RESUMO
The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
Assuntos
Células/metabolismo , Edição de Genes/métodos , Genoma Humano/genética , National Institutes of Health (U.S.)/organização & administração , Animais , Terapia Genética , Objetivos , Humanos , Estados UnidosRESUMO
Gene-targeted therapies (GTTs) are therapeutic platforms that are in principle applicable to large numbers of monogenic diseases. The rapid development and implementation of GTTs have profound implications for rare monogenic disease therapy development. This article provides a brief summary of the primary types of GTTs and a brief overview of the current state of the science. It also serves as a primer for the articles in this special issue.
Assuntos
Terapia Genética , HumanosRESUMO
Most rare diseases are caused by single-gene mutations, and as such, lend themselves to a host of new gene-targeted therapies and technologies including antisense oligonucleotides, phosphomorpholinos, small interfering RNAs, and a variety of gene delivery and gene editing systems. Early successes are encouraging, however, given the substantial number of distinct rare diseases, the ability to scale these successes will be unsustainable without new development efficiencies. Herein, we discuss the need for genomic newborn screening to match pace with the growing development of targeted therapeutics and ability to rapidly develop individualized therapies for rare variants. We offer approaches to move beyond conventional "one disease at a time" preclinical and clinical drug development and discuss planned regulatory innovations that are necessary to speed therapy delivery to individuals in need. These proposals leverage the shared properties of platform classes of therapeutics and innovative trial designs including master and platform protocols to better serve patients and accelerate drug development. Ultimately, there are risks to these novel approaches; however, we believe that close partnership and transparency between health authorities, patients, researchers, and drug developers present the path forward to overcome these challenges and deliver on the promise of gene-targeted therapies for rare diseases.
Assuntos
Edição de Genes , Doenças Raras , Recém-Nascido , Humanos , Doenças Raras/diagnóstico , Doenças Raras/genética , Doenças Raras/terapia , Terapia Genética/métodos , GenômicaRESUMO
It is a commonly held view that oxidatively induced DNA lesions are repaired by the base excision repair (BER) pathway, whereas DNA lesions induced by UV light and other "bulky" chemical adducts are repaired by the nucleotide excision repair (NER) pathway. While this distinction is generally accurate, the 8,5'-cyclopurine deoxynucleosides represent an important exception, in that they are formed in DNA by the hydroxyl radical, but are specifically repaired by NER, not by BER. They are also strong blocks to nucleases and polymerases, including RNA polymerase II in human cells. In this review, I will discuss the evidence that these lesions are in part responsible for the neurodegeneration that occurs in some XP patients, and what additional evidence would be necessary to prove such a role. I will also consider other DNA lesions that might be involved in XP neurologic disease. Finally, I will also discuss how our recent studies of these lesions have generated novel insights into the process of transcriptional mutagenesis in human cells, as well as the value of studying these lesions not only for a better understanding of NER but also for other aspects of human health and disease.
Assuntos
Dano ao DNA , Reparo do DNA , Desoxirribonucleosídeos/química , Doenças Neurodegenerativas/genética , Transcrição Gênica , Xeroderma Pigmentoso/genética , Adutos de DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Xeroderma Pigmentoso/metabolismoRESUMO
The classic model for neurodegeneration due to mutations in DNA repair genes holds that DNA damage accumulates in the absence of repair, resulting in the death of neurons. This model was originally put forth to explain the dramatic loss of neurons observed in patients with xeroderma pigmentosum neurologic disease, and is likely to be valid for other neurodegenerative diseases due to mutations in DNA repair genes. However, in trichiothiodystrophy (TTD), Aicardi-Goutières syndrome (AGS), and Cockayne syndrome (CS), abnormal myelin is the most prominent neuropathological feature. Myelin is synthesized by specific types of glial cells called oligodendrocytes. In this review, we focus on new studies that illustrate two disease mechanisms for myelin defects resulting from mutations in DNA repair genes, both of which are fundamentally different than the classic model described above. First, studies using the TTD mouse model indicate that TFIIH acts as a co-activator for thyroid hormone-dependent gene expression in the brain, and that a causative XPD mutation in TTD results in reduction of this co-activator function and a dysregulation of myelin-related gene expression. Second, in AGS, which is caused by mutations in either TREX1 or RNASEH2, recent evidence indicates that failure to degrade nucleic acids produced during S-phase triggers activation of the innate immune system, resulting in myelin defects and calcification of the brain. Strikingly, both myelin defects and brain calcification are both prominent features of CS neurologic disease. The similar neuropathology in CS and AGS seems unlikely to be due to the loss of a common DNA repair function, and based on the evidence in the literature, we propose that vascular abnormalities may be part of the mechanism that is common to both diseases. In summary, while the classic DNA damage accumulation model is applicable to the neuronal death due to defective DNA repair, the myelination defects and brain calcification seem to be better explained by quite different mechanisms. We discuss the implications of these different disease mechanisms for the rational development of treatments and therapies.
Assuntos
Encefalopatias/genética , Dano ao DNA , Reparo do DNA/genética , Mutação , Encefalopatias/imunologia , Encefalopatias/patologia , Encefalopatias/terapia , Humanos , Imunidade Inata , Fator de Transcrição TFIIH/metabolismoRESUMO
According to a recent IARC Working Group report, alcohol consumption is causally related to an increased risk of cancer of the upper aerodigestive tract, liver, colorectum, and female breast [R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, V. Bouvard, A. Altieri, V. Cogliano, Carcinogenicity of alcoholic beverages, Lancet Oncol. 8 (2007) 292-293]. Several lines of evidence indicate that acetaldehyde (AA), the first product of alcohol metabolism, plays a very important role in alcohol-related carcinogenesis, particularly in the esophagus. We previously proposed a model for alcohol-related carcinogenesis in which AA, generated from alcohol metabolism, reacts in cells to generate DNA lesions that form interstrand crosslinks (ICLs) [J.A. Theruvathu, P. Jaruga, R.G. Nath, M. Dizdaroglu, P.J. Brooks, Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde, Nucleic Acids Res. 33 (2005) 3513-3520]. Since the Fanconi anemia-breast cancer associated (FANC-BRCA) DNA damage response network plays a crucial role in protecting cells against ICLs, in the present work we tested this hypothesis by exposing cells to AA and monitoring activation of this network. We found that AA exposure results in a concentration-dependent increase in FANCD2 monoubiquitination, which is dependent upon the FANC core complex. AA also stimulated BRCA1 phosphorylation at Ser1524 and increased the level of gammaH2AX, with both modifications occurring in a dose-dependent manner. However, AA did not detectably increase the levels of hyperphosphorylated RPA34, a marker of single-stranded DNA exposure at replication forks. These results provide the initial description of the AA-DNA damage response, which is qualitatively similar to the cellular response to mitomycin C, a known DNA crosslinking agent. We discuss the mechanistic implications of these results, as well as their possible relationship to alcohol-related carcinogenesis in different human tissues.
Assuntos
Acetaldeído/toxicidade , Proteína BRCA1/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Histonas/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Linhagem Celular , Reagentes de Ligações Cruzadas/toxicidade , Dano ao DNA/efeitos dos fármacos , Etanol/toxicidade , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Humanos , Técnicas In Vitro , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Mitomicina/toxicidade , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.
Assuntos
Proteínas de Ciclo Celular/análise , Núcleo Celular/metabolismo , DNA Topoisomerases Tipo I/análise , Proteínas de Ligação a DNA/análise , Proteínas Nucleares/análise , Proteínas Serina-Treonina Quinases/análise , Células de Purkinje/metabolismo , Proteínas Supressoras de Tumor/análise , Hidrolases Anidrido Ácido , Adolescente , Adulto , Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The 8,5'-cyclopurine-2'-deoxynucleosides (cPu) are unique oxidatively induced DNA lesions in that they are specifically repaired by NER. In the absence of NER, a possible mechanism for cPu removal is spontaneous glycosidic bond hydrolysis followed by enzymic processing. Such a mechanism could be significant if the glycosidic bond in cPu were substantially destabilized, as shown for other DNA lesions. Therefore, we investigated the stability of the glycosidic bond in a cPu, (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) against acid hydrolysis. For comparison, we also studied 8-hydroxy-2'-deoxyadenosine (8-OH-dA). We found that the glycosidic bond in S-cdA is approximately 40-fold more resistant to glycosidic bond hydrolysis compared to dA. Interestingly, under the same conditions, the glycosidic bond in 8-OH-dA was even more stable than in S-cdA. These studies effectively rule out any mechanism for the removal of S-cdA or 8-OH-dA from DNA that requires spontaneous glycosidic bond hydrolysis, and further support the proposed role of cPu in the neurodegeneration observed in xeroderma pigmentosum patients who lack NER. Of broader significance, since NER does not function in non-transcribed DNA sequences of terminally differentiated cells, including neurons, cPu are expected to accumulate in such sequences even in individuals with normal NER, which could be important in the ageing process.
Assuntos
Dano ao DNA , Desoxiadenosinas/química , Formiatos/química , Estresse Oxidativo , Envelhecimento/genética , Cromatografia Líquida de Alta Pressão , Reparo do DNA , Desoxiadenosinas/metabolismo , Humanos , Hidrólise , Cinética , Espectrometria de Massas , Temperatura , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genéticaRESUMO
Patients with the genetic disease xeroderma pigmentosum (XP) lack the capacity to carry out a specific type of DNA repair process called nucleotide excision repair (NER). The NER pathway plays a critical role in the repair of DNA damage resulting from ultraviolet (UV) radiation. A subset of XP patients develops a profound neurodegenerative condition known as XP neurological disease. Robbins and colleagues [Andrews A, Barrett S, Robbins J (1978) Xeroderma pigmentosum neurological abnormalities correlate with the colony forming ability after ultraviolet irradiation. Proc Natl Acad Sci U S A 75:1984-1988] hypothesized that since UV light cannot reach into the human brain, XP neurological disease results from some form of endogenous DNA damage that is normally repaired by the NER pathway. In the absence of NER, the damage accumulates, causing neuronal death by blocking transcription. In this manuscript, I consider the evidence that a particular class of oxidative DNA lesions, the 8,5'-cyclopurine-2'-deoxynucleosides, fulfills many of the criteria expected of neurodegenerative DNA lesions in XP. Specifically, these lesions are chemically stable, endogenous DNA lesions that are repaired by the NER pathway but not by any other known process, and strongly block transcription by RNA polymerase II in cells from XP patients. A similar set of criteria might be used to evaluate other candidate DNA lesions responsible for neurological diseases resulting from defects in other DNA repair mechanisms as well.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Nucleosídeos de Purina/metabolismo , Xeroderma Pigmentoso/genética , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/fisiopatologia , Dano ao DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Humanos , Estrutura Molecular , Nucleosídeos de Purina/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/fisiopatologiaRESUMO
Alcoholic beverage consumption is associated with an increased risk of upper gastrointestinal cancer. Acetaldehyde (AA), the first metabolite of ethanol, is a suspected human carcinogen, but the molecular mechanisms underlying AA carcinogenicity are unclear. In this work, we tested the hypothesis that polyamines could facilitate the formation of mutagenic alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (Cr-PdG) adducts from biologically relevant AA concentrations. We found that Cr-PdG adducts could be formed by reacting deoxyguanosine with muM concentrations of AA in the presence of spermidine, but not with either AA or spermidine alone. The identities of the Cr-PdG adducts were confirmed by both liquid and gas chromatography-mass spectrometry. Using a novel isotope-dilution liquid chromatography-mass spectrometry assay, we found that in the presence of 5 mM spermidine, AA concentrations of 100 microM and above resulted in the formation of Cr-PdG in genomic DNA. These AA levels are within the range that occurs in human saliva after alcoholic beverage consumption. We also showed that spermidine directly reacts with AA to generate crotonaldehyde (CrA), most likely via an enamine aldol condensation mechanism. We propose that AA derived from ethanol metabolism is converted to CrA by polyamines in dividing cells, forming Cr-PdG adducts, which may be responsible for the carcinogenicity of alcoholic beverage consumption.
Assuntos
Acetaldeído/química , Adutos de DNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Mutagênese , Espermidina/química , Acetaldeído/metabolismo , Aldeídos/química , Adutos de DNA/metabolismo , Desoxiguanosina/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espermidina/farmacologiaRESUMO
The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N2-ethyl-2'-deoxyguanosine (N2-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N2-ethyl-dG adducted oligonucleotides. However, the N2-ethyl-dG adduct enhanced CPT-induced Top1-DNA cleavage complexes depending on the relative position of the N2-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N2-ethyl-dG adduct was present immediately 3' from the Top1 site (position +1). In addition, when the N2-ethyl-dG adduct was located at the -5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N2-ethyl-dG adduct located at the 5' end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1-DNA complex, thereby inhibiting further the religation (closing) reaction.
Assuntos
Camptotecina/farmacologia , Adutos de DNA/química , DNA Topoisomerases Tipo I/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Inibidores Enzimáticos/farmacologia , DNA/química , DNA/metabolismo , Substâncias Macromoleculares , Modelos MolecularesRESUMO
Hip resurfacing has been proposed as an alternative to traditional total hip arthroplasty in young, active patients. Much has been learned following the introduction of metal-on-metal resurfacing devices in the 1990s. The triad of a well-designed device, implanted accurately, in the correct patient has never been more critical than with these implants. Following Food and Drug Administration approval in 2006, we studied the safety and effectiveness of one hip resurfacing device (Birmingham Hip Resurfacing) at our hospital in a large, single-surgeon series. We report our early to mid-term results in 1333 cases followed for a mean of 4.3 years (2 to 5.7) using a prospective, observational registry. The mean patient age was 53.1 years (12 to 84); 70% were male and 91% had osteoarthritis. Complications were few, including no dislocations, no femoral component loosening, two femoral neck fractures (0.15%), one socket loosening (0.08%), three deep infections (0.23%), and three cases of metallosis (0.23%). There were no destructive pseudotumours. Overall survivorship at up to 5.7 years was 99.2%. Aseptic survivorship in males under the age of 50 was 100%. We believe this is the largest United States series of a single surgeon using a single resurfacing system.
Assuntos
Artroplastia de Quadril/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/educação , Artroplastia de Quadril/instrumentação , Criança , Feminino , Cabeça do Fêmur , Humanos , Curva de Aprendizado , Masculino , Pessoa de Meia-Idade , Tratamentos com Preservação do Órgão , Estudos Prospectivos , Resultado do Tratamento , Estados Unidos , Adulto JovemRESUMO
We measured stimulation of c-fos and oxytocin gene expression during excitation of oxytocin cells induced by systemic or local morphine withdrawal. Female rats were made morphine-dependent by intracerebroventricular morphine infusion over 5 d. Morphine withdrawal, induced by systemic injection of the opioid antagonist naloxone (5 mg/kg) in conscious or anesthetized rats, increased the density of c-fos messenger RNA and of oxytocin heterogeneous nuclear RNA in supraoptic nucleus cells compared with those of nonwithdrawn rats; c-fos messenger RNA was also increased in the magnocellular and parvocellular paraventricular nuclei of withdrawn rats. Morphine withdrawal increased the number of Fos-immunoreactive cells in the supraoptic and magnocellular paraventricular nuclei of conscious or pentobarbitone-anesthetized rats. Morphine withdrawal also increased Fos-immunoreactive cell numbers in the parvocellular paraventricular nucleus of conscious but not anesthetized rats. Central administration of the alpha(1)-adrenoreceptor antagonist benoxathian (5 microg/min) did not prevent morphine withdrawal-induced increases in the numbers of Fos-immunoreactive neurons in the supraoptic or magnocellular paraventricular nucleus. Unilateral microdialysis administration of naloxone (10(-5) M) into the supraoptic nucleus of anesthetized morphine-dependent rats increased Fos-immunoreactive cell numbers compared with the contralateral nucleus. Finally, we investigated whether dependence could be induced by chronic unilateral infusion of morphine into a supraoptic nucleus; systemic naloxone (5 mg/kg) increased Fos-immunoreactive cell numbers in the morphine-infused nucleus compared with the contralateral nucleus. Thus, morphine withdrawal excitation increases c-fos and oxytocin gene expression in supraoptic nucleus neurons. This occurs independently from excitation of their ascending noradrenergic inputs, and both dependence and withdrawal can be induced within the supraoptic nucleus.
Assuntos
Expressão Gênica , Hipotálamo/fisiologia , Morfina/efeitos adversos , Entorpecentes/efeitos adversos , Ocitocina/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Síndrome de Abstinência a Substâncias/genética , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Feminino , Hipotálamo/citologia , Hipotálamo/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/farmacologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/fisiologia , Núcleo Hipotalâmico Paraventricular/metabolismo , RNA Nuclear Heterogêneo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Núcleo Supraóptico/metabolismoRESUMO
8,5'-Cyclo-2'-deoxypurine (cPu) lesions result from the action of the hydroxyl radical on DNA. These lesions represent a unique class of oxidative DNA lesions in that they are repaired by the nucleotide excision repair (NER) pathway but not by base excision repair (BER) or direct repair. Previous work has shown that cyclopurines can block mammalian DNA and RNA polymerases. Thus, these lesions are of interest because of their potential role in the neurodegeneration as well as internal cancers observed in patients with xeroderma pigmentosum (XP) who lack the capacity to carry out NER. In the present work, we found that the S-isomer of 8,5'-cyclo-2'-deoxyadenosine (cA) can prevent binding of the TATA binding protein (TBP) to the TATA box from the CMV promoter. To assess the functional importance of this effect in living cells, we transfected constructs containing a single cA in the CMV TATA box into XP cells to determine the effect of the lesion on gene expression in vivo. Using this approach, we found that the lesion reduced gene expression by approximately 75%. This effect was comparable to the effect of an inactivating mutation of the TATA box in the same promoter. These findings identify an additional biological effect of cyclopurine lesions in mammalian cells, which is the ability to interfere with transcription by preventing transcription factor binding to cognate recognition sequences. In addition, the approach we used in this study represents a novel method for assessing the effects of DNA lesions in non-transcribed sequences on gene expression in living cells.
Assuntos
Reparo do DNA/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , TATA Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Xeroderma Pigmentoso/genética , Células Cultivadas , Citomegalovirus/genética , Dano ao DNA/efeitos dos fármacos , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Estresse Oxidativo , Plasmídeos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Xeroderma Pigmentoso/metabolismoRESUMO
Previous studies demonstrating the presence of immunoreactive insulin-like growth factors (IGFs) and their receptors in the brain suggest a role of the IGFs in the central nervous system. IGF-II has been implicated as the predominant IGF in brain of mature animals based on studies of immunoreactive peptide and of IGF-II mRNAs. To obtain information about the sites of synthesis of IGF-II in adult rat brain, a 32P-labeled 31 base long synthetic oligodeoxyribonucleotide complementary in sequence to trailer peptide coding sequences in rat IGF-II mRNA (IGF-II 31 mer) was hybridized with coronal sections of fixed rat brain. The IGF-II 31 mer showed specific hybridization with the choroid plexus throughout rat brain, whereas in other brain regions, structures or cells, hybridization was not discernibly above background. These findings suggest that the choroid plexus is a primary site of synthesis of IGF-II, a probable source of IGF-II in cerebrospinal fluid, and a potential source of IGF-II for actions on target cells within the adult rat brain.
Assuntos
Plexo Corióideo/metabolismo , Fator de Crescimento Insulin-Like II/genética , RNA Mensageiro/biossíntese , Somatomedinas/genética , Transcrição Gênica , Animais , Autorradiografia , Encéfalo/citologia , Encéfalo/metabolismo , Plexo Corióideo/citologia , Hibridização de Ácido Nucleico , Radioisótopos de Fósforo , RNA Mensageiro/genética , RatosRESUMO
The GH dependence of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and insulin like growth factor II (IGF-II) mRNAs was investigated by Northern blot hybridizations of polyadenylated RNAs from liver, pancreas, and brain of normal rats, untreated hypophysectomized rats, and hypophysectomized rats 4 h or 8 h after an ip injection of human GH (hGH). Using a 32P-labeled human Sm-C/IGF-I cDNA as probe, four Sm-C/IGF-I mRNAs of 7.5, 4.7, 1.7, and 1.2 kilobases (kb) were detected in rat liver and pancreas but were not detectable in brain. In both liver and pancreas, the abundance of these Sm-C/IGF-I mRNAs was 8- to 10-fold lower in hypophysectomized rats than in normal rats. Within 4 h after injection of hGH into hypophysectomized animals, the abundance of liver and pancreatic Sm-C/IGF-I mRNAs was restored to normal. A human IGF-II cDNA was used as a probe for rat IGF-II mRNAs which were found to be very low in abundance in rat liver and showed no evidence of regulation by GH status. In pancreas, IGF-II mRNA abundance was below the detection limit of the hybridization procedures. The brain contained two IGF-II mRNAs of 4.7 and 3.9 kb that were 5-fold lower in abundance in hypophysectomized rats than in normal rats. These brain IGF-II mRNAs were not, however, restored to normal abundance at 4 or 8 h after ip hGH injection into hypophysectomized animals. To investigate further, the effect of GH status on abundance of Sm-C/IGF-I and IGF-II mRNAs in rat brain, a second experiment was performed that differed from the first in that hypophysectomized rats were given an injection of hGH into the lateral ventricle (intracerebroventricular injection) and a rat Sm-C/IGF-I genomic probe was used to analyze Sm-C/IGF-I mRNAs. In this experiment, a 7.5 kb Sm-C/IGF-I mRNA was detected in brain polyadenylated RNAs. The abundance of the 7.5 kb mRNA was 4-fold lower in hypophysectomized rats than in normal rats and was increased to 80% of normal within 4 h after icv administration of hGH to hypophysectomized animals. As in the first experiment, the abundance of the 4.7 and 3.9 kb brain IGF-II mRNAs was lower than normal in hypophysectomized rats. Brain IGF-II mRNAs were increased to 50% of normal in hypophysectomized rats given an icv injection of hGH but within 8 h after the injection rather than at 4 h as with Sm-C/IGF-I mRNAs.
Assuntos
Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/efeitos dos fármacos , Somatomedinas/genética , Animais , Encéfalo/efeitos dos fármacos , Hormônio do Crescimento/administração & dosagem , Hipofisectomia , Injeções Intraperitoneais , Injeções Intraventriculares , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like II/análise , Fígado/efeitos dos fármacos , Masculino , Pâncreas/efeitos dos fármacos , RNA Mensageiro/análise , Ratos , Ratos EndogâmicosRESUMO
8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region.
Assuntos
Antioxidantes/metabolismo , Encéfalo/metabolismo , Dano ao DNA , Reparo do DNA , 8-Hidroxi-2'-Desoxiguanosina , Animais , Biomarcadores , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Radicais Livres/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Superóxido Dismutase/metabolismo , Distribuição TecidualRESUMO
Two separate forms of glutamic acid decarboxylase, now termed GAD65 and GAD67, are the rate limiting enzymes for synthesis of gamma-aminobutyric acid (GABA). Because of the significance of GABA to neuroendocrine processes, numerous attempts have been made to determine the impact of gonadal steroids on enzyme functioning with inconclusive results. Therefore, we attempted to determine the impact of estradiol on mRNA levels for each form of GAD by quantitative in situ hybridization histochemistry in various brain regions. Ovariectomized rats were treated with estradiol benzoate or oil vehicle on 2 consecutive days and the brains collected on the third day. DNA probes specific for GAD65 and GAD67 were radiolabeled with CTP32 using asymmetric polymerase chain reaction. Results of in situ hybridizations for each probe on alternate sections from the same animals were analyzed for magnocellular preoptic area (McPOA), dorsal medial nucleus of the hypothalamus (DMN), zona incerta (ZI), and midbrain central gray (MCG). In the McPOA, estradiol exerted opposite effects on the frequency distribution of pixels per cell for two GAD mRNA probes, significantly increasing GAD65 (P < .05) and decreasing GAD67 (P < .01; Kolmogorov-Smirnov). In the DMN, estradiol treatment significantly increased GAD67 by 60% (P < .05; two-way ANOVA) but decreased GAD65 mRNA by 73% (P < .01). Note the direction of effects are opposite between McPOA and DMN. In MCG, analysis showed no estradiol effect on GAD mRNA levels/cells, but the proportion of cells expressing detectable levels of GAD65 or GAD67 increased by 33-40% in estradiol-treated rats (chi 2, P < .001).