RESUMO
Belizatinib (BZB; TSR-011) is a next-generation anaplastic lymphoma kinase inhibitor that also inhibits tropomyosin-related kinases A/B/C. In this in-vitro study, we examined the formation of reactive metabolites from BZB using rat liver microsomes or human liver microsomes in the presence of a trapping agent (potassium cyanide) to generate iminium reactive intermediates. Identification of the in vitro BZB metabolites indicated that the major in-vitro metabolic reaction involved hydroxylation of the piperidine moiety. We identified eight in-vitro phase I metabolites and three iminium reactive intermediates, suggesting two possible BZB-bioactivation pathways. We propose that the tertiary nitrogen in the piperidine ring activates the attached benzyl carbon in addition to the two α carbons inside the ring. To our knowledge, this is the first report on the structural identification of reactive metabolites derived from BZB.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/metabolismo , Benzamidas/metabolismo , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas In Vitro , Estrutura Molecular , Cianeto de Potássio/química , RatosRESUMO
Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point Tm, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein's thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of Tm shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from Tm shifts.
Assuntos
Vírus do Sarampo/química , Vírus da Caxumba/química , Estabilidade Proteica , Proteínas do Envelope Viral/química , Albuminas/química , Células Cultivadas , Composição de Medicamentos , Estabilidade de Medicamentos , Guanidina/química , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Vírus do Sarampo/patogenicidade , Simulação de Dinâmica Molecular , Vírus da Caxumba/patogenicidade , Ovalbumina/química , Polimerização , Cianeto de Potássio/química , Desnaturação Proteica/efeitos dos fármacos , Temperatura de Transição , Ureia/químicaRESUMO
Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.
Assuntos
Cisteína/química , Proteínas de Escherichia coli/química , Proteínas Ferro-Enxofre/metabolismo , Transativadores/química , Catálise , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/metabolismo , Ferro/metabolismo , Ligantes , Metionina/química , Cianeto de Potássio/química , Ligação Proteica , Prótons , Energia Solar , Tirosina/químicaRESUMO
We have previously reported cyanide at concentrations of up to 150 µM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 µM: 47% fall, p<0.0012; 75 µM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 µM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway.
Assuntos
Cílios/metabolismo , Cianetos/química , Fibrose Cística/metabolismo , Mucosa Nasal/metabolismo , Escarro/metabolismo , Trifosfato de Adenosina/química , Sobrevivência Celular , Células Cultivadas/efeitos dos fármacos , Fibrose Cística/microbiologia , Voluntários Saudáveis , Humanos , Muco/fisiologia , Mucosa Nasal/microbiologia , Cianeto de Potássio/química , Prognóstico , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa , Testes de Função Respiratória , Sistema Respiratório/fisiopatologiaRESUMO
Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.
Assuntos
Cistina/química , Globinas/química , Substituição de Aminoácidos , Monóxido de Carbono/química , Citoglobina , Globinas/genética , Heme/química , Humanos , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Oxirredução , Cianeto de Potássio/química , Ligação Proteica , Multimerização Proteica , Análise Espectral RamanRESUMO
MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.
Assuntos
Sondas de DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Sequência de Bases , Linhagem Celular Tumoral , Sondas de DNA/química , Perfilação da Expressão Gênica/métodos , Ouro/química , Humanos , Nanopartículas Metálicas/química , MicroRNAs/metabolismo , Cianeto de Potássio/química , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.
Assuntos
Antagonistas de Receptores de Canabinoides/química , Iminas/química , Piperidinas/química , Pirazóis/química , Antagonistas de Receptores de Canabinoides/metabolismo , Antagonistas de Receptores de Canabinoides/toxicidade , Radioisótopos de Carbono/química , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/metabolismo , Humanos , Íons/química , Metaboloma/efeitos dos fármacos , Piperidinas/metabolismo , Piperidinas/farmacologia , Piperidinas/toxicidade , Cianeto de Potássio/química , Cianeto de Potássio/farmacologia , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Pirazóis/metabolismo , Pirazóis/toxicidade , Rimonabanto , Ritonavir/química , Ritonavir/farmacologiaRESUMO
Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O(2) (-)), which was added exogenously. Our results suggest that the metabolism of O(2) (-) has an important role in the development of P. cinnamomi.
Assuntos
Proteínas Fúngicas/química , Micélio/enzimologia , Phytophthora/enzimologia , Superóxido Dismutase/química , Parede Celular/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Oxidantes/farmacologia , Persea/microbiologia , Phytophthora/efeitos dos fármacos , Phytophthora/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Cianeto de Potássio/química , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo , Superóxidos/farmacologiaRESUMO
RATIONALE: Although para-phenylenediamine (PPD) is known to cause severe allergic contact dermatitis in consequence of autoxidation and/or skin metabolism pathways, it is commonly utilized as an ingredient in permanent hair dyes. The aim of this work was to simultaneously accelerate the autoxidation process and to simulate the metabolic activation of PPD using a purely instrumental system. METHODS: Electrochemistry (EC) in combination with electrospray ionization mass spectrometry (ESI-MS) was used in this study to assess the skin-sensitizing potential of PPD. Online and offline coupled EC/ESI-MS experiments were carried out and the emerging oxidation products were investigated. In a second approach, these primary species were allowed to react with the nucleophiles glutathione (GSH), cysteine (Cys), potassium cyanide (KCN) and lysine (Lys) in order to evaluate their reactivity. RESULTS: The reactive p-phenylene quinone diimine (PPQD), which can form upon autoxidation and/or skin metabolism of PPD, was effectively generated in a simple EC cell next to further oxidation products, including the trimeric product Bandrowski's Base (BB). Conjugation with GSH and Cys was successfully proven, but no adducts with KCN or Lys were observed. Furthermore, the application of different concentration ratios between PPD and nucleophile was shown to play a crucial role concerning the type of oxidation products and adducts being formed. CONCLUSIONS: It was found that EC/MS is a well-suited approach for the targeted generation of reactive haptens such as PPQD while avoiding detection problems due to the complexity of matrices encountered when conducting conventional in vitro or in vivo experiments.
Assuntos
Alérgenos/química , Cisteína/química , Técnicas Eletroquímicas/métodos , Glutationa/química , Fenilenodiaminas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Lisina/química , Oxirredução , Cianeto de Potássio/química , Testes de Irritação da Pele/métodosRESUMO
Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scans-dependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH- and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.
Assuntos
Descoberta de Drogas/métodos , Glutationa/análise , Espectrometria de Massas/métodos , Cianeto de Potássio/análise , Animais , Mineração de Dados , Glutationa/química , Ensaios de Triagem em Larga Escala , Microssomos Hepáticos/metabolismo , Modelos Químicos , Peso Molecular , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Cianeto de Potássio/química , RatosRESUMO
The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Embrião não Mamífero/metabolismo , Mitocôndrias/enzimologia , Rhipicephalus/crescimento & desenvolvimento , Hidrolases Anidrido Ácido/química , Animais , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético , Heparina/química , Heparina/metabolismo , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Oxirredução , Polifosfatos/química , Polifosfatos/farmacologia , Cianeto de Potássio/química , Cianeto de Potássio/metabolismoRESUMO
Bacteriophages are viruses that infect bacteria and contribute significant changes in the overall bacterial community. Prophages are formed when temperate bacteriophages integrate their DNA into the bacterial chromosome during the lysogenic cycle of the phage infection to bacteria. The prophage (phage DNA integrated into bacterial genome) on the bacterial genome remains dormant, but can cause cell lysis under certain environmental conditions. This research examined the effect of various environmental stress factors on the ammonia oxidation and prophage induction in a model ammonia oxidizing bacteria Nitrosospira multiformis ATCC 25196. The factors included in the study were pH, temperature, organic carbon (COD), the presence of heavy metal in the form of chromium (VI) and the toxicity as potassium cyanide (KCN). The selected environmental factors are commonly encountered in wastewater treatment processes, where ammonia oxidizing bacteria play a pivotal role of converting ammonia into nitrite. All the factors could induce prophage from N. multiformis demonstrating that cell lysis due to prophage induction could be an important mechanism contributing to the frequent upset in ammonia oxidation efficiency in full scale treatment plants. Among the stress factors considered, pH in the acidic range was the most detrimental to the nitrification efficiency by N. multiformis. The number of virus like particles (VLPs) increased by 2.3E+10 at pH 5 in 5h under acidic pH conditions. The corresponding increases in VLPs at pH values of 7 and 8 were 9.67E+9 and 1.57E+10 in 5h respectively. Cell lysis due to stress resulting in phage induction seemed the primary reason for deteriorated ammonia oxidation by N. multiformis at lower concentrations of Cr (VI) and potassium cyanide. However, direct killing of N. multiformis due to the binding of Cr (VI) and potassium cyanide with cell protein as demonstrated in the literature at higher concentrations of these toxic compounds was the primary mechanism of cell lysis of N. multiformis. Organics represented by the chemical oxygen demand (COD) did not have any effect on the phage induction in N. multiformis. This AOB remained dormant at low temperature (4 degrees C) without any phage induction. Significant decrease in the number of live N. multiformis cells with a corresponding increase in the number of VLPs was recorded when the temperature was increased to 35 degrees C. Death of N. multiformis at 45 degrees C was attributed to the destruction of cell wall rather than to the phage induction.
Assuntos
Nitrosomonadaceae/virologia , Prófagos/fisiologia , Vírion/fisiologia , Poluentes Químicos da Água/toxicidade , Amônia/metabolismo , Antibióticos Antineoplásicos/toxicidade , Bacteriólise/efeitos dos fármacos , Cromo/química , Cromo/toxicidade , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Microscopia de Fluorescência , Mitomicina/toxicidade , Nitrosomonadaceae/metabolismo , Compostos Orgânicos/química , Compostos Orgânicos/toxicidade , Oxirredução/efeitos dos fármacos , Cianeto de Potássio/química , Cianeto de Potássio/toxicidade , Temperatura , Vírion/ultraestrutura , Ativação Viral/efeitos dos fármacosRESUMO
A heteroditopic receptor having crown ether and trifluoroacetylcarboxanilide groups selectively recognizes both potassium and cyanide ions in acetonitrile with an association constant of as high as Ka = 1.9 x 10(7) M(-1) through a highly cooperative ion-pair interaction, resulting in two orders of magnitude enhancement in the binding affinity.
Assuntos
Ácidos Carboxílicos/análise , Éteres de Coroa/química , Compostos Ferrosos/química , Cianeto de Potássio/química , Ácido Trifluoracético/química , Anilidas/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Metalocenos , Estrutura Molecular , Cianeto de Potássio/análiseRESUMO
Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 microm KCN. It significantly reduced neuronal cell death induced by 300 microm KCN from 83.6 to 43.5%. However, at 1-100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Neurônios/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular , Córtex Cerebral/metabolismo , DNA/metabolismo , Densitometria , Relação Dose-Resposta a Droga , Regulação para Baixo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Raios Infravermelhos , Membranas Intracelulares/metabolismo , Luz , Substâncias Macromoleculares , Cianeto de Potássio/química , Cianeto de Potássio/farmacologia , Propídio/química , Ratos , Espectrofotometria , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
Red palm oil was tested for the reactivity of its components with CN-, and alkaline picrate as the color developing reagent. Palm oil components have a low-level absorbance at 490 nm which is reduced significantly (p< or =0.01) after reaction with CN-. Hydrolysis of palm oil components, and reaction of the hydrolysis products with CN- significantly increased the absorbance at 490 nm. In contrast, after reaction of palm oil with alkaline picrate, the absorbance at 490 nm is very high; this is reduced significantly by reaction with CN-, hydrolysis with 0.2 M NaOH, and reaction of the hydrolysis products with CN- before treatment with alkaline picrate. The results indicate that palm oil component(s) sequester CN- into a complex which may not be correctly estimated during cyanide quantification, resulting in the absence, or low levels of cyanide in palm oil-fried gari as earlier reported.
Assuntos
Manihot/química , Nitrilas/análise , Óleos de Plantas/química , Cianeto de Potássio/química , Manipulação de Alimentos , Hidrólise , Óleo de Palmeira , Picratos/química , Hidróxido de Sódio/química , EspectrofotometriaRESUMO
The question of whether the production of ammonia, from the reduction of nitrite by iron(II), is compatible with its use in the Strecker synthesis of amino acids, or whether the iron and the cyanide needed for the Strecker synthesis interfere with each other, is addressed. Results show that the presence of iron(II) appears to have little, or no, effect on the Strecker synthesis. The presence of cyanide does interfere with reduction of nitrite, but the reduction proceeds at cyanide/iron ratios of less than 4:1. At ratios of about 2:1 and less there is only a small effect. The reduction of nitrite and the Strecker can be combined to proceed in each other's presence, to yield glycine from a mixture of nitrite, Fe+2, formaldehyde, and cyanide.
Assuntos
Amônia/síntese química , Compostos Ferrosos/química , Nitritos/química , Formaldeído/química , Glicina/síntese química , Dióxido de Nitrogênio/química , Cianeto de Potássio/químicaRESUMO
The thiol groups of beta-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]-cyanylated-beta-lactoglobulins A and B which we prepared had signals at 109.7 p.p.m. and 114.4 p.p.m. We conclude that the thiocyanate carbon having a chemical shift of 109.7 p.p.m. is in an apolar environment similar to a cyclohexane solvent, whereas the thiocyanate carbon having a chemical shift of 114.4 p.p.m. is in a polar environment similar to water. The signals with chemical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons of the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We deduce that the signal at 114.4 p.p.m. is due to an irreversibly denatured/unfolded species produced by alkaline denaturation, which is caused by intramolecular thiol/disulphide exchange occurring during our cyanylation procedure. We propose that Cys-119 is cyanylated in the irreversibly denatured species and Cys-121 is cyanylated in the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers [McKenzie, Ralston and Shaw (1972) Biochemistry 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native beta-lactoglobulins is due to the thiol of Cys-121 and is not due to an equimolar mixture of Cys-119 and Cys-121 as they suggested.
Assuntos
Cisteína/química , Lactoglobulinas/química , Tiocianatos/química , Animais , Bovinos , Cromatografia em Gel , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Lactoglobulinas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Polímeros , Cianeto de Potássio/química , Desnaturação Proteica , Compostos de Sulfidrila/químicaRESUMO
Both of the thiol groups of Megasphaera elsdenii flavodoxin have been cyanylated using 13C-enriched cyanide. This chemical modification increases the dissociation constant of the apoflavodoxin-flavin mononucleotide (FMN) complex from 0.4 nM to 2 microM. The thiocyanate carbons of the cyanylated cysteine residues in apoflavodoxin had 13C chemical shifts of 109.4 ppm and 112.2 ppm, which were replaced by signals at 115.5 ppm and 109.6 ppm when FMN was bound. The signals at 109.4 ppm and 112.2 ppm due to the cyanylated apoflavodoxin were unstable at 28 degrees C, and they were slowly replaced signals at 114.5 ppm and 115.3 ppm which are attributed to an inactive form of the apoprotein, which does not bind FMN. At alkaline pH values or after prolonged incubation at neutral pH, the signals at 114.5 ppm and 115.3 ppm were replaced by signals at approximately 171 ppm. On the basis of results obtained with model compounds, the signals at 171 ppm are assigned to the 2-imino carbon of the 2-iminothiazolidine ring formed by the cyclization of the appropriate thiocyanate group. After determining the chemical shift of the thiocyanate carbon of model compounds in a range of solvents, we conclude that the thiocyanate carbons will have a minimal chemical shift of approximately 109 ppm in apolar solvents which do not contain hydrogen bond donors. In water, a more polar hydrogen-bonding solvent, the chemical shift increases to approximately 115 ppm. We also conclude that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Flavodoxina/química , Espectroscopia de Ressonância Magnética , Cianeto de Potássio/química , Tiocianatos/química , Veillonellaceae/química , Fenômenos Químicos , Físico-Química , Estabilidade de Medicamentos , Mononucleotídeo de Flavina/metabolismo , Flavodoxina/metabolismo , Estrutura MolecularRESUMO
The reaction of beta-N-methylaminoalanine (BMAA) with L-amino acid oxidase (L-AAO) in the presence of catalase yields ammonia and beta-N-methylaminopyruvate, which was trapped as its 2,4-dinitrophenylhydrazone, as products. Incubation of BMAA with L-AAO in the presence of semicarbazide led to the formation of a semicarbazone, indicating intermediate iminium ion formation; when potassium cyanide (5 mM) was added, semicarbazone formation was blocked. The formation of beta-N-methylaminopyruvate was decreased by omission of catalase and was reduced in the presence of hydrogen peroxide (100 mM). These results indicate that BMAA is converted by L-AAO to the corresponding alpha-imino acid, which undergoes hydrolysis to beta-N-methylaminopyruvate. The alpha-keto acid is readily oxidized to N-methylglycine by hydrogen peroxide.