Molecular genetic studies on voltage-gated ion channels

Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K⁺) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant para^ts. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.

In Drosophila, diverse voltage-gated K⁺ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K⁺-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K⁺-channel gene family probably diversified prior to the mammalian radiation. Functional K⁺ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K⁺ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K⁺-channel genes may efficiently co-assemble into heterooligomeric K⁺ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K⁺-channel diversity.

Trimethylsilyl anions: stereospecific deoxygenation of epoxides and trimethylsilyldehalogenation of aryl halides

I. Trimethylsilylpotassium reacts with epoxides to give olefins with inversion of stereochemistry. The reaction appears to proceed via the potassium β-silyl alkoxide (2) formed from the S_N2 attack of the silyl anion on the epoxide. Subsequent stereospecific synelimination of 2 affords the olefin of inverted stereo-chemistry. The reaction is convenient and preparatively useful.

The byproduct of the reaction, potassium trimethylsilanolate (17), effectively cleaves hexamethyldisilane to yield trimethylsilylpotassium. Since the latter reagent is generated and reacted in situ with epoxides, the overall reaction can be carried out with less than one equivalent of potassium methoxide.

II. The reaction of aryl halides with trimethylsilyl anions in HMPT provides good yields of aryltrimethylsilanes, useful synthetic intermediates. The choice of metal cation is unimportant. Chlorides and bromides give high yields of silylated products, while iodides give lower yields, with correspondingly increased amounts of reduced products. Arylammonium and arylphosphonium salts also undergo the reaction.

We have permissive evidence for the reaction proceeding via both aryl radical and aryl anion intermediates.

III. Trimethylsilyl and trimethylstannyl methoxycarbene complexes of chromium and tungsten have been prepared. One of these, (CO)_5WC(OMe)SnMe_3, reacts with norbornene at 80° to afford a new olefin polymer. Efforts to effect the alpha-elimination of the nonmetallic carbene ligands have not yet been successful. Reactions of these carbene complexes with acetone have been investigated.

Oxy anion-accelerated rearrangements

The alkali metal salts of 1,5-hexadien-3-ols undergo accelerated Cope rearrangements to the enolates of δ, ε-unsaturated carbonyl compounds. The generality of the rearrangement was investigated in numerous systems, particularly acyclic cases, and the effect of changes in substituents, counterions, solvents, and geometrical structures were noted and discussed. Applications of this methodology in synthesis included the synthesis of the insect pheromone frontalin, the preparation of selectively monoprotected 1,6-dicarbonyl compounds from 4-methoxy- and 4-phenylthio-1,5-hexadien-3-ols, and the construction of complex ring structures such as a D-homo-estratetraenone derivative.

Thermochemical estimates of the energetics of anionpromoted alkoxide fragmentations were made, and in all cases heterolytic cleavage was favored over hemolytic cleavage by 8.5-53 kcal/mol. The implication of these and other thermochemical estimates is that the anionic oxy-Cope rearrangement occurs via a concerted mechanism rather than a dissociation-recombination process. The concepts of anion-induced bond weakening were successfully applied to an accelerated [1,3]-shift of a dithiane fragment in a cyclohexenyl system. Trapping experiments demonstrated that > 85% of the [1,3]-shift occurred within a solvent cage. Attempts at promoting an intramolecular ene reaction using the potassium salts of 2,7-octadien-1-o1 and 2,8-nonadien-1-o1 were unsuccessful. A general review of anion-promoted bond reorganizations and anion substituent effects is also presented.

Síntese e caracterização de biodiesel obtido via catálises homogênea e heterogênea de óleo extraído de sementes de Helianthus annuus

O biodiesel é definido como um mono alquil éster de ácidos graxos de cadeia longa derivado de fontes renováveis tais como óleos vegetais e gorduras animais. Sua importância esta associada ao uso como um combustível alternativo para motores do ciclo Diesel podendo ser utilizado puro ou em misturas com o diesel representando economia de petróleo e menor poluição ambiental. Em geral é obtido por meio da reação de transesterificação na qual os triacilgliceróis, principais constituintes dos óleos e gorduras reagem com álcool, em presença de um catalisador ácido ou básico, produzindo ésteres de ácidos graxos e glicerol. A transesterificação pode ser conduzida por catálise homogênea ou heterogênea. O grande desafio da indústria é otimizar o processo a fim de alcançar um produto e uma rota de produção tecnologicamente eficiente e ambientalmente correta. O objetivo desta pesquisa foi estudar a síntese do biodiesel utilizando o processo de transesterificação do óleo de girassol por catálises homogênea e heterogênea. Foram realizadas reações de transesterificação via rotas metílica e etílica, empregando como catalisador homogêneo alcóxido de potássio e como catalisador heterogêneo a resina comercial de troca iônica Amberlyst 26

Estudo da correlação entre as características da matéria-prima graxa e as propriedades físico-químicas do biodiesel

A possibilidade de utilização de várias matérias graxas na produção do biodiesel nacional é um fator diferencial em relação aos outros países e requer um conhecimento mais profundo das particularidades do biodiesel obtido a partir de matérias-primas tão distintas. Neste contexto, se insere a presente pesquisa, em que se buscou estabelecer correlações entre algumas propriedades do biodiesel e a fonte oleaginosa que lhe deu origem. O biodiesel foi obtido a partir da reação de transesterificação etílica de dez diferentes matérias-primas graxas (óleos refinados de soja, arroz, canola, girassol, milho, oliva, cyclus (mistura contendo óleos de canola, milho e girassol); óleos brutos de mamona e murumuru, além de resíduo de fritura), utilizando etanolato de potássio como agente catalítico, em um reator com sistema de refluxo a 70C durante uma hora. Após purificação, foram determinadas algumas propriedades do biodiesel (massa específica, viscosidade, índice de acidez e índice de iodo) e da matéria-prima (massa específica, viscosidade, índice de acidez, índice de iodo e composição) que lhe deu origem. Os resultados obtidos nas caracterizações geraram gráficos de correlações entre os diversos parâmetros e os dados foram analisados estatisticamente pelo método de correlações canônicas. Para todas as amostras de biodiesel, os valores obtidos nas caracterizações foram compatíveis com as especificações estabelecidas pela ANP para o produto, com exceção do biodiesel de murumuru (índice de acidez elevado e viscosidade baixa) e do biodiesel de mamona (viscosidade e massa específica elevadas). Os dados estatísticos demonstram altas correlações entre o biodiesel (massa específica (87,4%), viscosidade (98,5%) e índice de acidez (82,8%)) e as matérias-primas (massa específica (92,1%) e viscosidade (99,3%)). Além disso, existe uma grande correlação entre o índice de iodo (84,5%) do biodiesel e o índice de iodo (77,2%) e a massa molar dos ésteres na faixa de C16-C18 (MMTG) presentes na matéria-prima (89,1%). Estes resultados estatísticos ratificam as observações analíticas

I. Studies on alkaline phosphatase activity in developing sea urchin embryos. II. Studies on the activation of protein biosynthesis in sea urchin eggs at fertilization

I. Alkaline phosphatase activity in the developing sea urchin Lytechinus pictus has been investigated with respect to intensity at various stages, ionic requirements and intracellular localization. The activity per embryo remains the same in the unfertilized egg, fertilized egg and cleavage stages. At a time just prior to gastrulation (about 10 hours after fertilization) the activity per embryo begins to rise and increases after 300 times over the activity in the cleavage stages during the next 60 hours.

The optimum ionic strength for enzymatic activity shows a wide peak at 0.6 to 1.0. Calcium and magnesium show an additional optimum at a concentration in the range of 0.02 to 0.07 molar. EDTA at concentrations of 0.0001 molar and higher shows a definite inhibition of activity.

The intracellular localization of alkaline phosphatase in homogenates of 72-hour embryos has been studied employing the differential centrifugation method. The major portion of the total activity in these homogenates was found in mitochondrial and microsomal fractions with less than 5% in the nuclear fraction and less than 2% in the final supernatant. The activity could be released from all fractions by treatment with sodium deoxycholate.

II. The activation of protein biosynthesis at fertilization in eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus has been studied in both intact eggs and cell-free homogenates. It is shown that homogenates from both unfertilized and fertilized eggs are dependent on potassium and magnesium ions for optimum amino acid incorporation activity and in the case of the latter the concentration range is quite narrow. Though the optimum magnesium concentrations appear to differ slightly in homogenates of unfertilized and fertilized eggs, in no case was it observed that unfertilized egg homogenates were stimulated to incorporate at a level comparable to that of the fertilized eggs.

An activation of amino acid incorporation into protein has also been shown to occur in parthenogenetically activated non-nucleate sea urchin egg fragments or homogenates thereof. This activation resembles that in the fertilized whole egg or fragment both in amount and pattern of activation. Furthermore, it is shown that polyribosomes form in these non-nucleate fragments upon artificial activation. These findings are discussed along with possible mechanisms for activation of the system at fertilization.

Experimental and theoretical studies of the electrothermal instability in a nonequilibrium plasma

Experimental and theoretical studies have been made of the electrothermal waves occurring in a nonequilibrium MHD plasma. These waves are caused by an instability that occurs when a plasma having a dependence of conductivity on current density is subjected to crossed electric and magnetic fields. Theoretically, these waves were studied by developing and solving the equations of a steady, one-dimensional nonuniformity in electron density. From these nonlinear equations, predictions of the maximum amplitude and of the half width of steady waves could be obtained. Experimentally, the waves were studied in a nonequilibrium discharge produced in a potassium-seeded argon plasma at 2000°K and 1 atm. pressure. The behavior of such a discharge with four different configurations of electrodes was determined from photographs, photomultiplier measurements, and voltage probes. These four configurations were chosen to produce steady waves, to check the stability of steady waves, and to observe the manifestation of the waves in a MHD generator or accelerator configuration.

Steady, one-dimensional waves were found to exist in a number of situations, and where they existed, their characteristics agreed with the predictions of the steady theory. Some extensions of this theory were necessary, however, to describe the transient phenomena occurring in the inlet region of a discharge transverse to the gas flow. It was also found that in a discharge away from the stabilizing effect of the electrodes, steady waves became unstable for large Hall parameters. Methods of prediction of the effective electrical conductivity and Hall parameter of a plasma with nonuniformities caused by the electrothermal waves were also studied. Using these methods and the values of amplitude predicted by the steady theory, it was found that the measured decrease in transverse conductivity of a MHD device, 50 per cent at a Hall parameter of 5, could be accounted for in terms of the electrothermal instability.

Avaliação da influência do material de confecção das matrizes no teste de conversão monomérica por espectrometria no infravermelho de um compósito

Este estudo teve por objetivo avaliar, in vitro, a influência do material de confecção das matrizes, traçando um perfil da conversão monomérica de um compósito micro-híbrido, além de avaliar qual dos materiais testados mais se assemelha a uma matriz de dentina. A avaliação foi feita através da análise do grau de conversão (GC). Foram confeccionadas 3 matrizes bipartidas, sendo estas de teflon negro, tefon branco e aço inoxidável, ambas com 10mm de diâmetro e 2 mm de profundidade. Para o grupo controle foi utilizado um incisivo central bovino, o qual teve sua face vestibular aplainada em uma lixadeira sob refrigeração constante, com o auxílio de uma lixa de carbeto de silício, número 800. Após, este dente foi preparado com uma broca diamantada número 2294 (KG Sorensen) em alta rotação, própria para a preparação de cavidades padronizadas para ensaios de laboratório, apresentando um limitador de penetração. Em seguida, com um motor de baixa rotação foi realizado o acabamento das paredes, obtendo-se uma cavidade de 2,0 mm de profundidade por 9,0 mm de diâmetro. Pela palatina desse dente, com uma broca carbide cilíndrica de numeração 2056 (KG Sorensen), fez-se uma penetração até se obter uma parede de dentina extremamente fina, porém sem que esta fosse rompida. Assim, com uma agulha, fez-se uma pequena perfuração no centro dessa dentina para que este instrumental servisse como um pino para remoção do corpo de prova de dentro da matriz de dente. Os corpos de prova (CP) foram obtidos a partir da inserção do compósito no interior da perfuração das matrizes em um único incremento e cobertos na superfície externa com uma matriz de poliéster mais uma lamínula de vidro. Os CP foram fotopolimerizados por 40 s pela fonte de luz halógena Optilux 501 (Demetron), com 500 mW/cm. Imediatamente após a polimerização, os corpos de prova eram submetidos no topo e na base para a análise de espectrometria no infravermelho para a determinação da profundidade de polimerização, pela técnica do filme vazado para o compósito não polimerizado e pela técnica da pastilha de brometo de potássio (KBr) para o compósito polimerizado. Foram confeccionados 5 CP de cada grupo. Em cada grupo, o compósito da base e do topo das amostras foi moído até se obter de 1,5 a 2,0 mg de pó e misturado com 70 mg de KBr, para obtenção da pastilha de KBr. Foi feita a análise de espectrofotometria no infravermelho por Transformada de Fourier (FTIR). As absorções selecionadas para o cálculo foram 1610 cm-1 e 1637 cm-1, os picos dos espectros das ligações dos carbonos aromáticos e alifáticos, respectivamente. Os dados obtidos foram tratados estatisticamente. Os grupos Gr1B, Gr2B, Gr3B e Gr4B representam, respectivamente, as bases dos CP confeccionados pelas matrizes de DB, TN, TB e AI. Já os Gr1T, Gr2T, Gr3T e Gr4T representam os topos. Médias (%) e DP: Gr1T (46,461,99), Gr2T (39,864,51), Gr3T (44,053,44) e Gr4T (38,045,08). Gr1B (40,441,49), Gr2B (36,153,81), Gr3B (40,093,18) e Gr4B (35,593,35). Em posse dos resultados, pôde-se concluir que os grupos do teflon negro, teflon branco e aço inoxidável não apresentaram diferenças entre o grau de conversão do topo e da base, enquanto que o grupo da dentina apresentou maior conversão do topo. Comparando as matrizes entre elas, pôde-se perceber que no topo, o GC do dente bovino é maior que o GC do aço inoxidável e do que o de teflon negro, o GC do teflon branco é maior que o GC do aço inoxidável e do que o de teflon negro. Já o topo dos grupos de dente bovino e teflon banco foram semelhantes. Nas bases dos CPs, não houve diferença significativa entre os grupos testados. De acordo com os resultados obtidos no experimento, pôde-se concluir que nos grupos do teflon negro, teflon branco e aço inoxidável não houve diferença entre 0 e 2 mm, ou seja, topo e base, o que mostra que o material de confecção da matriz não influênciou o grau de conversão do compósito. Já para o grupo da matriz de dentina, o topo apresentou valor de conversão monomérica maior, mostrando que, neste caso, o material da matriz interferiu no grau de conversão. Pode-se perceber também que existe uma tendência da matriz de teflon branco se assemelhar mais a matriz de dentina, pois foi o único grupo que apresentou semelhança nos valores de conversão monomérica no topo das amostras. Porém analisando a base das amostras, percebe-se que todos os grupos se comportaram de forma semelhante, obtendo valores do grau de conversão sem diferença significante.

Acid precipitation and its effects on aquatic systems in the English Lake District

There is no evidence of an increase in the acidity (lower pH or alkalinity) of water-bodies in the Lake District over the last 50 years. Brown trout occur in acid streams and upland tarns where pH is 4.5-5.2 throughout the year. Their occurrence in such waters in Britain and Ireland has been known for most of this century and there is no previous evidence of harmful effects on salmonid fisheries, though numbers of fish are naturally low. However, many benthic invertebrates that are common in hill-streams where pH is above 5.7 do not occur in more acid streams. This phenomenon occurs in the headwaters of several western rivers in Cumbria. It is not a recent response to "acid rain". Harmful effects of pH are undoubtedly more pronounced in waters that are poor in other dissolved ions. Low concentrations of sodium, potassium, calcium and chloride are especially important and may limit the distributions of some aquatic animals even where pH is above 5.7. The concentration of sulphate ions is usually relatively high but this is not important to the fauna; concentrations are at least two times higher in productive alkaline water-bodies than they are in unproductive acid waters.

The structure elucidation of a proposed intermediate in the stereoselective synthesis of dl-desoxypodocarpic acid

The hydroxyketone C-3, an intermediate in the stereo-selective total synthesis of dl-Desoxypodocarpic acid (ii), has been shown by both degradative and synthetic pathways to rearrange in the presence of base to diosphenol E-1 (5-isoabietic acid series). The exact spatial arrangements of the systems represented by formulas C-3 and E-1 have been investigated (as the p-bromobenzoates) by single-crystal X-ray diffraction analyses. The hydroxyketone F-1, the proposed intermediate in the rearrangement, has been synthesized. Its conversion to diosphenol E-1 has been studied, and a single-crystal analysis of the p-bromobenzoate derivative has been performed. The initially desired diosphenol C-6 has been prepared, and has been shown to be stable to the potassium t-butoxide rearrangement conditions. Oxidative cleavage of diosphenol E-1 and subsequent cyclization with the aid of polyphosphoric acid has been shown to lead to keto acid I-2 (benzobicyclo [3.3.1] nonane series) rather than keto acid H-2 (5-isoabietic acid series).

Bicyclo[3.2.0]hepta-1,4,6-triene: synthesis, thermal rearrangement, and anion formation

In order to determine the properties of the bicycloheptatrienyl anion (Ia) (predicted to be conjugatively stabilized by Hückel Molecular Orbital Theory) the neutral precursor, bicyclo[3. 2. 0] hepta-1, 4, 6-triene (I) was prepared by the following route.

Reaction of I with potassium-t-butoxide, potassium, or lithium dicyclohexylamide gave anion Ia in very low yield. Reprotonation of I was found to occur solely at the 1 or 5 position to give triene II, isolated as to its dimers.

A study of the acidity of I and of other conjugated hydrocarbons by means of ion cyclotron resonance spectroscopy resulted in determination of the following order of relative acidities:

H₂S ˃ C₅H₆ ˃ CH₃NO₂ ˃ 1, 4- C₅H₈ ˃ I ˃ C₂H₅OH ˃ H₂O; cyclo-C₇H₈ ˃ C₂ H₅OH; фCH₃ ˃ CH₃OH

In addition, limits for the proton affinities of the conjugate bases were determined:

350 kcal/mole ˂ PA(C₅ H₅^-) ˂ 360 kcal/mole

362 kcal/mole ˂ PA(C₅H₇^-, Ia, cyclo-C₇H₇^-) ˂ 377 kcal/mole PA(фCH₂^-) ˂ 385 kcal/mole

Gas phase kinetics of the trans-XVIII to I transformation gave the following activation parameters: E_a = 43.0 kcal/mole, log A = 15.53 and ∆S^ǂ (220°) = 9.6 cu. The results were interpreted as indicating initial 1,2 bond cleavage to give the 1,3-diradical which closed to I. Similar studies on cis-XVIII gave results consistent with a surface component to the reaction (E_a = 22.7 kcal/mole; log A = 9.23, ∆S^ǂ (119°) = -18.9 eu).

The low pressure (0.01 to 1 torr) pyrolysis of trans-XVIII gave in addition to I, fulvenallene (LV), ethynylcyclopentadiene (LVI) and heptafulvalene (LVII). The relative ratios of the C₇H₆ isomers were found to be dependent upon temperature and pressure, higher relative pressure and lower temperatures favoring formation of I. The results were found to be consistent with the intermediacy of vibrationally excited I and subsequent reaction to give LV and LVI.

Concentrations of major ions in the stream water of the River Duddon, England, 1970-1974 [Dataset]

This dataset provides raw data of chemical analyses made during studies on seasonal variations of some major ions in the stream water of the River Duddon in Cumbria. Measurements of sodium, calcium, potassium, magnesium and chloride ions and pH were taken at 5 stations in the River Duddon between January 1970 and August 1974.

Concentrations of major ions in the stream water of the upper basin of the River Duddon, England, 1972-1974 [Dataset]

This dataset provides raw data of chemical analyses made during studies on seasonal variations of some major ions in the stream water of the upper basin of the River Duddon in Cumbria. Measurements of sodium, calcium, potassium, magnesium and chloride ions and pH were taken at 26 stations in the River Duddon basin between 1972 and 1974.

Analysis of stream waters in the catchment of Lake Windermere (1975-1978), England [Dataset]

This dataset provides raw data of chemical analyses made during studies on seasonal variations of some major ions in the stream water of the catchment of Lake Windermere in Cumbria. Measurements of sodium, calcium, potassium, magnesium, chloride ions and pH were taken at 37 stations in the catchment between 1975 and 1978.

Analyses of treated sewage effluent from Grasmere Treatment Unit (1974-1976), England [Dataset]

This dataset provides raw data of chemical analyses made during studies on seasonal variations of treated sewage effluent from Grasmere Treatment Unit in Cumbria. Measurements of sodium, calcium, potassium, magnesium and chloride ions were taken between 1974 and 1976.