Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP-binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Amino Acid Residues Implicated in the Interaction of Melanocortin Ligands and their Receptors: A Study of MC2R Selectivity

Author: Kristopher D. Veo Title: Amino acid residues implicated in the interaction of Melanocortin ligands and their receptors: A study of MC2R selectivity Advisor: Dr. Robert M. Dores Degree Date: August 2009 ABSTRACT Melanocortin receptor ligand selectivity has been a question not easily answered. The inability to functionally express melanocortin 2 receptor (MC2R) has inhibited the study of why MC2R is only stimulated by ACTH, a melanocortin hormone. With the recent discovery of the MC2R accessory protein (MRAP), creating a heterologous system is now feasible. Using a general cell line like CHO-K1 cells, which do not express endogenous MCRs, we were able to create a heterologous expression system and test the selectivity of MC2R using analog variants of ACTH(1-24). Our results indicate an amino acid requirement in the C-terminal portion of ACTH(1-24) for activation, which supports the 2-step method of activation hypothesized for MC2R. This site, the tetra basic cleavage site, when altered does not stimulate cAMP production and does not compete with ACTH(1-24) for binding. We also demonstrate the potential for a non-mammalian MC2R system in cloning full length Silurana tropicalis MC2R and completed localization studies with this system with MRAP using CHO-K1 cells.

Degradação enzimática de clorofenol em microrreator.

O microrreator faz parte de conjunto de dispositivos de uma nova e promissora tecnologia, que podem ser chamados de micro fabricados, atuante em campos como a da química, biológica, farmacêutica, engenharia química e biotecnologia. Trata-se de um dispositivo que possibilita reação química, tais como os reatores convencionais, mas com dimensões menores, com canais na escala micrométrica. A tecnologia de miniaturização de dispositivos para reações químicas vem se expandindo promovendo uma importante evolução, com microssistemas que abrange dispositivos mais eficazes, com configuração e geometrias específicas e menor consumo de energia, onde reações com elevadas taxas de transporte podem ser usadas para muitas finalidades diferentes, tais como, reações rápidas, mistura, reações sensíveis à temperatura, temperatura de homogeneização, ou até mesmo precipitação de nano partículas. Devido sua escala ser extremamente reduzida em relação à escala macro, oferecem um sistema que permite uma investigação do processo em um curto espaço de tempo, sendo muito útil para o rastreio de substratos, enzimas, condições de reação, bem como a determinação de parâmetros cinéticos. O presente trabalho teve por objetivo estudar a biodegradação enzimática de 2,4,6-Triclorofenol, com a utilização das enzimas Lacase e Soybean Peroxidase em microrreator da Syrris com volume de 250 ?l, que permite o estudo de cinéticas muito rápidas. Para as análises de degradação utilizou-se duas enzimas, a Lacase em concentrações de 0,05; 0,1 e 0,2 mg/ml; e a Soybean Peroxidase em concentrações de 0,0005; 0,001 e 0,002 mg/ml com a adição de Peróxido de Hidrogênio. Através dos ensaios realizados obteve-se dados experimentais da reação enzimática, possibilitando a verificação da taxa inicial de reação e sua cinética. Posteriormente, realizou-se as análises em simulação utilizando os dados experimentais, que através de um sistema de EDOs estimando inicialmente as constantes cinéticas k1, k2 e k3 usando a ferramenta ESTIMA, onde apresentaram duas respostas, uma resposta típica de mínimos quadrados, e a outra resposta que a velocidade inicial, que foi melhor representada pelos parâmetros obtidos. O método empregado na degradação do substrato, o microrreator mostrou-se eficiente, permitindo a detecção de baixo consumo de substrato para a determinação da taxa inicial, em curto tempo de residência. Perante os ensaios realizados com Lacase e Soybean Peroxidase, o microrreator é também um equipamento eficaz na repetitividade e na reprodutibilidade dos dados obtidos em diferentes concentrações.

Estudo cinético da lixiviação de metais de placas de circuito impresso obsoletas.

O processo tradicional de recuperação de metais de resíduos de equipamentos eletroeletrônicos (REEE) geralmente envolve processamento pirometalúrgico. Entretanto, o uso desta tecnologia para processar placas de circuito impresso (PCI) obsoletas pode levar à liberação de dioxinas e furanos, devido à decomposição térmica de retardantes de chama e resinas poliméricas presentes no substrato das placas. Portanto, este trabalho propõe uma rota hidrometalúrgica para recuperação de metais. O comportamento dos metais, com destaque para cobre, zinco e níquel, durante a lixiviação ácida, foi estudado em três temperaturas diferentes (35ºC, 65ºC e 75ºC), com e sem adição de um agente oxidante (peróxido de hidrogênio H2O2). A cinética de dissolução ácida desses metais foi estudada baseada na análise química por ICP-OES (Espectrometria de emissão ótica por plasma acoplado indutivamente) e EDX (Espectroscopia de fluorescência de raios-X por energia dispersiva). O balanço de massa e a análise química indicaram que a etapa de lixiviação sem adição de oxidante é pouco eficaz na extração dos metais, sendo responsável pela dissolução de menos do que 6% do total extraído. A 65ºC e H2SO4 1 mol/L, com adição de 5 mL de H2O2 (30%) a cada quinze minutos e densidade de polpa de 1 g / 10 mL, 98,1% do cobre, 99,9% do zinco e 99,0% do níquel foram extraídos após 4 horas. A cinética de dissolução desses metais é controlada pela etapa da reação química, seguindo, dependendo da temperatura, a equação 1 (1 XB)1/3 = k1.t ou a equação ln (1 XB) = k4.t.

Determination of fat-soluble vitamins in vegetable oils through microwave-assisted high-performance liquid chromatography

In this manuscript, a study of the effect of microwave radiation on the high-performance liquid chromatography separation of tocopherols and vitamin K1 was conducted. The novelty of the application was the use of a relatively low polarity mobile phase in which the dielectric heating effect was minimized to evaluate the nonthermal effect of the microwave radiation over the separation process. Results obtained show that microwave-assisted high-performance liquid chromatography had a shorter analysis time from 31.5 to 13.3 min when the lowest microwave power was used. Moreover, narrower peaks were obtained; hence the separation was more efficient maintaining or even increasing the resolution between the peaks. This result confirms that the increase in mobile phase temperature is not the only variable for improving the separation process but also other nonthermal processes must intervene. Fluorescence detection demonstrated better signal-to-noise compared to photodiode arrayed detection mainly due to the independent effect of microwave pulses on the baseline noise, but photodiode array detection was finally chosen as it allowed a simultaneous detection of nonfluorescent compounds. Finally, a determination of the content of the vitamin E homologs was carried out in different vegetable oils. Results were coherent with those found in the literature.

Anisotropy-magnetic susceptibility from the Krems Wachtberg archaeological Site (Austria) and high temperature susceptibility

The loess sediment embedding the main Gravettian layer at the Krems-Wachtberg archaeological site facilitates exceptional preservation. To gain insight in the sedimentation process before and after the Paleolithic settlement, the magnetic fabric (preferential orientation of magnetic particles) of loess of the Krems-Wachtberg site is investigated. Magnetic fabric properties clearly show an eolian origin of the loess, but may indicate some relocation in the meter above the cultural layer. The magnetic fabric properties can be divided into three intervals, the top interval shows lowest foliation and inconsistent magnetic fabric directions. The middle interval around the main cultural layer shows low foliation, but a clear preferential NW - SE direction of the lineation. This lineation is interpreted as preferential direction of the eolian loess accumulation from the South-East. The interval below ca. 0.5 m underneath the main find horizon shows a northeast-southwest lineation, but an imbrication suggesting that sediment accumulation occurred perpendicular to this direction, similar to the interval around the find horizon.

Magnetic properties of ODP Site 191-1179 sediments

As reported by Shipboard Scientific Party (2001b, doi:10.2973/odp.proc.ir.191.104.2001) in the Site 1179 chapter of the Initial Reports volume, Leg 191 Site 1179 is located on abyssal seafloor northwest of Shatsky Rise, ~1650 km east of Japan. This part of the Pacific plate was formed during the Early Cretaceous, as shown by northeast-trending M-series magnetic lineations that become younger toward the northwest (Larson and Chase, 1972, doi:10.1130/0016-7606(1972)83[3627:LMEOTW]2.0.CO;2; Sager et al., 1988, doi:10.1029/JB093iB10p11753; Nakanishi et al., 1989, doi:10.1029/1999JB900002). The site is situated on magnetic Anomaly M8 (Nakanishi et al., 1999, doi:10.1029/1999JB900002), corresponding to an age of ~129 Ma and the Hauterivian stage of the Early Cretaceous (Gradstein et al., 1994, doi:10.1029/94JB01889; 1995). The sediments recovered at Site 1179 are split into four lithostratigraphic units based on composition and color (Shipboard Scientific Party, 2001b, doi:10.2973/odp.proc.ir.191.104.2001). Unit I (0-221.52 meters below seafloor [mbsf]) is a dominantly olive-gray clay- and radiolarian-bearing diatom ooze. Unit II (221.52-246.0 mbsf) is a yellowish brown to light brown clay-rich and diatom-bearing radiolarian ooze. Unit III (246.0-283.53 mbsf) is composed of brown pelagic clay. Unit IV (283.53-377.15 mbsf) is composed of chert and some porcellanite; any softer sediments present were washed out of the core barrel by the fluid circulating during the coring process.

Calcium carbonate content and anisotropy of magnetic susceptibility of ODP Leg 172 sites

Sediments from Sites 1057 and 1061 of Ocean Drilling Program Leg 172 on the Blake Outer Ridge exhibit nearly isotropic magnetic susceptibility. Resolving the degree of anisotropy of magnetic susceptibility proved difficult in many samples because of the generally weak magnetic susceptibility of the sediments relative to the noise level of the susceptibility meters used. Lineation varies from 1.0 to 1.013 and foliation varies from 1.0 to 1.08 in the samples that pass rejection criteria. In general the foliation is better resolved than the lineation, particularly at Site 1061, where the foliation exhibits long-term trends that mimic the mean susceptibility. The changes in the foliation at this site are likely the result of changes in the magnetic mineralogy of the sediment. The poorly developed or absent magnetic fabric in the sediments overall can be attributed to high carbonate concentrations and to a circulation regime that was diffuse or with currents too weak to effectively align magnetic particles.

De termino Michaelis anno XXXIIII. regni Regis Henrici Sexti.

Numbered as leaves, printed as pages.

Catalecta rabbinica in usum scholarum privatarum edita /

Mode of access: Internet.

Isbrandi de Diemerbroeck ... Opera omnia anatomica et medica : partim jam antea excusa, sed plurimis locis ab ipso auctore emendata, & aucta, partim nondum edita /

Mode of access: Internet.

Elementos de medicina practica /

Mode of access: Internet.

Oeuvres de Monsieur de Montesquieu.

Contiene : Avertissement sur cette nouvelle edition ; Éloge de M. de Montesquieu par M. D'Alembert ; Analise de l'Esprit des Loix par M. D'Alembert ; Discours prononcé le 24 Janvier 1728 par M. de Montesquieu à l'Académie Françoise ; Livres I-XII inclusivement de l'Esprit des Loix