spleen/radiation effects , Survival , T-Lymphocytes, Regulatory/radiation effects , Transcriptional Activation/genetics , Tumor Suppressor Protein p53/genetics
17.
Exp Cell Res ; 288(2): 313-23, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12915122

ABSTRACT

Efficient high-level expression of transfected genes in stem cells is difficult to achieve. Using P19 embryonal carcinoma stem cells, we previously showed that cotransfection with a fragment of the Pgk-1 gene increased the number of integrated copies of genes on cotransfected plasmids and resulted in enhanced expression of these genes. Active transcription through introns and exons of the Pgk-1-derived sequence was required for elevated expression of the cotransfected reporter genes. We show here that the enhanced expression appears to be due to two effects: (1) increased ligation of plasmid DNAs prior to integration into the host genome and (2) maintenance of an active domain in the region comprising the cluster of transgenes. We found that individual clones of stably transformed cells had highly variable numbers of transgenes integrated into their genomes but the ratio of genes comprising each transgene was proportional to the ratio of genes on different plasmids in the transfection mixture. The presence of the Pgk-1 gene fragment ensured that the expression of cotransfected genes in each clone was proportional to the numbers of integrated gene copies and selection for high-level expression of one cotransfected gene ensures high-level expression of the other cotransfected genes.


Subject(s)
Gene Dosage , Gene Expression Regulation, Neoplastic , Gene Silencing , Neoplastic Stem Cells/physiology , Transcription, Genetic , Transgenes , Animals , Embryonal Carcinoma Stem Cells , Exons , Genes, Reporter , Introns , Plasmids/genetics , Plasmids/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Statistics as Topic
18.
Mol Cancer Res ; 1(5): 402-9, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12651913

ABSTRACT

The yeast sir2 gene plays a central role in mediating gene silencing and DNA repair in this organism. The mouse sir2alpha gene is closely related to its yeast homologue and encodes a nuclear protein expressed at particularly high levels in embryonic stem (ES) cells. We used homologous recombination to create ES cells null for sir2alpha and found that these cells did not have elevated levels of acetylated histones and did not ectopically express silent genes. Unlike yeast sir2 mutants, our sir2alpha null ES cells had normal sensitivity to insults such as ionizing radiation and heat shock, and they were able to silence invading retroviruses normally. These sir2alpha null cells were able to differentiate in culture normally. Our results failed to provide evidence that the mammalian SIR2alpha protein plays a role in gene silencing and suggest that the physiological substrate(s) for the SIR2alpha deacetylase may be nuclear proteins other than histones.


Subject(s)
Carcinoma, Embryonal , Gene Silencing/physiology , Sirtuins/genetics , Sirtuins/metabolism , Stem Cells/physiology , Acetylation , Animals , DNA Repair/physiology , Gene Expression Regulation, Neoplastic , Histones/metabolism , Mice , Proviruses/genetics , Retroviridae/genetics , Retroviridae Infections/genetics , Sirtuin 1 , Stem Cells/cytology , Stem Cells/radiation effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , X-Rays
19.
Mol Cell Biol ; 23(1): 38-54, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12482959

ABSTRACT

The yeast Sir2p protein has an essential role in maintaining telomeric and mating type genes in their transcriptionally inactive state. Mammalian cells have a very large proportion of their genome inactive and also contain seven genes that have regions of homology with the yeast sir2 gene. One of these mammalian genes, sir2alpha, is the presumptive mammalian homologue of the yeast sir2 gene. We set out to determine if sir2alpha plays a role in mammalian gene silencing by creating a strain of mice carrying a null allele of sir2alpha. Animals carrying two null alleles of sir2alpha were smaller than normal at birth, and most died during the early postnatal period. In an outbred background, the sir2alpha null animals often survived to adulthood, but both sexes were sterile. We found no evidence for failure of gene silencing in sir2alpha null animals, suggesting that either SIR2alpha has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing in confined to a small subset of mammalian genes. The phenotype of the sir2alpha null animals suggests that the SIR2alpha protein is essential for normal embryogenesis and for normal reproduction in both sexes.


Subject(s)
Embryo, Mammalian/physiology , Germ Cells/physiology , Sirtuins/genetics , Sirtuins/metabolism , Animals , Animals, Newborn , Animals, Outbred Strains , Euchromatin/genetics , Euchromatin/metabolism , Female , Fetal Death/genetics , Gene Expression Regulation, Developmental , Gene Silencing , Infertility, Female/genetics , Infertility, Male/genetics , Male , Mammals , Mice , Mice, Inbred Strains , Mice, Knockout , Mice, Mutant Strains , Sirtuin 1 , Telomere/genetics , Testis/abnormalities , Testis/physiology
20.
Exp Cell Res ; 274(1): 1-8, 2002 Mar 10.
Article in English | MEDLINE | ID: mdl-11855851

ABSTRACT

Foreign DNA can be readily integrated into the genomes of mammalian embryonic cells by retroviral infection, DNA microinjection, and transfection protocols. However, the transgenic DNA is frequently not expressed or is expressed at levels far below expectation. In a number of organisms such as yeast, plants, Drosophila, and nematodes, silencing of transfected genes is triggered by the interaction between adjacent or dispersed copies of genes of identical sequence. We set out to determine whether a mechanism similar to repeat-induced gene silencing (RIGS) is responsible for the silencing of transgenes in murine embryonal carcinoma stem cells. We compared the expression of identical reporter gene constructs in cells carrying single or multiple copies and found that the level of expression per integrated copy was more than 10-fold higher in single-copy integrants. In cells carrying tandem copies of the transgene, many copies were methylated and clones frequently failed to express both copies of near-identical integrated alleles. Addition of extra copies of the reporter gene coding sequence reduced the level of expression from the same reporter driven by a eukaryotic promoter. We also found that inhibitors of histone deacetylase such as trichostatin A forestall the silencing of multicopy transgenes, suggesting that chromatin mediates the silencing of transfected genes. This evidence is consistent with the idea that RIGS does occur in mammalian embryonic stem cells although silencing of single-copy transgenes also occurs, suggesting that RIGS is only one of the mechanisms responsible for triggering transgene silencing.


Subject(s)
Tandem Repeat Sequences/genetics , Transgenes/genetics , Animals , Cells, Cultured/cytology , Cells, Cultured/metabolism , Chromatin/genetics , Chromatin/physiology , Embryo, Mammalian/cytology , Gene Dosage , Gene Expression Regulation , Gene Silencing/drug effects , Genes, Reporter , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Mammals , Mice , Stem Cells/cytology , Stem Cells/metabolism , Stem Cells/ultrastructure , Tumor Cells, Cultured
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