RESUMO
BACKGROUND: The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants. RESULTS: Informatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes. CONCLUSIONS: An in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.
Assuntos
Sequenciamento do Exoma/métodos , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Camundongos , Ratos , Análise de Sequência de DNA/métodos , Fixação de TecidosRESUMO
Sustainable fertilizer production is a pressing challenge due to a growing human population. The manufacture of synthetic nitrogen fertilizer involves intensive emissions of greenhouse gases. The synthetic nitrogen that ends up in biowaste such as animal waste perturbs the nitrogen cycle through significant nitrogen losses in the form of ammonia volatilization, a major human health and environmental hazard. Low-temperature air-plasma treatment of animal waste holds promise for sustainable fertilizer production on farmlands by enabling nitrogen fixation via ionization, forming nitrogen oxyacids. Although the formation of nitrogen oxyacids in plasma treatment of water is well-established, the extent of nitrogen oxyanion enrichment in animal waste and its downstream effects on acidifying the waste remain elusive because many compounds found in complex biowaste media may interfere with absorbed NOx species. This work aims to establish that plasma treatment of dairy manure can suppress ammonia loss by volatilization via acidification of animal waste while enriching the waste in total nitrogen due to nitrogen retained in ammonia as well as adding nitrogen oxyacids by reacting NOx with the aqueous slurry. To this end, air-plasma effluent containing NOx is bubbled through dairy manure, which is then analyzed for changes in the nitrogen oxyanion content and pH. Increasing the plasma treatment time results in more acidic manure, reduced ammonium content in the downstream acid trap, and increased nitrogen oxyanion content, where the yield of nitrogen oxyanion from absorbed NOx species is approximately 100%. Increased plasma treatment also led to an increase in the total Kjeldahl nitrogen and the total nitrogen. These results indicate that plasma treatment of animal waste can significantly suppress ammonia pollution from animal husbandry facilities such as dairy farms while upcycling animal waste as a rich organic source of nitrogen.
RESUMO
Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinant α-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reduced α-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca(2+)-responsiveness of filament sliding speed was increased either by increased pCa(50) (V95A), reduced cooperativity n (D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.
Assuntos
Actinas/metabolismo , Cardiomiopatia Hipertrófica Familiar/genética , Mutação , Tropomiosina/genética , Tropomiosina/metabolismo , Actinas/química , Animais , Cálcio/metabolismo , Cardiomiopatia Hipertrófica Familiar/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Coelhos , Temperatura , Tropomiosina/químicaRESUMO
College students lack fact-checking skills, which may lead them to accept information at face value. We report findings from an institution participating in the Digital Polarization Initiative (DPI), a national effort to teach students lateral reading strategies used by expert fact-checkers to verify online information. Lateral reading requires users to leave the information (website) to find out whether someone has already fact-checked the claim, identify the original source, or learn more about the individuals or organizations making the claim. Instructor-matched sections of a general education civics course implemented the DPI curriculum (N = 136 students) or provided business-as-usual civics instruction (N = 94 students). At posttest, students in DPI sections were more likely to use lateral reading to fact-check and correctly evaluate the trustworthiness of information than controls. Aligning with the DPI's emphasis on using Wikipedia to investigate sources, students in DPI sections reported greater use of Wikipedia at posttest than controls, but did not differ significantly in their trust of Wikipedia. In DPI sections, students who failed to read laterally at posttest reported higher trust of Wikipedia at pretest than students who read at least one problem laterally. Responsiveness to the curriculum was also linked to numbers of online assignments attempted, but unrelated to pretest media literacy knowledge, use of lateral reading, or self-reported use of lateral reading. Further research is needed to determine whether improvements in lateral reading are maintained over time and to explore other factors that might distinguish students whose skills improved after instruction from non-responders.
Assuntos
Leitura , Estudantes , Currículo , Humanos , AprendizagemRESUMO
Genetic defects in DNA repair are increasingly recognized as being able to cause degenerative ataxia syndromes. It remains a mystery, however, why disruption of a process fundamental to proliferating cells can be selectively toxic to postmitotic neurons. Recent studies now reveal that an ataxia gene, tyrosyl phosphodiesterase 1 (TDP1), repairs single-stranded DNA breaks in nondividing cells. Here we review the implications of this and other findings for a growing list of hereditary ataxias.
Assuntos
Reparo do DNA/fisiologia , Degenerações Espinocerebelares/genética , Animais , DNA de Cadeia Simples/fisiologia , Humanos , Diester Fosfórico Hidrolases/genética , Degenerações Espinocerebelares/fisiopatologiaRESUMO
Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid beta precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Abeta1-40 transgenes in APP mouse models. Expression of BRI2-Abeta1-40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Abeta deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Abeta aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Abeta deposition in vivo.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/fisiologia , Encéfalo/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Amiloide/genética , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/administração & dosagem , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Galinhas , Cricetinae , Dependovirus/genética , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Glicoproteínas de Membrana , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , FenótipoRESUMO
Instabilities play a prominent role in determining the inherent structure and properties of magnetized plasma jets spanning both laboratory and astrophysical settings. The manner in which prominent unstable modes dynamically evolve remains key to understanding plasma behavior and control. In astrophysical phenomena, self-similar jets are observed to propagate over vast distances while avoiding breakup caused by unstable mode growth. However, the production of stable dense plasma jets in the laboratory has been limited by the onset of unstable modes that restrict jet lifetime, collimation, and scalability. In this work, we visualize the formation of stable laboratory-generated, dense, super-magnetosonic plasma jets in real time, and we identify an underlying mechanism that contributes to this behavior. The current-driven plasma jets generated in our experiments form a flowing Z-pinch, which is generally unstable to the m = 1 kink instability. Our results indicate that a stable dense plasma jet can be maintained for timescales over which a steady pinch current can be sustained, even at levels which would otherwise lead to rapid unstable mode growth and resultant pinch disassembly.
RESUMO
BACKGROUND & AIMS: Oxidative stress and depletion of antioxidants may play a role in the pathogenesis of Crohn's disease (CD). The aim of this study was to determine the effect of exclusive enteral nutrition, which is increasingly being used as primary therapy for CD, on plasma antioxidant concentrations in children with active CD. METHODS: In a double-blind randomised controlled trial, 15 children with active CD (mean age, 11.3 years, range 6.8-15.7) attending a paediatric gastroenterology referral centre, were assigned to receive either a standard polymeric diet (Group S, n=8) or a glutamine-enriched polymeric diet (Group G, n=7) as primary therapy for active CD. Plasma concentrations of selenium, urates, vitamin A, vitamin E, vitamin C, glutathione, and also malondialdehyde (MDA) were measured at baseline and after 4 weeks of exclusive enteral nutritional treatment. RESULTS: Mean (95% CI) selenium concentration of the cohort increased significantly from 0.82 micromol/l (0.72, 0.91) to 1.14 micromol/l (0.98, 1.3), P<0.001. There were, however, significant reductions in mean concentrations of vitamin C {11.8 mg/l (7.7, 15.8) to 6.5 mg/l (4.5, 8.7), P=0.01} and vitamin E {11.3 mg/l (10.3, 12.4) to 9.4 mg/l (8.7, 10.1), P=0.03}. The concentrations of vitamin A, urates, glutathione and MDA did not change significantly over the study period. Glutamine supplementation did not have any significant effect on plasma antioxidant concentrations. CONCLUSIONS: Significant changes in circulating antioxidant concentrations occurred in children with active CD receiving exclusive enteral nutritional treatment. Glutamine supplementation was not beneficial in improving plasma antioxidant status.
Assuntos
Antioxidantes/metabolismo , Doença de Crohn/sangue , Doença de Crohn/terapia , Nutrição Enteral , Glutamina/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Adolescente , Ácido Ascórbico/sangue , Criança , Método Duplo-Cego , Feminino , Glutamina/metabolismo , Glutationa/sangue , Humanos , Masculino , Malondialdeído/sangue , Selênio/sangue , Ácido Úrico/sangue , Vitamina A/sangue , Vitamina E/sangueRESUMO
Huntington's disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As both a cochaperone and ubiquitin ligase, the C-terminal Hsp70 (heat shock protein 70)-interacting protein (CHIP) links the two major arms of protein quality control, molecular chaperones, and the ubiquitin-proteasome system. Here, we demonstrate that CHIP suppresses polyQ aggregation and toxicity in transfected cell lines, primary neurons, and a novel zebrafish model of disease. Suppression by CHIP requires its cochaperone function, suggesting that CHIP acts to facilitate the solubility of mutant polyQ proteins through its interactions with chaperones. Conversely, HD transgenic mice that are haploinsufficient for CHIP display a markedly accelerated disease phenotype. We conclude that CHIP is a critical mediator of the neuronal response to misfolded polyQ protein and represents a potential therapeutic target in this important class of neurodegenerative diseases.
Assuntos
Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Peptídeos/metabolismo , Ubiquitina-Proteína Ligases/farmacologia , Animais , Western Blotting/métodos , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Modelos Animais de Doenças , Embrião de Mamíferos , Embrião não Mamífero , Imunofluorescência/métodos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Doença de Huntington/tratamento farmacológico , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Neurônios/citologia , Peptídeos/genética , Radioimunoensaio/métodos , Ratos , Transfecção/métodos , Peixe-ZebraRESUMO
Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.
Assuntos
Alelos , Doença de Alzheimer/genética , Mutação/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Sequência de Bases , Células COS , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Reporter/genética , Células HeLa , Humanos , Especificidade por Substrato , Proteínas Virais , Proteínas tau/genéticaRESUMO
The temporomandibular opening index (TOI) is a more useful measure of mandibular movement than linear mouth opening, since it is independent of age, gender, ramus length, and gonial angle. It is also useful when categorizing temporomandibular disorder (TMD) patients into diagnostic groups. Two subgroups of myogenous patients have been identified, one with a high and one with a low temporomandibular opening index. This study examined initial symptom severity in these two subgroups. Thirty-three (33) patients with a myogenous temporomandibular disorder were recruited. Twenty-six (26) were female and seven male. Eleven were found to be in the high temporomandibular opening index group and the remaining 22 in the low group. Symptom severity scores were determined prior to the start of treatment. Pain, joint sounds, headache, and neck pain were all rated by patients on a four-point verbal response scale. These symptom scores were compared between the two subgroups using the Wilcoxon two sample test. There appeared to be a significant difference between the two groups (p = 0.0025). TMD patients with high temporomandibular opening index appeared to have more severe signs and symptoms of TMD than patients with a low index.
Assuntos
Amplitude de Movimento Articular/fisiologia , Transtornos da Articulação Temporomandibular/diagnóstico , Adolescente , Adulto , Idoso , Criança , Dor Facial/fisiopatologia , Feminino , Cefaleia/fisiopatologia , Humanos , Masculino , Mandíbula/fisiopatologia , Pessoa de Meia-Idade , Cervicalgia/fisiopatologia , Medição da Dor , Som , Articulação Temporomandibular/fisiopatologia , Transtornos da Articulação Temporomandibular/fisiopatologia , Síndrome da Disfunção da Articulação Temporomandibular/diagnóstico , Síndrome da Disfunção da Articulação Temporomandibular/fisiopatologiaRESUMO
OBJECTIVE: The cardinal signs and symptoms of temporomandibular disorder (TMD) are pain in joints and/or muscles, joint sounds, and limitation of movement. They are also associated with other complaints, one of which is headache. Myogenous TMD patients can be divided into those with a high and low temporomandibular opening index (TOI). These two subgroups appear to vary in several ways, including symptom severity. The objective was to assess the relationship between reported headache and TMD patients and a control group with no TMD and to compare the report of headache in high- and low-TOI myogenous TMD patients. METHOD AND MATERIALS: Sixty-six patients with TMD were included in the study. Fortythree were diagnosed with myogenous TMD, 23 with arthrogenous TMD, and 20 with no TMD were included as a control. Patients reported a history of headache using a four-point Verbal Rating Scale for both severity and frequency. Multiple logistic regression analysis was performed, after adjusting for confounders of sex and age. This helped investigate the association between the study groups and reported headache. Seventeen of the myogenous TMD patients were studied further. Seven were assigned to the high and 10 to the low-TOI group. Mean ages were 38.43 years and 33.00 years respectively. The Mann Whitney test was used to examine the difference in report of headache between these two groups. RESULTS: 76.7% of the myogenous group, 26.1% of the arthrogenous group, and 35% of the control group reported headache. Age and myogenous TMD were significantly associated with reported headache (P = .001 and .01, respectively). Myogenous TMD is a significant risk factor (OR = 5.20, P = .01) for reported headache while arthrogenous TMD is not (OR = 0.75, P = .69) A significant difference in report of headache between the two myogenous TMD groups was found (P = .0067). CONCLUSION: The risk for reported headache is 5.20-times greater for myogenous TMD patients compared to the control group, but no difference was noted between the arthrogenous TMD and the control group. Age serves as a mild protective for reported headache. Younger patients tend to report more headaches. More frequent and severe headache occurred in the high-TOI group. This study serves as a reminder for clinicians in general practice to consider the effect of comorbidity when faced with TMD patients with headache.
Assuntos
Cefaleia/complicações , Transtornos da Articulação Temporomandibular/fisiopatologia , Articulação Temporomandibular/fisiopatologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos da Articulação Temporomandibular/complicações , Adulto JovemRESUMO
OBJECTIVE: The temporomandibular opening index (TOI) is endfeel distance divided by active and passive mouth opening. The asymmetry index (AI) is a measure of difference in left and right condylar heights. This study examined the relationship between AI and TOI in myogenous TMD and non-TMD patients. METHOD AND MATERIALS: Fourteen myogenous TMD patients diagnosed by the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) were recruited (1 man and 13 women) and 14 non-TMD patients (6 men and 8 women) were included as controls. Differences between the TMD patients and control group of non-TMD patients were determined by the two-tailed t test, while multiple linear regression analysis was used to examine the correlation between AI and TOI adjusting for sex and age. RESULTS: AI and TOI were significantly higher (P = .001 and P = .045, respectively) among TMD patients. A significant positive correlation was found between TOI and AI for the TMD group (r = 0.84, P = .01) but not for the control group. CONCLUSION: A positive correlation was found between TOI and AI in the myogenous TMD group.
Assuntos
Côndilo Mandibular/fisiopatologia , Amplitude de Movimento Articular/fisiologia , Síndrome da Disfunção da Articulação Temporomandibular/fisiopatologia , Articulação Temporomandibular/fisiopatologia , Adolescente , Adulto , Cefalometria/métodos , Feminino , Humanos , Masculino , Côndilo Mandibular/patologia , Pessoa de Meia-Idade , Projetos Piloto , Radiografia Panorâmica , Articulação Temporomandibular/patologia , Adulto JovemRESUMO
BACKGROUND: Alzheimer's disease (AD) and a host of other neurodegenerative central nervous system (CNS) proteinopathies are characterized by the accumulation of misfolded protein aggregates. Simplistically, these aggregates can be divided into smaller, soluble, oligomeric and larger, less-soluble or insoluble, fibrillar forms. Perhaps the major ongoing debate in the neurodegenerative disease field is whether the smaller oligomeric or larger fibrillar aggregates are the primary neurotoxin. Herein, we propose an integrative hypothesis that provides new insights into how a variety of misfolded protein aggregates can result in neurodegeneration. RESULTS: We introduce the concept that a wide range of highly stable misfolded protein aggregates in AD and other neurodegenerative proteinopathies are recognized as non-self and chronically activate the innate immune system. This pro-inflammatory state leads to physiological senescence of CNS cells. Once CNS cells undergo physiological senescence, they secrete a variety of pro-inflammatory molecules. Thus, the senescence of cells, which was initially triggered by inflammatory stimuli, becomes a self-reinforcing stimulus for further inflammation and senescence. Ultimately, senescent CNS cells become functionally impaired and eventually die, and this neurodegeneration leads to brain organ failure. CONCLUSION: This integrative hypothesis, which we will refer to as the proteinopathy-induced senescent cell hypothesis of AD and other neurodegenerative diseases, links CNS proteinopathies to inflammation, physiological senescence, cellular dysfunction, and ultimately neurodegeneration. Future studies characterizing the senescent phenotype of CNS cells in AD and other neurodegenerative diseases will test the validity of this hypothesis. The implications of CNS senescence as a contributing factor to the neurodegenerative cascade and its implications for therapy are discussed.
RESUMO
In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.
Assuntos
Proteínas de Drosophila/metabolismo , Glicoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Chlorocebus aethiops , Primers do DNA , Proteínas de Drosophila/genética , Glicoproteínas/genética , Glicosilação , Humanos , Cinética , Dados de Sequência Molecular , Neurônios/fisiologia , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Ubiquitina/genéticaRESUMO
Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca(2+) activation. To determine whether Ca(2+) sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and alpha-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca(2+) sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca(2+) sensitivity.
Assuntos
Citoesqueleto de Actina/fisiologia , Cálcio/metabolismo , Coração/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miosinas/fisiologia , Animais , Miosinas Cardíacas/fisiologia , Humanos , Isoenzimas/fisiologia , Cinética , Subfragmentos de Miosina/fisiologia , Coelhos , Proteínas Recombinantes/metabolismo , Suínos , Tropomiosina/metabolismo , Troponina/metabolismo , Troponina T/metabolismoRESUMO
1.RNA interference (RNAi) is a recently discovered biological pathway that mediates post-transcriptional gene silencing. The process of RNAi is orchestrated by an increasingly well-understood cellular machinery. 2. The common entry point for both natural and engineered RNAi are double stranded RNA molecules known as short interfering RNAs (siRNAs), that mediate the sequence-specific identification and degradation of the targeted messenger RNA (mRNA). The study and manipulation of these siRNAs has recently revolutionized biomedical research. 3. In this review, we first provide a brief overview of the process of RNAi, focusing on its potential role in brain function and involvement in neurological disease. We then describe the methods developed to manipulate RNAi in the laboratory and its applications to neuroscience. Finally, we focus on the potential therapeutic application of RNAi to neurological disease.
Assuntos
Biologia Molecular/tendências , Neurociências/tendências , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Animais , Química Encefálica/genética , Inativação Gênica/fisiologia , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Biologia Molecular/métodos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurociências/métodos , RNA Mensageiro/genéticaRESUMO
PURPOSE: To assess magnetic resonance (MR) safety for a newly developed, fiber-optic cardiac pacing lead. MATERIALS AND METHODS: MR safety was assessed for the fiber-optic cardiac pacing lead by evaluating magnetic field interactions and heating. Translational attraction and torque were evaluated using a 1.5-Tesla MR system and previously described, standardized techniques. MR imaging-related heating was assessed using a 1.5-Tesla MR system and a transmit/receive, body radiofrequency (RF) coil with the fiber-optic lead positioned to simulate an in vivo condition in a saline-filled phantom. The phantom had dimensions similar to a human subject's torso and head. A fluoroptic thermometry system was used to record temperatures on and near the electrodes of the fiber-optic pacing lead at five-second intervals immediately before and during 20 minutes of MR imaging performed at a whole-body-averaged specific absorption rate (SAR) of 1.5 W/kg. Temperatures were also recorded from a reference site during this experiment. RESULTS: Magnetic field interactions for the fiber-optic lead were minimal (deflection angle, 23 degrees; torque, +2). The highest temperature change recorded for the fiber-optic cardiac pacing lead and reference site was +0.8 degrees C. CONCLUSION: The minor magnetic field interactions and relative lack of heating for the fiber-optic pacing lead indicate that it should be safe for patients with this device to undergo MR imaging procedures using MR systems operating at 1.5-T or less and at a whole-body-averaged SARs up to 1.5 W/kg.
Assuntos
Imageamento por Ressonância Magnética/normas , Marca-Passo Artificial , Eletrodos , Segurança de Equipamentos , Temperatura Alta/efeitos adversos , Humanos , Imageamento por Ressonância Magnética/instrumentação , Imagens de FantasmasRESUMO
Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.
Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Animais , Ataxina-3 , Células COS , Proteína de Ligação a CREB , Proteínas de Fluorescência Verde , Transtornos Heredodegenerativos do Sistema Nervoso/etiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Neurológicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Transfecção , Repetições de TrinucleotídeosRESUMO
OBJECTIVE: To describe a treatment strategy for paradoxical vocal-cord dysfunction (PVCD) as it applies to an athletic population. BACKGROUND: Paradoxical vocal-cord dysfunction has been identified as a cause of dyspnea and stridor in athletes. The basic element of PVCD is an inappropriate closure of the vocal cords during respiration, resulting in airway obstruction. This condition is familiar to speech-language pathologists and otolaryngologists yet remains poorly understood in the sports medicine community. Treatment strategies are even less understood. A therapeutic exercise program designed to promote diaphragmatic breathing may allow an athlete to gain control during episodes of dyspnea. Elimination of contributing or concomitant conditions is critical to resolution of the condition. DESCRIPTION: The treatment of PVCD requires an understanding of the pathoanatomy of the condition. The focus of the exercise program is on relaxation of the larynx and conscious activation of the diaphragm and abdominal muscles during respiration. The athlete must have a sense of laryngeal control while performing the exercises. In addition, the patient and practitioner must realize the amount of neuromuscular reeducation required to change breathing patterns. CLINICAL ADVANTAGES: This therapy may allow the athlete to gain control over episodic dyspnea, participate in athletic activities with fewer complications, and, perhaps, reduce or eliminate medications prescribed to treat suspected bronchospasm.