Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase 1

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C1 fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto. The enzyme preparation was extremely stable in the presence of folate or L-serine. Pyridoxal 5'-phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55°C. The Km values for serine were 1.25 and 68 millimolar, corresponding to Vmax values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K0.5 value for L-tetrahydrofolate (H4folate) was 0.98 millimolar. Glycine, the product of the reaction and D-cycloserine, a structural analog of D-alanine, were linear competitive inhibitors with respect to L-serine with Ki values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H4folate was a competitive inhibitor when H4folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H4folate (nH = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (nH = 1.0) and the increase in sigmoidicity by NAD+ and adenine nucleotides, negative allosteric effectors (nH = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, DL-{alpha}-methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the nH value from 2.0 for H4folate to 1.5 in presence of L-serine. These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.

Assessing taxpayer response to legislative changes: A case study of ‘in-house’ fringe benefits rules

On 22 October 2012, the Australian Federal Government announced the removal of the $1,000 in-house fringe benefits concession when used as part of a salary packaging arrangement. At the time of the announcement, the Federal Government predicted that the removal of the concession would contribute additional tax revenue of $445 million over the following four years as well as an increase of GST payments to the States and Territories. However, anecdotal evidence at the same time indicated that the Australian employer response was to immediately stop providing employees with such in-house fringe benefits via salary sacrificing arrangements. Data presented in this article, collected from a combination of interviews with tax managers of four Australian entities as well as a review of the published archival data, confirms that the abolition of the $1,000 in-house fringe benefits concession was perceived as a negative change, whereby employees were considered the ‘big losers’ despite assertions by the Federal Government to the contrary. Using a conceptual map of tax rule change developed by Oats and Sadler, this article seeks to understand the reasons for this fringe benefits tax change and taxpayer response. In particular, the economic and political factors, and the responses of the relevant taxpayers (employers) are explored. Drawing on behavioural economic concepts, the actions, attitudes and response of employers to the rule change are also examined. The research findings suggest that the decision by Australian employers to cease providing the in-house fringe benefits as part of a salary-packaging arrangement after the legislative amendment was impacted by more than simple rational behaviour.

The effects of the 2004 reduction in the price of alcohol on alcohol-related harm in Finland : A natural experiment based on register data

Changes in alcohol pricing have been documented as inversely associated with changes in consumption and alcohol-related problems. Evidence of the association between price changes and health problems is nevertheless patchy and is based to a large extent on cross-sectional state-level data, or time series of such cross-sectional analyses. Natural experimental studies have been called for. There was a substantial reduction in the price of alcohol in Finland in 2004 due to a reduction in alcohol taxes of one third, on average, and the abolition of duty-free allowances for travellers from the EU. These changes in the Finnish alcohol policy could be considered a natural experiment, which offered a good opportunity to study what happens with regard to alcohol-related problems when prices go down. The present study investigated the effects of this reduction in alcohol prices on (1) alcohol-related and all-cause mortality, and mortality due to cardiovascular diseases, (2) alcohol-related morbidity in terms of hospitalisation, (3) socioeconomic differentials in alcohol-related mortality, and (4) small-area differences in interpersonal violence in the Helsinki Metropolitan area. Differential trends in alcohol-related mortality prior to the price reduction were also analysed. A variety of population-based register data was used in the study. Time-series intervention analysis modelling was applied to monthly aggregations of deaths and hospitalisation for the period 1996-2006. These and other mortality analyses were carried out for men and women aged 15 years and over. Socioeconomic differentials in alcohol-related mortality were assessed on a before/after basis, mortality being followed up in 2001-2003 (before the price reduction) and 2004-2005 (after). Alcohol-related mortality was defined in all the studies on mortality on the basis of information on both underlying and contributory causes of death. Hospitalisation related to alcohol meant that there was a reference to alcohol in the primary diagnosis. Data on interpersonal violence was gathered from 86 administrative small-areas in the Helsinki Metropolitan area and was also assessed on a before/after basis followed up in 2002-2003 and 2004-2005. The statistical methods employed to analyse these data sets included time-series analysis, and Poisson and linear regression. The results of the study indicate that alcohol-related deaths increased substantially among men aged 40-69 years and among women aged 50-69 after the price reduction when trends and seasonal variation were taken into account. The increase was mainly attributable to chronic causes, particularly liver diseases. Mortality due to cardiovascular diseases and all-cause mortality, on the other hand, decreased considerably among the-over-69-year-olds. The increase in alcohol-related mortality in absolute terms among the 30-59-year-olds was largest among the unemployed and early-age pensioners, and those with a low level of education, social class or income. The relative differences in change between the education and social class subgroups were small. The employed and those under the age of 35 did not suffer from increased alcohol-related mortality in the two years following the price reduction. The gap between the age and education groups, which was substantial in the 1980s, thus further broadened. With regard to alcohol-related hospitalisation, there was an increase in both chronic and acute causes among men under the age of 70, and among women in the 50-69-year age group when trends and seasonal variation were taken into account. Alcohol dependence and other alcohol-related mental and behavioural disorders were the largest category in both the total number of chronic hospitalisation and in the increase. There was no increase in the rate of interpersonal violence in the Helsinki Metropolitan area, and even a decrease in domestic violence. There was a significant relationship between the measures of social disadvantage on the area level and interpersonal violence, although the differences in the effects of the price reduction between the different areas were small. The findings of the present study suggest that that a reduction in alcohol prices may lead to a substantial increase in alcohol-related mortality and morbidity. However, large population group differences were observed regarding responsiveness to the price changes. In particular, the less privileged, such as the unemployed, were most sensitive. In contrast, at least in the Finnish context, the younger generations and the employed do not appear to be adversely affected, and those in the older age groups may even benefit from cheaper alcohol in terms of decreased rates of CVD mortality. The results also suggest that reductions in alcohol prices do not necessarily affect interpersonal violence. The population group differences in the effects of the price changes on alcohol-related harm should be acknowledged, and therefore the policy actions should focus on the population subgroups that are primarily responsive to the price reduction.

Les fleuves dans l'œuvre romanesque de Jean-Marie Gustave Le Clézio

Relying on Merleau-Ponty's phenomenology of perception and on Mircea Eliade's works on the Sacred and the Profane, this study explores the river as a perceptual space and as the sacred Center in a cosmic vision of the world in twelve of Jean-Marie Gustave Le Clézio's fictional works, from The Interrogation (1963) to Revolutions (2003). In the first chapter, after introducing the field of study, I discuss the relation between the radical subjectivity and the evasiveness of perceiving subjects in Le Clézio's fiction. Next are some thoughts on the relation between Merleau-Ponty's and Le Clézio's ideas. The second chapter studies the river as an experience in the text, first as a topographical space, then as a sound world. The investigations move on to its water as a visual and a tactile phenomenon. Then follows the human use of the river, the (absence of) baths, and the river as a traveling space. The chapter closes with the study of the metaphorical use of the word, occurring mainly in urban space and for phenomena in the sky. The third chapter is organized around the river as the Center of the world in a religious cosmogony, where the river represents the origin of the world and of the human race. The core analysis shows how the middle of the river is a symbolic space of a new beginning. As a sacred space, the river abolishes time as the object of contemplation and as relative immobility from the point of view of a person drifting downstream. The functions of a new beginning and of abolition of time are combined in the symbolic immersions in the water. Finally, the dissertation explores other symbolical spaces, such as the unknown destination of the drift, and the river as the Center of a utopia. The chapter closes with the existential agony as a result of the elimination of the Center in the urban environment. In the final chapter, the river is compared to other watercourses : the creek, the brook and the rapids. The river is more of a spatial entity, whereas the actual water is more important in the smaller watercourses. The river is more common than the other watercourses as a topographical element in the landscape, whereas the minor watercourses invite the characters to a closer contact with their element, in immersions and in drinking their water. Finally, the work situates the rivers in a broader context of different fictional spaces in Le Clézio's text.

Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata)

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 micrograms/day release rate), via an Alzet pump implant (s.c.) led to a 60-80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3.3 +/- 2.0 micrograms/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P < 0.01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P < 0.001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone. In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by > 90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P < 0.01) the elongate spermatid (HC) population showed an increase of 52% (P < 0.05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased.

In vitro and in vivo effects of methyl isocyanate on rat brain mitochondrial respiration

The present study deals with the in vitro and in vivo effects of methyl isocyanate (MIC) on rat brain mitochondrial function. Addition of MIC to tightly coupled brain mitochondria in vitro resulted in a mild stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/0 ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fourfold) to the inhibitory action of MIC than succinate while cytochrome oxidase was unaffected. Administration of MIC subcutaneously at a lethal dose affected respiration only with glutamate + malate as the substrate (site I) and caused a 20% decrease in state 3 oxidation leading to a significant decrease in respiratory control index while state 4 respiration and ADP/O ratio remained unaffected. As both the malondialdehyde and iron contents of brain mitochondria were not altered, it may be inferred that the observed in vivo inhibition of state 3 oxidation is induced by MIC through systemic stagnant hypoxia leading to ischemia of brain, which further contributes to the cerebral hypoxia.

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.

Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions

A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

El mito de Safo en el relato decadente

[FR] La femme homosexuelle, reflet d’un désir mythique hermaphrodite, est, pendant la fin du siècle une figure littéraire paradoxale et en même temps obsédante. Sapho est, comme la femme fatale, une des protagonistes de la fin-du-siècle qui répond à une misogynie récurrente dans l’imaginaire créateur. Il s’agit d’un désir artificiel, éros intangible (Péladan 1910: 74), contre nature, un idéal impossible: Sexe initial,sexe définitif, absolu de l’amour, absolu de la forme, sexe qui nies le sexe, sexe d’éternité! Los à toi, Androgyne! (Péladan 1910: 76). Sapho, figure archétypique, androgyne, constitue en définitive le symbole de l’abolition des sexes, de la confusion des genres, de la crise du couple traditionnel. Elle met en évidence la solitude, la peur et un désir inassouvi.

João de Mattos: as aventuras de um padeiro na luta pela liberdade e pela dignidade da profissão (1876-1912)

Este trabalho dissertativo é um estudo sobre as memórias deixadas por João de Mattos em seu Manuscrito, intitulado, Histórico Social de 1876 a 1912. Tal autor viveu, inicialmente, em Santos até 1876, a partir de 1877 em São Paulo e após 1878 no Rio de Janeiro. No referido Manuscrito, João de Mattos relata suas aventuras pela conquista da liberdade e sua luta por uma vida digna para a categoria dos padeiros. A dissertação examina a trajetória de Mattos e de seus companheiros na organização de três associações criadas pelos empregados de padarias no Rio de Janeiro: O Bloco de Combate dos Empregados de Padarias; a Sociedade Cooperativa dos Empregados de Padarias no Brasil; e a Sociedade Cosmopolita Protetora dos Empregados de Padaria. Além disso, analisa a presença dos trabalhadores da categoria em três momentos singulares da História: Na abolição, nas campanhas republicanas e no movimento operário.

Equilibrando a vida: os enunciados sobre os malabaristas da subsistência do pós-abolição carioca

Esta dissertação inscreve a história de personagens presentes nas ruas da cidade do Rio de Janeiro: os trabalhadores informais. Uma história da diferença que esses indivíduos, ao afetarem e serem afetados pelo controle urbano e pela sociedade, engendraram (e engendram) na urbe carioca, é o que se propõe. O momento privilegiado é o do pós-abolição, por ser um período de tensão entre descontinuidade (fim da mão-de-obra escravista) e continuidade (das pequenas profissões amorfas à classificação assalariada de trabalho); e, ainda, marcado pela modernização da cidade (as reformas urbanas e higiênicas dos governos Rodrigues Alves e Pereira Passos). Para ir ao encalço dessas vidas-equilibristas, problematizo alguns enunciados produzidos sobre eles: o historiográfico (pesquisas que tocam nesse tema), o jurídico (por meio da Contravenção da Vadiagem e sua reincidência artigo 399 e 400 do Código Penal de 1890) e o caricatural (dois álbuns intitulados Cenas da Vida Carioca, de Raul Pederneiras). Que ditos sobre os trabalhadores informais compõem a memória dizível da cidade? Ao compará-los, por que e quais são as semelhanças e diferenças entre esses enunciados? Que interfaces entre eles o controle urbano e os transeuntes foram possíveis?

Pilares negros: educação, fé e política na Diocese de Duque de Caxias (1988-2000)

A presente pesquisa tem como enfoque a ação educativa do movimento negro no Brasil constituído na base católica. Os marcos temporais e espaciais se localizam entre 1988 e 2000 na Diocese de Duque de Caxias e sua paróquia em São João de Meriti, ambas situadas na Baixada Fluminense, região metropolitana do Rio de Janeiro. A escolha do ano de 1988 se deve ao papel protagonista que os atores coletivos tiveram na Campanha da Fraternidade da CNBB (Confederação Nacional dos Bispos do Brasil) no ano de 1988, centenário da abolição da escravatura no Brasil. A principal fonte pesquisada o periódico diocesano Pilar, publicado em Duque de Caxias desde 1990 apontou que, ao longo da década em estudo, o movimento negro de base católica operou práticas formativas que buscavam alcançar a consciência da negritude no mundo leigo e eclesial. Os movimentos sociais unem a intencionalidade político-formativa ao projeto de transformação de realidades e, neste sentido, a construção do conhecimento atua para a emancipação dos atores. A luta anti-racista, que ainda afeta patrimônios sociais e culturais, empreendeu uma ação contra-hegemônica interior e exterior à Igreja Católica, aqui analisada à luz do jornal Pilar.

Iluminar a terra pela inteligência: Trajetória do Aprendizado Agrícola de Barbacena, MG (1910 - 1933)

Esta tese objetiva percorrer a trajetória da criação, implantação e mudanças, ao longo do processo histórico do Aprendizado Agrícola de Barbacena, desde seu início, em 1910, até a época do presidente Getúlio Vargas, 1933. Como preliminares e pano de fundo, as iniciativas do Brasil, que, premido pela carência de mão de obra qualificada para atender à demanda das fazendas, após a abolição da escravatura, busca conhecimentos e experiências em outros países, na área da educação agrícola. Com o advento da República, verifica-se uma guinada do Estado brasileiro no sentido de assumir o ensino elementar público, a fim de ir diminuindo o percentual de analfabetos e de ensaiar a implantação do ensino agrícola, tendo em vista a crise que surgira no setor agrícola. É nesse cenário que se criam os aprendizados agrícolas. O Aprendizado Agrícola de Barbacena surge em 1910. Com foco nessa Instituição, abordam-se: o papel de lideranças políticas mineiras para trazer para Barbacena o primeiro Aprendizado Agrícola de Minas Gerais; as mudanças por que o Aprendizado passou, indo de seu início até a época de Getúlio, quando foi transformado em Escola Agrícola; o método de ensino, prevalentemente teórico-prático; a integração do Aprendizado com seu meio; o sistema de administração que incluía participação dos alunos nos lucros; a estrutura didático-pedagógica e o regime de internato. O estudo destaca o trabalho do seu primeiro Diretor, Diaulas Abreu, por 45 anos à frente da Instituição. Como metodologia de pesquisa, analisam-se decretos relativos à criação do Aprendizado, regulamentos, relatórios, dados do arquivo da Instituição e a troca de correspondência entre a direção e órgãos do governo. A pesquisa se encerra na era do presidente Getúlio Vargas, após a Revolução de 1930.

A delimitação das cláusulas pétreas tributárias e financeiras na Constituição de 1988

O texto constitucional brasileiro de 1988 sofreu diversas alterações em matéria tributária e financeira. Praticamente todas as emendas constitucionais tiveram sua constitucionalidade questionada em razão de alegadas violações aos limites materiais do poder de reforma constitucional. O presente trabalho procura delinear alguns parâmetros para a construção de contornos mais precisos dos limites do poder de reforma constitucional em matéria tributária e financeira. A delimitação de tais parâmetros de reforma, além de contribuir para o exercício da jurisdição constitucional na análise da constitucionalidade das emendas constitucionais, permite que sejam mais bem delineadas quais as decisões fundamentais que a Constituição adotou na seara financeira e tributária, relacionadas ao federalismo, aos direitos fundamentais e à separação de poderes.