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1.
Mol Genet Metab ; 141(1): 108111, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38103461

RESUMO

Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.


Assuntos
Melanoma , Metionina , Humanos , Metionina/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Racemetionina/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Homocisteína/metabolismo , Vitamina B 12/metabolismo , Oxirredutases/metabolismo
2.
CNS Neurosci Ther ; 28(3): 364-371, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34984839

RESUMO

INTRODUCTION: About 20%-35% of multiple sclerosis (MS) patients fail to respond to high-dose corticosteroids during a relapse. Repository corticotropin injection (RCI, Acthar® Gel) is a naturally sourced complex mixture of adrenocorticotropic hormone analogs and pituitary peptides that has anti-inflammatory and immunomodulatory effects. AIMS: The study objective was to determine the efficacy and safety of RCI in patients with MS relapse that inadequately responded to corticosteroids. This was a multicenter, double-blind, placebo-controlled study. Nonresponders to high-dose corticosteroids were randomized to receive RCI (80 U) or placebo daily for 14 days. Assessments included improvements on the Expanded Disability Status Scale (EDSS), Multiple Sclerosis Impact Scale (MSIS-29), Clinical Global Impression of Improvement (CGI-I), and adverse events (AEs). RESULTS: Eighteen patients received RCI, and 17 received placebo. A greater proportion of EDSS responders was observed in the RCI group at Day 7, 21, and 42 compared with the placebo group. Qualitative CGI-I showed that more patients receiving RCI were much improved or very much improved than with placebo. No meaningful differences were observed between treatment groups for MSIS-29. No serious AEs or deaths were reported. CONCLUSION: RCI is safe and effective for MS relapse patients who do not respond to high-dose corticosteroids.


Assuntos
Esclerose Múltipla , Corticosteroides/uso terapêutico , Hormônio Adrenocorticotrópico/uso terapêutico , Doença Crônica , Método Duplo-Cego , Humanos , Esclerose Múltipla/tratamento farmacológico , Recidiva
3.
Artigo em Inglês | MEDLINE | ID: mdl-36926525

RESUMO

Long-term alteration of dopaminergic neurotransmission is known to modulate the D2/D3 receptor expression in the brain. The modulation can occur as a response to pathological processes or pharmacological intervention. The receptor density can be monitored by in vivo positron emission tomography (PET) of [11C] raclopride. To obtain accurate measurements of receptor-ligand interaction, it is essential to estimate binding parameters at true (if transient) equilibrium of bound and unbound ligand quantities. We designed this study as a comparison of two quantitative approaches to transient equilibrium, the TRansient EquilibriuM BoLus Estimation (TREMBLE) method and the Transient Equilibrium Model (TEM) method, to determine binding parameters at transient equilibrium with bolus injection of the radioligand. The data demonstrates that TREMBLE unlike TEM identified the time at which equilibrium existed. TREMBLE revealed that equilibrium prevailed at one or more times after bolus injection and identified differences of receptor density among regions such as putamen and caudate nucleus. We demonstrated that TREMBLE is a quantitative approach suitable for the study of pathophysiological conditions of certain types of neurotransmission the brain.

4.
Cold Spring Harb Protoc ; 2021(12)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853120

RESUMO

Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (ß-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.


Assuntos
Peptídeos , Proteínas , Eletroforese em Gel de Poliacrilamida , Géis , Peso Molecular , Proteínas/química , Dodecilsulfato de Sódio
5.
Cold Spring Harb Protoc ; 2021(12)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853121

RESUMO

Many variations of the original Coomassie Brilliant Blue staining procedure are in use. This protocol describes some selected variations on the standard procedure that give comparable and consistent staining results for proteins in the 20- to 200-kDa range.


Assuntos
Eletroforese em Gel de Poliacrilamida , Resinas Acrílicas , Corantes de Rosanilina , Dodecilsulfato de Sódio , Coloração e Rotulagem
6.
Cold Spring Harb Protoc ; 2021(12)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853122

RESUMO

This protocol describes silver staining procedures to detect low-abundance proteins in sodium dodecyl sulfate-polyacrylamide gels.


Assuntos
Sais , Prata , Resinas Acrílicas , Eletroforese em Gel de Poliacrilamida , Coloração pela Prata/métodos , Dodecilsulfato de Sódio , Coloração e Rotulagem
7.
Cold Spring Harb Protoc ; 2021(12)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853123

RESUMO

In immunoblotting (western blotting), proteins are first separated by SDS-PAGE and then transferred electrophoretically from the gel onto a support membrane that binds proteins tightly. After the unreacted binding sites of the membrane are blocked to suppress nonspecific adsorption of antibodies, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen-antibody complexes are visualized using chromogenic, fluorescent, or chemiluminescent reactions. Immunoblotting protocols are reagent specific and, owing to the wide assortment of equipment, reagents, and antibodies available, highly diverse. Presented here is an example of a workable protocol for developing a blot using horseradish peroxidase (HRP)-conjugated secondary antibody and enhanced chemiluminescence (ECL). ECL is based on the emission of light during the HRP-catalyzed oxidation of luminal or other substrates. Emitted light is captured on film or by a CCD camera, for qualitative or semiquantitative analysis. Because ECL is so sensitive, it has become a popular detection method. This protocol can be modified for different membranes, antibodies, and detection systems. Optimal dilutions of the primary and secondary antibodies need to be determined empirically, but recommendations provided by the manufacturer are usually a good starting point.


Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Western Blotting , Peroxidase do Rábano Silvestre , Immunoblotting , Indicadores e Reagentes , Coloração e Rotulagem
8.
Front Psychol ; 12: 701189, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34367026

RESUMO

In adapting to remote emergency teaching modes during pandemic-imposed conditions, teachers' instruction has changed dramatically. Early research indicates that the well-being of music teachers has suffered during the COVID-19 pandemic and that high levels of depression are widespread. The purpose of this survey study was to assess the continued psychological well-being of music teachers working amid a global pandemic based upon previous research we conducted during the Spring 2020 semester when most teachers in the United States were forced into emergency remote teaching. A secondary purpose was to explore the ways that pandemic conditions have affected music teachers' sense of safety at work and their current teaching situations. Our questionnaire consisted of sections pertaining to (1) demographic and institutional information, (2) well-being and depression, (3) instructional format and preparedness, (4) teaching efficacy compared to the start of the pandemic, and (5) potential positive outcomes of the pandemic-imposed adjustments. In total, 1,325 music teachers responded to our survey. Overall, the participants reported poorer well-being than both published norms and the sample of participants in our previous study. In addition, 17% reported mild depression, 25% reported moderate depression, and 24% reported severe extremely severe levels of depression. Summaries of the participants instructional experiences and their implications for music education are discussed within.

9.
PLoS One ; 16(8): e0256224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34388216

RESUMO

The impacts of autonomous vehicles (AV) are widely anticipated to be socially, economically, and ethically significant. A reliable assessment of the harms and benefits of their large-scale deployment requires a multi-disciplinary approach. To that end, we employed Multi-Criteria Decision Analysis to make such an assessment. We obtained opinions from 19 disciplinary experts to assess the significance of 13 potential harms and eight potential benefits that might arise under four deployments schemes. Specifically, we considered: (1) the status quo, i.e., no AVs are deployed; (2) unfettered assimilation, i.e., no regulatory control would be exercised and commercial entities would "push" the development and deployment; (3) regulated introduction, i.e., regulatory control would be applied and either private individuals or commercial fleet operators could own the AVs; and (4) fleets only, i.e., regulatory control would be applied and only commercial fleet operators could own the AVs. Our results suggest that two of these scenarios, (3) and (4), namely regulated privately-owned introduction or fleet ownership or autonomous vehicles would be less likely to cause harm than either the status quo or the unfettered options.


Assuntos
Automação/ética , Veículos Autônomos/ética , Modelos Estatísticos , Propriedade/economia , Acidentes de Trânsito/prevenção & controle , Atitude , Automação/legislação & jurisprudência , Condução de Veículo/psicologia , Veículos Autônomos/legislação & jurisprudência , Técnicas de Apoio para a Decisão , Humanos , Princípios Morais , Inquéritos e Questionários
10.
Biopolymers ; 112(6): e23427, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33792032

RESUMO

Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.


Assuntos
Bivalves/metabolismo , Oxiemoglobinas/química , Multimerização Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Sequência de Aminoácidos , Animais , Bivalves/genética , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Heme/química , Heme/metabolismo , Sulfeto de Hidrogênio/metabolismo , Oxiemoglobinas/genética , Oxiemoglobinas/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem/métodos
11.
Struct Dyn ; 8(2): 020402, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33728362
12.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526417

RESUMO

Many Escherichia coli expression vectors make use of the lac operon. In general, the lac operator (lacO) is located downstream from the promoter of the target gene, so that binding of the lac repressor blocks transcription initiation until lactose or the isopropyl-ß-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the ara operon offer an alternative to expression systems based on the lac operon; guidance for their use is also provided.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Isopropiltiogalactosídeo/farmacologia , Regiões Promotoras Genéticas , DNA Recombinante/genética , Escherichia coli/efeitos dos fármacos , Vetores Genéticos/metabolismo , Proteínas Recombinantes/metabolismo , Solubilidade
13.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526418

RESUMO

For expression of some proteins in Escherichia coli, export to the periplasmic space is preferred over conventional expression in the cytosol. Export can be accomplished by fusing the coding sequence to DNA encoding a signal peptide (e.g., using pET-22b), which is cleaved by the bacterial signal peptidase as the protein is exported into the space between the inner and outer membranes of E. coli This protocol uses osmotic shock to release polypeptides from the periplasm. Although not quantitative, it should provide preliminary information on the cellular location of signal peptide fusion proteins.


Assuntos
Escherichia coli/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Clonagem Molecular , Transporte Proteico , Esferoplastos/metabolismo , Frações Subcelulares/metabolismo
14.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526419

RESUMO

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. For cells enveloped in cell walls (such as Escherichia coli), vigorous methods are often required. This protocol focuses on E. coli lysis by sonication. Also included are methods for lysis by freeze-thaw and enzymatic treatments.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sonicação/métodos , Extratos Celulares , Fracionamento Celular , Congelamento , Muramidase/metabolismo , Solubilidade , Sonicação/instrumentação
15.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526420

RESUMO

The expression of foreign proteins at high levels in Escherichia coli often results in the formation of cytoplasmic granules or inclusion bodies composed of insoluble aggregates of the expressed protein. These inclusion bodies can be seen with a phase-contrast microscope and are readily separated from most soluble and membrane-bound bacterial proteins, as described in this protocol.


Assuntos
Fracionamento Celular/métodos , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/metabolismo , Redobramento de Proteína , Proteínas Recombinantes/química , Solubilidade
16.
Cold Spring Harb Protoc ; 2021(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397779

RESUMO

Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its sole carbon source. Growth in methanol-containing medium results in dramatic induction of genes in the alcohol oxidation pathway including alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD), and dihydroxyacetone synthase (DHAS). These proteins may comprise up to 30% of the biomass. Investigators have exploited these methanol-dependent genes to generate tightly regulated expression vectors. Most Pichia vectors use the strong and tightly regulated AOX1 promoter to drive heterologous protein expression. Obtaining integrated Pichia transformants requires more DNA than transformations into Saccharomyces cerevisiae, where the gene is expressed from episomal plasmids; however, transformants are extremely stable and can be stored for many years.


Assuntos
Clonagem Molecular/métodos , Regulação Fúngica da Expressão Gênica , Metanol/química , Pichia/genética , Regiões Promotoras Genéticas , Fracionamento Celular , DNA/genética , Eletroporação , Recombinação Homóloga/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Transformação Genética
17.
Cold Spring Harb Protoc ; 2021(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397780

RESUMO

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. Disruption methods vary depending on the type of cells, the total volume, and the number of samples being processed. For cells enveloped in cell walls (such as yeast), mild techniques such as hypotonic shock are not sufficient to achieve adequate lysis. More vigorous methods are often required. Although the preferred medium- or large-scale method of breaking yeast cells is mechanical shearing, lysis with the aid of glass beads in a BeadBeater is described here.


Assuntos
Extratos Celulares/química , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Ácidos , Microesferas , Pichia/citologia
18.
Cold Spring Harb Protoc ; 2021(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397781

RESUMO

Isolating membrane proteins from their native cells while maintaining structural and functional integrity is challenging. Many detergents have been developed over the years that interact favorably with membrane proteins and mimic the physical properties of the lipid bilayer. Choosing the appropriate detergent is crucial for the successful extraction of a protein in its properly folded, active conformation.


Assuntos
Bioquímica/métodos , Proteínas de Membrana/isolamento & purificação , Detergentes/química , Proteínas de Membrana/química , Solubilidade
19.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33272973

RESUMO

Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this obstacle, many researchers take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily cultured cells, such as Escherichia coli, Pichia pastoris (yeast), and several varieties of insect and mammalian cells. Heterologous expression systems also allow for easy modification of the protein to optimize expression, mutational analysis of specific sites within the protein and facilitate their purification with engineered affinity tags. Some degree of purification of the target protein is usually required for functional analysis. Purification to near homogeneity is essential for characterization of protein structure by X-ray crystallography or nuclear magnetic resonance (NMR) and characterization of the biochemical and biophysical properties of a protein, because contaminating proteins almost always adversely affect the results. Methods for producing and purifying proteins in several different expression platforms and using a variety of vectors are introduced here.


Assuntos
Clonagem Molecular/métodos , Proteínas/genética , Proteínas/isolamento & purificação , Expressão Gênica , Vetores Genéticos/metabolismo , Proteômica , Proteínas Recombinantes de Fusão/metabolismo
20.
Sci Eng Ethics ; 26(5): 2411, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32990876

RESUMO

Due to an unfortunate miscommunication with the copy editor an important reference was omitted from this recently published article. The reference that should be included is.

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