RESUMO
Cadmium-113 nuclear magnetic resonance spectroscopy has been used in studies of the structure and dynamics of inorganic and bioinorganic molecules. Chemical dynamics play an important role in the analysis of relaxation and chemical shift data. Naïve interpretations of relaxation data can be checked by performing these experiments at a variety of temperatures and magnetic field strengths. A combination of solid- and liquid-state nuclear magnetic resonance measurements can provide the user with unambiguous data on chemical shielding. These data can be used to characterize zinc and calcium ion binding sites in metalloproteins.
RESUMO
Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Magnésio/química , Magnésio/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Sequência de Bases , Humanos , Cinética , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression is strongly activated by a variety of extracellular stimuli, acting through the Raf/MEK/MAP kinase pathway. To study the elements that respond to this pathway, we have isolated and sequenced a fragment of the rat HB-EGF gene promoter. By transfection of a series of promoter/reporter constructs into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, gave a fold-stimulation of activity no greater than that seen for the parental pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced little or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB-EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5'-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two species approximately 260 bp upstream of the transcriptional start site. PCR analysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in the HB-EGF gene.
Assuntos
Fator de Crescimento Epidérmico/genética , Regiões Promotoras Genéticas/genética , Células 3T3 , Animais , Sequência de Bases , DNA/análise , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência do Ácido Nucleico , TATA BoxRESUMO
The interactions of thymidylate synthase (TS) with deoxyuridylate (dUMP), deoxythymidylate (dTMP) and 5-fluorodeoxyuridylate (FdUMP) were examined by 31P-NMR. Single 31P resonances appeared at 3.3 ppm, 3.2 ppm and 3.0 ppm from the standard, 85% phosphoric acid, for unbound dUMP, dTMP, and FdUMP, respectively. Incubation of the enzyme with either dUMP or dTMP, alone, resulted in new resonances at 3.9 and 3.6 ppm, respectively, which were assigned to noncovalent complexes with the enzyme. The same experiment employing FdUMP as the ligand gave two new resonances appearing at 3.6 and 4.6 ppm, which were attributed to noncovalent and covalent binary complexes, respectively. When the cofactor, CH2H4 folate, was present in the solution with enzyme and FdUMP, a new resonance appeared at 5.1 ppm, corresponding to the covalent inhibitory ternary complex. The ternary complex comprised of the enzyme, dUMP and the quinazoline folate CB 3731 produced a resonance at 5.0 ppm at the expense of the resonance due to the enzyme-dUMP binary complex at 3.9 ppm. Similarly, the ternary complex consisting of TS with dTMP and CB 3731 showed a deshielding of the resonance at 3.6 ppm by 0.8 ppm. A maximum binding of 1.5 nucleotides per enzyme dimer was found for dUMP and dTMP in both the presence and the absence of the quinazoline folate. The deshielding observed was attributed to changes in the interaction of the phosphate group with the nearby residues of the active site of the enzyme.
Assuntos
Espectroscopia de Ressonância Magnética , Timidilato Sintase/metabolismo , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Fluordesoxiuridilato/química , Fluordesoxiuridilato/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Ácido Fólico/metabolismo , Isótopos de Fósforo , Quinazolinas/química , Quinazolinas/metabolismo , Tetra-Hidrofolatos/farmacologia , Timidina Monofosfato/química , Timidina Monofosfato/metabolismo , Timidilato Sintase/químicaRESUMO
A general strategy for the observation of low gamma half-integer quadrupolar nuclides in biological systems is presented. The methodology combines low-temperature (4-100 K) techniques with cross-polarization (CP) experiments while employing a so-called Carr-Purcell-Meiboom-Gill spin-echo sequence (CPMG). This combined approach is termed CP/QCPMG. Also discussed are data processing issues that are unique to the induced signals that result from the QCPMG pulse sequence. Central to this strategy is the development of a stable low-temperature (4 to 250 K) NMR double-resonance probe. The probe is robust enough to handle multiple contact experiments and long acquisition periods with 1H decoupling. This approach is illustrated with low-temperature solid-state 67Zn and 25Mg NMR CP/QCPMG experiments on model compounds. The conclusion reached is that the strategy affords sufficient sensitivity to examine Zn2+ and/or Mg2+ binding sites in metalloproteins.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Análise de Fourier , Magnésio , Radioisótopos , Temperatura , Radioisótopos de ZincoRESUMO
The design of a broadband 4-mm magic-angle spinning (MAS) X-(1)H/(19)F double resonance probe for cross-polarization (CP)/MAS NMR studies at 21.15 T ((1)H at 900 MHz) is described. The high-frequency (1)H/(19)F channel employs a new and efficient transmission line tuning design. The first (13)C CP/MAS NMR spectra recorded at 21.15 T have been obtained with this probe and exhibit the best S/N per milligram sample of hexamethylbenzene achieved so far for a 4-mm rotor.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Derivados de Benzeno/química , Desenho de Equipamento , Espectroscopia de Ressonância Magnética/métodosRESUMO
By combination of fast magic-angle spinning (MAS) and detection of the free-induction decay during a rotor-synchronized quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) train of refocusing pulses, the sensitivity of quadrupolar-echo MAS NMR spectra for the central transition of half-integer quadrupolar nuclei exhibiting large quadrupolar couplings may be significantly enhanced. Enhancements by an order of magnitude may easily be realized while maintaining information about the anisotropic interactions. In the present study the so-called QCPMG-MAS experiment is demonstrated experimentally and by numerical simulations for the two 87Rb sites in Rb2SO4.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Anisotropia , Fenômenos Químicos , Físico-Química , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Aumento da Imagem/métodos , Modelos Químicos , Rubídio/química , Radioisótopos de Rubídio/química , Sulfatos/química , Difração de Raios XRESUMO
We have developed a "virtual NMR facility" (VNMRF) to enhance access to the NMR spectrometers in Pacific Northwest National Laboratory's Environmental Molecular Sciences Laboratory (EMSL). We use the term virtual facility to describe a real NMR facility made accessible via the Internet. The VNMRF combines secure remote operation of the EMSL's NMR spectrometers over the Internet with real-time videoconferencing, remotely controlled laboratory cameras, real-time computer display sharing, a Web-based electronic laboratory notebook, and other capabilities. Remote VNMRF users can see and converse with EMSL researchers, directly and securely control the EMSL spectrometers, and collaboratively analyze results. A customized Electronic Laboratory Notebook allows interactive Web-based access to group notes, experimental parameters, proposed molecular structures, and other aspects of a research project. This paper describes our experience developing a VNMRF and details the specific capabilities available through the EMSL VNMRF. We show how the VNMRF has evolved during a test project and present an evaluation of its impact in the EMSL and its potential as a model for other scientific facilities. All Collaboratory software used in the VNMRF is freely available from www.emsl.pnl.gov:2080/docs/collab.
Assuntos
Internet , Espectroscopia de Ressonância Magnética , Comunicação , SoftwareRESUMO
(1)H dynamic nuclear polarization (DNP) has been measured in supercritical ethylene in the pressure range 60-300 bar in an external field of 1.4 T. A single-cell sapphire tube was used as a high-pressure cell, and powdered 1,3-bisdiphenylene-2-phenyl allyl (BDPA) free radicals were added and distributed at the wall of the cell. At all pressures the dominant DNP mechanism was a positive Overhauser enhancement, caused by proton-electron contact interactions at the fluid/solid radical interface. The observed enhancements varied from 12 at 60 bar to 17 at 300 bar. Besides the Overhauser enhancement, small solid state and thermal mixing enhancements also were observed, indicating that part of the ethylene was adsorbed at the radical surface for a prolonged time. The impacts of the experimental conditions on the Overhauser enhancement factors are discussed, and enhancements of at least 40-60 are estimated when the EPR saturation factor and the leakage factor become maximal. These data indicate that DNP-enhanced NMR has the potential of extending the impact of NMR in research areas involving supercritical fluids.
Assuntos
Etilenos/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of health and disease at a cellular level, particularly when data acquired with different methodologies can be correlated in both time and space. In this Communication, a brief description of a novel instrument capable of simultaneously performing confocal optical and magnetic resonance microscopy is presented, and the first combined images of live Xenopus laevis oocytes are shown. Also, the potential benefits of combined microscopy are discussed, and it is shown that the a priori knowledge of the high-resolution optical images can be used to enhance the boundary resolution and contrast of the MR images.
Assuntos
Imageamento por Ressonância Magnética , Microscopia Confocal , Oócitos/ultraestrutura , Animais , Desenho de Equipamento , Microscopia de Fluorescência , XenopusRESUMO
Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.
Assuntos
Fosfopiruvato Hidratase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Cádmio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Fosfopiruvato Hidratase/antagonistas & inibidores , Conformação Proteica , Especificidade por SubstratoRESUMO
Recurrences after surgery for thyroglossal cysts and fistulas were common until, in 1920, Sistrunk described his technique for removing the whole thyroglossal tract. Since that time there has been considerable argument concerning the exact path taken by the tract particularly in relation to the hyoid bone. In an attempt to elucidate this problem, studies have been made of embryos at varying stages of development, of serially step-sectioned adult larynges and of surgical specimens. These studies suggest that the thyroglossal tract occupies a constant and embryologically predictable position in relation to the hyoid bone and they support the rationale of Sistrunk's procedure.
Assuntos
Glândula Tireoide/embriologia , Língua/embriologia , Feminino , Humanos , Osso Hioide/embriologia , Gravidez , Cisto Tireoglosso/cirurgia , Glândula Tireoide/cirurgia , Língua/cirurgiaRESUMO
Ascites, hepatic encephalopathy, and bleeding esophageal and gastric varices are frequent consequences of hepatic disease and portal hypertension. Variceal bleeding, however, is the most dangerous sequel and the most yielding to surgical intervention. Portal decompression by the shunting procedure is instituted in order to decrease the risk of future gastrointestinal hemorrhage. The presence of hepatic disease and alterations in hepatic blood flow following the procedure present implications for postoperative management of fluid and electrolyte balance, mental status, ventilation, and coagulation.
Assuntos
Varizes Esofágicas e Gástricas/cirurgia , Hemorragia Gastrointestinal/cirurgia , Hipertensão Portal/cirurgia , Varizes Esofágicas e Gástricas/complicações , Varizes Esofágicas e Gástricas/etiologia , Hemorragia Gastrointestinal/etiologia , Hemodinâmica , Humanos , Hipertensão Portal/complicações , Fígado/irrigação sanguínea , Métodos , Cuidados Pós-Operatórios , Cuidados Pré-OperatóriosRESUMO
We describe our experience in the surgical treatment of 32 patients with severe snoring. In the first 16 patients we used the standard surgical procedure of uvulopalatopharyngoplasty (UPP) but found this unsatisfactory because of its radical nature, complications and uncertain outcome. We therefore investigated the mechanics of snoring in the laboratory as a help in devising a more effective operation. These studies have shown that there are several methods by which snoring can be generated but that palatal flutter is probably the most important. The dominant parameters in the generation of flutter of the palate are its length and stiffness. Any removal of tissue to shorten the palate as in UPP inevitably risks impairing its function, so we chose the stiffening alternative. Using the laser, a central longitudinal strip of mucosa was removed from the surface of the soft palate which healed by fibrosis, producing the required stiffening. The early results of this new procedure show that snoring has been eliminated or greatly reduced in 14 of 16 patients and that apart from local pain there have been no complications.
Assuntos
Terapia a Laser/métodos , Palato Mole/cirurgia , Ronco/cirurgia , Adulto , Idoso , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Palato Mole/fisiopatologia , Faringe/cirurgia , Complicações Pós-Operatórias , Ronco/fisiopatologia , Úvula/cirurgia , VibraçãoRESUMO
We report our experience in the out-patient triage of 100 patients presenting with a lump in the neck. The out-patient visit consisted of a general history and examination, assessment of the upper air and food passages and, where indicated, ultrasound and core needle biopsy of the lump. Other investigations were performed as appropriate. One hundred neck lump patients were seen in a 9 month period. Ninety-six of these lumps were diagnosed on an out-patient basis, only four requiring admission for formal excision biopsy. Among the diagnoses were eleven lymphomas, nine parotid neoplasms, nine lymph node metastases, five thyroglossal cysts, and four branchial cysts. Almost half the patients seen had either a reactive lymphadenopathy, or no abnormality. The establishment of a tissue diagnosis on an out-patient basis allowed appropriate referrals to be made and management plans to be formulated. The theoretical risk of seeding of malignant cells in the needle tract is acknowledged and discussed.
Assuntos
Neoplasias de Cabeça e Pescoço/diagnóstico , Triagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Assistência Ambulatorial/métodos , Biópsia por Agulha , Criança , Pré-Escolar , Inglaterra , Feminino , Neoplasias Hematológicas/diagnóstico , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Parotídeas/diagnóstico , Cisto Tireoglosso/diagnósticoRESUMO
Voice changes developing after endotracheal intubation have been found to be due to a recurrent laryngeal nerve palsy in four patients. In none of these patients was there any obvious cause and this posed the question whether endotracheal intubation itself could result in a paralysis of the nerve. Cadaver dissection has suggested a way in which which paralysis could occur and this and other possible aetiologies are discussed.
Assuntos
Intubação Intratraqueal/efeitos adversos , Nervos Laríngeos , Nervo Laríngeo Recorrente , Paralisia das Pregas Vocais/etiologia , Adulto , Cadáver , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pressão , Nervo Laríngeo Recorrente/anatomia & histologiaRESUMO
Haemangiomas are the commonest benign tumours of the head and neck in childhood, but they are extremely rate on the palate. We report a case of a palatal haemangioma presenting with feeding difficulties in a young infant. Clinical diagnosis and pathological features are discussed and we suggest a classification which may help in the diagnosis of these cases.
Assuntos
Hemangioma Capilar/patologia , Neoplasias Palatinas/patologia , Feminino , Humanos , LactenteRESUMO
Abscesses of the pterygopalatine fossa are extremely rare. The cases reported in the literature are all secondary to dental or periodontal disease. We present a case of an abscess in this site which was a complication of chemotherapy and was the result of haematogenous spread.
Assuntos
Abscesso/etiologia , Antibacterianos , Quimioterapia Combinada/efeitos adversos , Imunossupressores/efeitos adversos , Seio Maxilar , Infecções Estreptocócicas/complicações , Neoplasias da Mama , Quimioterapia Combinada/uso terapêutico , Feminino , Humanos , Imunossupressores/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Pessoa de Meia-Idade , Mitomicina/administração & dosagem , Mitomicina/efeitos adversos , Cuidados Paliativos , Vimblastina/administração & dosagem , Vimblastina/efeitos adversosRESUMO
A large proportion of the population either snores or suffers the snoring of others. Recent advances with the use of fibre-optic endoscopes have enabled surgeons to observe the inside of the pharynx while a patient is asleep and snoring. In this article we look at the underlying structure of the upper airway and explain, with the use of simple mechanical models, the aerodynamic events occurring inside the upper airway during snoring.