bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Agricultural Extension in Cocoa Production and Livelihood of Farmers in Meme Division, Cameroon
Authors: Taku J. Defang , Njoh R. N. , Meliki N. S. , Amungwa F. A. , Manu I. N.
Published: December 2020

Agriculture is important not only for its supply of food but also for its provision of raw materials. Cocoa is a lucrative cash crop grown in kumba, Meme Division of Cameroon. Given the importance of the crop to farm communities and the economy of Cameroon, agricultural extension has key roles to enhance production through information dissemination and adoption of innovations. This survey thus assess the role of agricultural extension in cocoa production and the livelihood of farmers in Meme Division. Through simple random sampling technique, 137 farmers were selected from the population of 210 registered cocoa farmers in the study area. A questionnaire made up of open and close ended questions was the instrument for primary data collection. The collected data was analyzed using SPSS and results revealed that 90% of the farmers were married men and 60% were of the age 35-49 years. Also, 74% of the respondents had been into cocoa farming for more than 10 years, signifying that their livelihood depend on it. A low level of education is observed with 55% of them attaining only primary education. Moreover, 64% are small farm holders with 68% acknowledging use of family labor for production activities. Results further show that agricultural extension plays an essential role in cocoa production as 92% affirmed that extension provided information, knowledge and technical skills for capacity building and empowerment using Farmers Field School (FFS) and Farmers Business School (FBS) approaches. To 96%, extension initiates diffusion and adoption of innovations and organizes credits and market linkages (87%). Another 89%, were confirmative that extension liaise projects under the Program for the Improvement of Competitiveness of Family Agro-Pastoral Farms (with French acronym ACEFA) to accompany the farmers. Conclusively, extension service delivery empowers and ensures livelihood of farmers.
Role of Tax Revenue, Non-tax Revenue, and Foreign Aid to Increase the Size of Budget in Nepal
Authors: Dahal Arjun Kumar , Dhakal Ghanshyam , Thapa Khagendra Kumar
Published: December 2020

Purpose: The purpose of the present study is to find the impact of tax revenue, non-tax revenue, and foreign aid to increase the size of the budget in Nepal.

Methods: This study is based on descriptive, analytical, and exploratory research designs. The Johnsen Co-integration Test, VECM, Wald Test, and Granger Causality Test are used to find long-run relation, impact, short-run causality, and granger cause between the pairs of variables.

Results: The tax revenue, non-tax revenue, foreign aid, and budget are co-integrated, or they have a long-run association ship. The result of VECM shows that tax revenue, non-tax revenue, foreign aid is nicely fitted, and they are jointly significant to explain the size of the budget in Nepal. Short-run causality was found between the size of budget and tax revenue and size of budget and foreign aid, but there was an absence of short-run causality between budget and non-tax revenue in Nepal. The granger cause was not found between the pair of variables.

Implications: It seems to increase the tax revenue and decrease the dependency on foreign aid.

Limitations: This study was based on the secondary data of 40 years from the fiscal year 1979/80 to 2018/19.  Only three variables, tax revenue, non-tax revenue, and foreign aid, are considered the effecting factor of the budget size. Hence, further study is necessary by employing other tools and variables.

Originality: The author was not affected by the study and findings of others.
Influence of Different Types of Organic Manure and Weeding Frequencies on Weed Parameters and Leafy Yield of Jute Mallow (Corchorus olitorious)
Authors: Olorukooba M. M. , Suleiman R. , Olukotun O. , Mohammed R. , Apene E. , Essien J. , Danbaki C. A. , Olafemi S. O.
Published: December 2020

Field trials were carried out during 2018 and during 2019 dry season period at Federal College of Forestry and Mechanization Afaka Kaduna, located between latitude 10° 35”N and longitude 007° 21”E at altitude 644 m above sea level, to determine the influence of different types of organic manure and weeding frequencies on weed parameters and leafy yield of jute mallow (Corchorus olitorious). The experimental treatments consists of ten combinations of  poultry manure + weed free, cow dung + weed free, goat dropping + weed free, poultry manure + weeding at 2 WAT, cow dung + weeding at 2 WAT, goat dropping + weeding at 2 WAT, poultry manure + weeding at 4 WAT, cow dung + weeding at 4 WAT, goat dropping + weeding at 4 WAT and no organic manure application + no weeding which were laid out in a randomize complete block design (RCBD) and replicated 3 times. The weed parameters and yield component observed and measured were common weeds that infested the plots, weed dry weight, weed control index, weed competition index and cumulative leaf yield at 2, 4, 6 WAT and at harvest. The result revealed that the plots were highly infested with three major weed species namely Synedralla nodiflora Gaertn, Gomphrena celosiodes and Cyperus esculentus. Highest weed completion index was obtained from poultry manure + weeding at 2 WAT while the least weed competition index was observed with application of cow dung + weeding at 4 WAT and goat dropping + weeding at 4 WAT respectively. The result also revealed that application of poultry manure + weed free plots had the highest mean cumulative yield value of 236 kg per hectare, closely followed by plots with application of poultry manure + weeding at 4 WAS (223 kg) per hectare compared to the control treatment that plots that have no organic manure + no weeding that had the least mean cumulative yield value of 111 kg per hectare. The study concludes that the use of poultry manure plus weed free gave the best yield of jute mallow compared to other types of organic manure and the control. It is therefore recommended that farmers in the study area should adopt the use of poultry manure plus weed free treatment to obtain bumper harvest from cultivation of jute mallow (Corchorus olitorious).
COVID-19 Pandemic and the 2020 Elections in Ghana
Authors: Ijon Frank Bitafir , Bingab B. B. B.
Published: December 2020

The paper explores the impact of the Coronavirus pandemic on Ghana’s election 2020 which will be conducted on the 7th December 2020. The paper argues that despite the fact that the pandemic has negatively affected the 2020 elections process, it also presents political parties and the Electoral Commission the opportunity to be innovative in their activities. The study adopts a content analysis approach and depends mainly on the desk review of related literature on the topic under investigation. The study found that the possible negative impacts of the virus on the 2020 elections include: low voter turnout because of the fear of contracting the virus due to human contacts at the voting centers and postponement of the 2020 elections if the spread goes out of hand. The impacts felt already include postponement of voter registration exercise from April 2020 to June 2020 and the ban on political activities such as mass campaigns.

Again, one positive impact of the virus is that it has made political parties innovative in the mobilization of voters through the use of various social media platforms. Now campaigns and other political activities are held via the internet. With such innovation, the paper proposes that the E.C. should consider online voting, postal voting, and early voting in the 2020 election as a way of reducing human conduct and overcrowding on the day of voting in order to reduce the spread of COVID-19.
Caracterização de pacientes com sequelas após Infecção pelo vírus chikungunya de acordo com a CIF
Authors: Da Silva Filho Jurandir , Silva Lídia Ferreira da , Lima Jullyane Maria , Almeida Mylena Cristina Ever de , Jacob Mellina Monteiro , Moraes Suellen Alessandra Soares de
Published: December 2020

Objetivo: Caracterizar segundo a Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF), as sequelas apresentadas por pacientes após infecção pelo vírus Chikungunya (CHIKV). Metodologia: Estudo Transversal e descritivo, realizado com 34 participantes, com diagnóstico sorológico e/ou clínico de infecção pelo CHIKV. As variáveis foram coletadas por meio da Escala Visual Analógica (EVA), Short Form Health Survey 36 (SF-36) e Medical Research Council (MRC), e  relacionadas com os domínios da CIF para então classificá-las. Os dados foram tabulados em planilha Microsoft Excel 2013 com estatística descritiva calculada pelo programa GraphPad Prism 6. Os resultados foram apresentados como média e desvio padrão ou mediana e intervalo interquartil. Resultados: Os resultados obtidos com a  EVA foram caracterizados como moderada a grave deficiência, no SF-36 os domínios variaram de moderada a completa deficiência e no MRC todos os movimentos analisados corresponderam a ligeira deficiência. Conclusão: Foi possível caracterizar alterações osteomioarticulares e a funcionalidade dos pacientes infectados pelo CHIKV e classificá-los dentro do contexto da CIF, garantindo uma padronização para a descrição dos componentes relacionados à saúde desses pacientes.
Impacto da severidade da oclusopatia na qualidade de vida em escolares de 12 anos da rede estadual de ensino em uma população brasileira
Authors: Hayne Mabel Cancio , Meira Ana Claudia Lima de Oliveira , Oliveira Márcio Campos
Published: December 2020

A má oclusão pode impactar a qualidade de vida dos indivíduos acometidos. O objetivo desse estudo foi avaliar a associação entre severidade das oclusopatias e o impacto na qualidade de vida em escolares de 12 anos da rede estadual de uma população brasileira. Através do Dental Aesthetic Index (DAI) e do questionário Oral Health Impact Profile (OHIP-14), numa amostra de 503 estudantes do município de Feira de Santana-BA, estimou-se a severidade das oclusopatias e a prevalência do impacto negativo da saúde oral na qualidade de vida, verificando associações entre essas variáveis e as condições sociodemográficas da população. Para testes estatísticos foram utilizados: o teste t de Student ou teste de Mann-Whitney, os testes de Fisher ou do qui-quadrado e variantes, além do programa estatístico computacional GraphPad Prism, versão 6.0.3, GraphPad Software, San Diego-CA, USA. A prevalência das oclusopatias 51,29% [45,98-54,60] IC95% e suas categorias: Leve (48,7%), Definida (27,8%), Severa (15,5%) e Incapacitante (8%) tiveram associação estatística com o impacto negativo na qualidade de vida, cuja prevalência foi de 62,23% [57,91-67,33%] IC95% (313/503). Características sociodemográficas não diferiram entre os grupos. A presença e a severidade das oclusopatias estão associadas ao impacto negativo na qualidade de vida. De acordo com o aumento do grau de severidade dos problemas oclusais, pior o impacto na qualidade de vida.
Avaliação da morfologia hepática em ratos norvegicus albinus após administração do ranelato de estrôncio
Authors: Oliveira Caio Dias , Câmara Pereira Eliana Dos Santos , Barreto Isabela Cerqueira , Campos Moraes Laise Monteiro
Published: December 2020

Introdução: o fármaco ranelato de estrôncio (RE) é muito utilizado na terapêutica profilática e no controle da osteoporose. Age sistemicamente diminuindo a reabsorção e aumentando a formação óssea.

Objetivo: avaliar a morfologia hepática em ratos norvegicus albinus após administração do RE.

Metodologia: estudo experimental com 10 ratos, divididos aleatoriamente em dois grupos, Grupo Controle (GC), sem administração do RE, e Grupo Ranelato de Estrôncio (GRE), ambos acompanhados durante 15 dias, e, em seguida, sacrificados e o fígado de cada animal colocado para fixação no formol a 4% durante 48 horas. Após essa etapa, foram realizados os procedimentos necessários à análise pela microscopia óptica, com lâminas coradas pela hematoxilina & eosina, e picrosirius red.

Resultados: nos GC e GRE foram encontradas alterações similares, como reação ductular, dilatação sinusoidal e fibrose perissinusoidal, com intensidades distintas entre os grupos, sendo a reação ductular mais proeminente no GC, e a dilatação sinusoidal e fibrose perissinusoidal mais pronunciada no GRE. Além disso, no GC foram evidenciados achados inflamatórios, como presença de infiltrado inflamatório misto e hiperplasia de células de Kupffer, não visualizados no GRE, implicando numa possível ação anti-inflamatória do RE.

Conclusão: pode-se concluir que foram encontrados achados morfológicos no parênquima hepático dos ratos tratados com o RE que os distinguem daqueles não tratados, seja por seu tipo ou intensidade, ainda que esses achados não sejam suficientes para se inferir sobre a incidência de um processo patológico característico.
A suplementação com óleo de peixe três vezes mais concentrado em epa (ácido eicosapentaenoico) modifica a função renal em ratos
Authors: Cassol Bruno Henrique , Peruffo Letícia Chaffin Barbosa , Arioni Sofia Tomaselli , Fernandez Ricardo
Published: December 2020

Objetivos: Óleo de peixe, rico em ácido eicosapentaenoico (EPA), mostrou benefícios renoprotetores em modelos animais de doenças crônicas. O objetivo deste trabalho foi avaliar os efeitos da suplementação com óleo de peixe três vezes mais concentrado em EPA (EPA 3X) na função renal de ratos e a eficiência desta suplementação por um curto período de tempo.

Metodologia: Ratos Wistar (n=16) foram divididos em dois grupos, controle (C) e suplementado (CO) por 14 dias com óleo de peixe (EPA 3X). Foram determinados os seguintes parâmetros: ganho de peso, ingesta de água e ração, fluxo urinário, proteinemia, trigliceridemia, osmolaridade, clearance de creatinina e fração de excreção de sódio.

Resultados: Os ratos suplementados tiveram níveis de triglicerídeos no plasma 58,4% menores que os ratos controle (p=0,0239), entretanto os restantes dos parâmetros sistemicos avaliados não apresentaram alterações. A suplementação com óleo de peixe não alterou o fluxo urinário. Entretanto, no grupo CO identificou-se um aumento na filtração glomerular, com um incremento em média de 67,8% no clearance de creatinina em relação ao grupo C (p=0,054). A excreção média de proteínas totais na urina e a fração de excreção de sódio foram semelhantes entre os grupos.

Conclusão: A suplementação com óleo de peixe três vezes mais concentrado em EPA por um período curto de tempo (quinze dias) provocou alterações significativas tanto em parâmetros sistêmicos (redução significativa na trigliceridemia) quanto na função renal de ratos controle (incremento na filtração glomerular) indicando a eficiência desta suplementação. 
Controle de qualidade de marcações de PSMA-11, utilizando um gerador itinerante de Ge68- Ga68 para estudos de PET-CT
Authors: Silva Daniel Coiro da , Nascimento Roberto Jose Meyer
Published: December 2020

Introdução: Este estudo trata do controle de qualidade do radiofármaco utilizado em estudos de PET-CT para diagnóstico e/ou estadiamento de pacientes acometidos pelo câncer de próstata. Objetivo: Avaliar a qualidade do radiofármaco PSMA-11, marcado com uso de gerador de Ge68-Ga68 itinerante. Método: Análise do aspecto visual, pH e pureza radioquímica do radiofármaco marcado a cada recebimento do gerador de Ge68-Ga68. Resultado: Todas as marcações realizadas se apresentaram límpidas quanto ao aspecto visual, o pH ficou entre 5,0 e 6,0 e a pureza radioquímica apresentou em 92% dos casos valores ³ 96%. Conclusão: Com os devidos controles de qualidade, pode ser uma opção para uso na clínica médica, em serviços que não disponham de condição para adquirir um sistema, por ser importado.
Avaliação do grau de corrosão de fios ortodônticos submetidos a desafio de pH
Authors: Santana Kalil Ayres , Araujo Danilo Barral de
Published: December 2020

Introdução: A movimentação dentária requer a aplicação de um sistema de forças que é orientado com o uso de acessórios, incluindo fios ortodônticos. Existem diversos materiais e fabricantes disponíveis no mercado, por isso é importante que o profissional conheça a composição deste material, uma vez que estará em contato com a mucosa oral, podendo sofrer corrosão e liberação de íons metálicos. Objetivo: Avaliar, in vitro, o grau de corrosão de duas ligas metálicas que compõem fios ortodônticos de três fabricantes diferentes, submetidos à ciclagem de pH. Metodologia: 60 corpos de prova foram confeccionados com dois tipos de ligas metálicas (NiTi e CrNi-aço 302) produzidas por três fabricantes diferentes, divididos em 6 Grupos de Testes com 10 unidades cada. Os corpos-de-prova foram submetidos à ciclagem ácida de pH 4,3, diariamente, e mantidos em estufa a 37 ° C por 14 dias. A massa foi pesada antes e após a ciclagem, por meio de balança analítica de precisão. A avaliação das características visuais dos fios foi realizada por um único observador em lupa estereomicroscópica. Resultados: A ciclagem de pH dos fios ortodônticos não promoveu perda significativa de massa ou corrosão, apesar da análise das características visuais terem mudado após a ciclagem. Conclusão: Com base nos resultados do protocolo experimental estabelecido no presente estudo, a análise do comportamento das ligas de aço NiTi e CrNi - 302 que compõem os fios ortodônticos, frente ao desafio ácido de pH 4,3, por duas semanas, concluiu-se que esses materiais não apresentaram perdas de massa, opacidade, brilho e lisura consideradas significativas. Outros estudos são recomendados para fornecer maiores esclarecimentos sobre o tema.
Cooperativity in the binding of 1α,25‐dihydroxyvitamin D3 to the chick intestinal receptor
Authors: Wilhelm Francois , Norman Anthony W.
Published: July 2002

The binding of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] to its chick intestinal receptor does not fit well to the linear regression line of Scatchard's model (r = −0.79; dissociation constant, K
d = 0.51 nM). In fact, a concave ‘hook’ curve describes the data better. By using the Hill analysis the linear fitting was improved (r= 0.99), the K
d was found to be 0.14 nM and the Hill coefficient (n
H) 1.42, which indicates a positive cooperativity in the binding of 1,25(OH)2D3 to its receptor. Further we found that K
d and n
H are strongly correlated (p < 0.001). These data suggest the existence of two ligand binding sites located in subunits for the 1,25(OH)2D3 receptor.
Diacylglycerol lipase activity in human platelet intracellular and surface membranes
Authors: Authi Kalwant S. , Lagarde Michel , Crawford Neville
Published: July 2002

Diacyl glycerol lipase activity has been examined of intracellular and surface membranes isolated from human blood platelets by free flow electrophoresis. Enzyme activity is present on both membranes but is activated at different substrate concentrations (K
m 14 μM and 140 μM for intracellular and surface membrane, respectively). Both enzyme activities are stimulated by EGTA and GSH, and inhibited by added Ca2+. The specificity of the intracellular membrane enzyme has been investigated using a range of diacylglycerol substrates differing only in their ‘2’ position fatty acid. Arachidonic acid is clearly the preferred ‘2’ position moiety with activities towards eicosatrienoic, linoleic, oleic and palmitic acid‐containing substrates, all substantially lower.
A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis
Authors: Lad Pramod M. , Olson Charles V. , Grewal Iqbal S.
Published: July 2002

Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet‐activating factor (PAF) or f‐Met‐Leu‐Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12‐myristate‐13‐acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non‐opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor‐initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion.
Low concentrations of the stable prostaglandin endoperoxide U44069 stimulate shape change in quin2‐loaded platelets without a measurable increase in [Ca2+]i
Authors: Simpson Alec W.M. , Hallam Trevor J. , Rink Timothy J.
Published: July 2002

U44069
Ionomycin Ca2+

Shape change
Platelet
Platelet‐activating factor
Synthetic magainin analogues with improved antimicrobial activity
Authors: Chen Hao-Chia , Brown Judith H. , Morell John L. , Huang C.M.
Published: July 2002

Based on modifications to enhance the α‐helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.
Tentative identification of RNA‐dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases
Authors: Koonin E.V. , Gorbalenya A.E. , Chumakov K.M.
Published: July 2002

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA‐dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage φ6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), λ3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA‐dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.
Biological activity of hemimethylated and single‐stranded DNA after direct gene transfer into tobacco protoplasts
Authors: Weber H. , Graessmann A.
Published: July 2002

In this investigation we analyzed the effect of DNA methylation on gene expression in tobacco protoplasts. Methylation of the chimeric GUS gene (CaMV 35s/GUS/NOS) with the HpaII methyltransferase had no effect on transient and on stable GUS gene expression. The chimeric gene was then cloned as single‐stranded DNA. In vitro second‐strand DNA synthesis was performed with 5‐methylcytosine, leading to methylation of every cytosine in one DNA strand. After transfection this DNA was totally inactive. Control experiments revealed that the single‐stranded DNA was as active as the double‐stranded form.
Mechanism of elongation factor 2 (EF‐2) inactivation upon phosphorylation Phosphorylated EF‐2 is unable to catalyze translocation
Authors: Ryazanov Alexey G. , Davydova Elena K.
Published: July 2002

Previously we have found that elongation factor 2 (EF‐2) from mammalian cells can be phosphorylated by a special Ca2+/calmodulin‐dependent protein kinase (EF‐2 kinase). Phosphorylation results in complete inactivation of EF‐2 in the poly(U)‐directed cell‐free translation system. However, the partial function of EF‐2 affected by phosphorylation remained unknown. Here we show that phosphorylated EF‐2, unlike non‐phosphorylated EF‐2, is unable to switch ribosomes carrying poly(U) and Phe‐tRNA in the A site to a puromycin‐reactive state. Thus, phosphorylation of EF‐2 seems to block its ability to promote a shift of the aminoacyl(peptidyl)‐tRNA from the A site to the P site i.e. translocation itself.
A second isoform of chicken brush border myosin I contains a 29‐residue inserted sequence that binds calmodulin
Authors: Halsall David J. , Hammer John A.
Published: July 2002

Chicken brush border myosin I (CBB‐MI) is a single‐headed, nonfilamentous, myosin‐like mechanoenzyme which, as isolated, has 3 mol of calmodulin (CAM) ‘light chains’ bound per mole of 119 kDa heavy chain. We have isolated a partial cDNA clone for CBB‐MI that encodes the C‐terminal ~ 35 kDa of the heavy chain. The sequence of this clone is identical to that of an authentic, near‐full‐length CBB‐MI cDNA clone reported recently, except for an 87‐bp/29‐residue insertion occurring ~32 kDa from the C‐terminus. This insert, which is probably generated by an alternate splicing event, is expressed in brush border as part of a message of the size predicted for the CBB‐MI heavy chain, although the steady state level of this transcript is ~8‐fold lower than for transcripts lacking the insert. 125I‐CAM overlays of this cDNA clone (expressed as a trpE fusion protein in E. coli) indicate that it binds one more calmodulin than does a second cDNA clone that lacks the 29‐residue insert. A synthetic peptide corresponding to the insert sequence binds tightly to CAM‐Sepharose, demonstrates a shift and enhancement of fluorescence in the presence of CAM, and binds CAM in solution with a K
D of 190 nM (in 100 mM KCl). We conclude that a second, low‐abundance isoform of CBB‐MI contains an additional (and possibly fourth) CAM binding site as a result of a 29‐residue peptide that is inserted into the tail domain by an apparent alternate splicing event.
Partial reversion of the electrogenic reaction in the ubiquinol
Authors: Mulkidjanian Armen Ya. , Mamedov Mahir D. , Semenov Alexey Yu. , Shinkarev Vladimir P. , Verkhovsky Michael I. , Drachev Lel A.
Published: July 2002

The interaction of the photosynthetic reaction center (RC)‐generated ubiquinol with the ubiquinone‐reducing center C of ubiquinol:cytochrome c
2‐oxidoreductase (bc
1 ‐complex) has been studied electrometrically in Rhodobacter sphaeroides chromatophores. The addition of myxothiazol inhibited the ubiquinol‐oxidizing center Z, suppressing the phases of membrane potential generation by the bc
1,‐complex, but at the same time induced an electrogenic phase of opposite polarity, sensitive to antimycin A, the inhibitor of center C. The rise time of this reverse phase varied from 3 ms at pH 6.0 to 1 ms at pH 9.5. At pH > 9.5 the reverse phase was limited by the rate of ubiquinol formation in RC. The magnitude of the reverse phase was constant within the pH range 7.5–10.0. It is assumed that the reverse phase is due to the electrogenic deprotonation reaction which takes place after the binding of the RC‐generated ubiquinol to center C.
Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent
Authors: Lo Bello Mario , Petruzzelli Raffaele , De Stefano Ester , Tenedini Cristiana , Barra Donatella , Federici Giorgio
Published: July 2002

A fluorescent maleimide derivative, N‐(4‐anilino‐1‐naphthyl) maleimide (ANM), a specific probe for thiol groups, reacted with human placental glutathione transferase (GST, EC 2.5.1.18), causing a complete inactivation of the enzyme in a few minutes. The modified enzyme was denatured, alkylated and digested with (L‐1‐tosylamide‐2‐phenylethyl chloromethyl ketone)‐trypsin. The tryptic digest was analysed by HPLC and a fluorescent peptide was obtained. The sequence of this peptide allowed us, by a comparison with a well known primary structure, to assign the position 47 to the most reactive cysteine of GST enzyme.
Identification of Lys190 as the primary binding site for pyridoxal 5′‐phosphate in human serum albumin
Authors: Bohney James P. , Fonda Margaret L. , Feldhoff Richard C.
Published: July 2002

The covalent binding of pyridoxal 5′‐phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high‐affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H]PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu‐Asp‐Glu‐Leu‐Arg‐Asp‐Glu‐Gly‐Xaa‐Ala‐Ser‐Ser‐Ala‐Lys which corresponds to residues 182–195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys325) and acetylation by aspirin (Lys199).
Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity
Authors: Gak Eva , Taylor William G. , Chan Andrew M.-L. , Rubin Jeffrey S.
Published: July 2002

Hepatocyte growth factor is a plasminogen‐like molecule with diverse biological effects. Although it is synthesized as a single chain polypeptide, it was originally purified as a disulfide‐linked haterodimer which was generated by an internal proteolytic event. Subsequent work indicated that preparations consisting largely of the monomeric form also exhibited potent activity. By using a combination of protease inhibition and site‐directed mutagenesis, we established that conversion of the single chain polypeptide to the hoterodimer occurred during the bioassay and was required ror mitogenic and motogenic activity.
Glycogen contains phosphodiester groups that can be introduced by UDPglucose:glycogen glucose 1‐phosphotransferase
Authors: Lomako Joseph , Lomako Wieslawa M. , Whelan William J. , Marchase Richard B.
Published: July 2002

Rabbit‐muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C‐6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose:glycogen glucose 1 ‐phosphotransferase that positions glucose 1 ‐phosphate on C‐6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1‐phosphate.
Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides
Authors: Hatta Toshifumi , Kim Sang-Gug , Nakashima Hideki , Yamamoto Naoki , Sakamoto Kensaku , Yokoyama Shigeyuki , Takaku Hiroshi
Published: July 2002

We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase‐associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription.
Identification of an essential glutamate residue in the active site of endoglucanase III from Trichoderma reesei
Authors: Macarron Ricardo , van Beeumen Jozef , Henrissat Bernard , de la Mata Isabel , Claeyssens Marc
Published: July 2002

n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.
Homology of the NifS family of proteins to a new class of pyridoxal phosphate‐dependent enzymes
Authors: Ouzounis Christos , Sander Chris
Published: July 2002

Iterative profile sequence analysis reveals a remote homology of peroxisomal serine‐pyruvate aminotransferases from mammals to the small subunit of soluble hydrogenases from cyanobacteria, an isopenicillin N epimerase, the NifS gene products from bacteria and yeast, and the phosphoserine aminotransferase family. All members of this new class whose function is known are pyridoxal phosphate‐dependent enzymes, yet they have distinct catalytic activities. Upon alignment, a lysine around position 200 remains invariant and is predicted to be the pyridoxal phosphate‐binding residue. Based on the detected homology, it is predicted that NifS has also a pyridoxal phosphate‐dependent serine (or related) aminotransferase function associated with nitrogen economy and/or protection during nitrogen fixation.
The N‐oligosaccharyltransferase complex from yeast
Authors: Knauer Roland , Lehle Ludwig
Published: July 2002

N‐Oligosaccharyltransferase catalyzes the N‐glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved in all eukaryotes. An enzymatically active complex was isolated from microsomal membranes from Saccharomyces cerevisiae, which is composed of four proteins: Wbplp and Swplp (previously found to be encoded by two essential genes necessary for N‐glycosylation in vivo and in vitro) and two additional proteins with a molecular mass of 60/62 kDa and 34 kDa. The 60/62 component represents differentially glycosylated forms of a protein that has sequence homology to ribophorin I. Wbplp and Swplp reveal homology to mammalian OST 48 and ribophorin II, respectively. Ribophorin I and II and OST 48 were recently shown to be constituents of the mammalian transferase from dog pancreas. The data reveal a high conservation of the organization of this enzyme activity.
Fluorescent differential display: Arbitrarily primed RT‐PCR fingerprinting on an automated DNA sequencer
Authors: Ito Takashi , Kito Keiji , Adati Naoki , Mitsui Yuko , Hagiwara Hisashi , Sakaki Yoshiyuki
Published: July 2002

We established robust, reliable protocols for ‘Differential Display (DD),’ an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967–971] using RT‐PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such ‘Fluorescent Differential Display (FDD)’ techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.
HMG‐X, a Xenopus gene encoding an HMG1 homolog, is abundantly expressed in the developing nervous system
Authors: Kinoshita Makoto , Hatada Seigo , Asashima Makoto , Noda Makoto
Published: July 2002

We used a PCR‐based subtraction cloning procedure with Concanavalin A‐treated and untreated animal caps from stage 9 Xenopus embryos to search for genes the expression of which is induced during neurogenesis. One of these genes was found to encode a homolog of mammalian HMG 1 and 2, hence named HMG‐X. HMG‐X mRNA was maternally transmitted, up‐regulated in neuroectoderm‐derived tissues throughout early development, and eventually down‐regulated in all adult tissues examined except ovary. Our data suggest that we have identified a gene for a member of the HMG1/2 family that could have an important role in neurogenesis.
Interaction of protein phosphatase type 1 with a splicing factor
Authors: Hirano Katsuya , Erdödi Ferenc , Patton James G. , Hartshorne David J.
Published: July 2002

A gizzard cDNA library was screened by the two‐hybrid hybtem using as bait the S isoform of the catalytic subunit of protein phosphatase 1 (PP1δ) Among the proteins identified was a fragment of the polypyrimidine tract‐binding protein‐associated splicing factor (PSF) and for 242 residues was 97.1% identical to the human isoforms. Binding of PSF and PP1δ was confirmed by inhibition of phosphatase activity and by an overlay technique. The PP1δ binding site was contained in the N‐terminal 82 residues of the PSF fragment. PSF may therefore act as a PPl target molecule in the spliceosome.
Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization
Authors: Michetti Mauro , Salamino Franca , Tedesco Ilaria , Averna Monica , Minafra Roberto , Melloni Edon , Pontremoli Sandro
Published: July 2002

The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E‐64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca2+, membrane‐bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+‐induced translocation to the inner surface of plasma membranes. In the membrane‐bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.
Domain 1 of the urokinase receptor (uPAR) is required for uPAR‐mediated cell binding to vitronectin
Authors: Sidenius Nicolai , Blasi Francesco
Published: July 2002

In the present paper we have analyzed uPAR‐mediated cellular binding to vitronectin using the murine erythroid progenitor cell line 32D. We show that expression of uPAR in 32D cells promotes cellular binding to vitronectin, but fails to support cell spreading. The strength of binding is correlated to the expression level of uPAR and is strongly stimulated by the presence of uPAR ligands. Using a truncated variant of uPAR lacking domain 1 and by antibody inhibition experiments, we demonstrate that domain 1 plays a crucial role in uPAR‐mediated cellular binding. The failure of the mutant uPAR to promote cellular binding is paralleled by a strong reduction in the affinity for vitronectin in vitro.
Protein translocation pathways of the mitochondrion
Authors: Koehler Carla M
Published: July 2002

The biogenesis of mitochondria depends on the coordinated import of precursor proteins from the cytosol coupled with the export of mitochondrially coded proteins from the matrix to the inner membrane. The mitochondria contain an elaborate network of protein translocases in the outer and inner membrane along with a battery of chaperones and processing enzymes in the matrix and intermembrane space to mediate protein translocation. A mitochondrial protein, often with an amino‐terminal targeting sequence, is escorted through the cytosol by chaperones to the TOM complex (translocase of the outer membrane). After crossing the outer membrane, the import pathway diverges; however, one of two TIM complexes (translocase of inner membrane) is generally utilized. This review is focused on the later stages of protein import after the outer membrane has been crossed. An accompanying paper by Lithgow reviews the early stages of protein translocation.
The gene for hepatocyte nuclear factor (HNF)‐4α is activated by glucocorticoids and glucagon, and repressed by insulin in rat liver
Authors: Oyadomari Seiichi , Matsuno Fumihiko , Chowdhury Shoaib , Kimura Tatsuya , Iwase Katsuro , Araki Eiichi , Shichiri Motoaki , Mori Masataka , Takiguchi Masaki
Published: July 2002

The gene for a transcription factor hepatocyte nuclear factor‐4α (HNF‐4α) is responsible for maturity‐onset diabetes of the young, type 1. We examined hormonal regulation of the HNF‐4α gene in the liver. Stimulation of primary‐cultured rat hepatocytes with dexamethasone or glucagon led to induction of HNF‐4α mRNA, being antagonized by insulin. In the liver of streptozotocin‐induced diabetic rat, mRNA and protein levels for HNF‐4α were elevated, and were normalized by insulin treatment. Therefore, HNF‐4α in the liver is likely to be involved in the regulation of glucose metabolism in response to these hormones.
RasGAP is involved in signal transduction triggered by FGF1 in Xenopus oocytes expressing FGFR1
Authors: Cailliau Katia , Browaeys-Poly Edith , Vilain Jean Pierre
Published: July 2002

The role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2‐SH3‐SH2 domains of RasGAP suppressed Ras activity, extracellular signal‐regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2‐SH3‐SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet‐derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF‐BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes involves RasGAP as a co‐activator of Src to stimulate the Ras/mitogen‐activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction.
Anti‐peptide antibodies sensitive to the ‘active’ state of the β2‐adrenergic receptor
Authors: Mijares Alfredo , Lebesgue Diane , Argibay Jorge , Hoebeke Johan
Published: July 2002

Antibodies directed against a peptide corresponding to the second loop of the human β
2‐adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freund's adjuvant. The resulting antibodies were affinity‐purified and shown to be monospecific for the target receptor. They were able to stimulate the L‐type Ca2+ channels in whole‐cell patch‐clamp experiments on isolated adult guineapig cardiomyocytes. This effect was similar to that obtained by the specific β
2‐adrenergic agonist zinterol. The antibody effects could be blocked with the specific β
2‐adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the β
2‐adrenergic receptor.
The structure, function and distribution of the mouse TWIK‐1 K+ channel
Authors: Lesage Florian , Lauritzen Inger , Duprat Fabrice , Reyes Roberto , Fink Michel , Heurteaux Catherine , Lazdunski Michel
Published: July 2002

The two P domain K+ channel mTWIK‐1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK‐1 currents are K+‐selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK‐1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK‐1 subunits dimerize via a disulfide bridge. TWIK‐1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK‐1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.
Identification of sds22 as an inhibitory subunit of protein phosphatase‐1 in rat liver nuclei
Authors: Dinischiotu Anca , Beullens Monique , Stalmans Willy , Bollen Mathieu
Published: July 2002

sds22 was originally identified in yeast as a regulator of protein phosphatase‐1 that is essential for the completion of mitosis. We show here that a structurally related mammalian polypeptide (41.6 kDa) is part of a 260‐kDa species of protein phosphatase‐1. This holoenzyme, designated PP‐1Nsds22, could be immunoprecipitated with sds22 antibodies and was retained by microcystin‐Sepharose. PP‐1Nsds22 is a latent phosphatase, but its activity could be revealed by the proteolytic destruction of the noncatalytic subunit(s). PP‐1Nsds22 accounted for only 5–10% of the total activity of PP‐1 in rat liver nuclear extracts. A synthetic 22‐mer peptide, corresponding to a leucine‐rich repeat of sds22, specifically inhibited the catalytic subunit of PP‐1, showing that at least part of the latency stems from the interaction of the sds22 repeat(s) with PP‐1C.
Requirement of nitric oxide and calcium mobilization for the induction of apoptosis in adrenal vascular endothelial cells
Authors: López-Collazo Eduardo , Mateo Jesús , Miras-Portugal Marı́a T , Boscá Lisardo
Published: July 2002

Exposure of adrenal vascular endothelial cells (AVEC) to pharmacological nitric oxide (NO) donors, pro‐inflammatory cytokines or lipopolysaccharide was unable to induce apoptosis as occurred when macrophages were treated under identical experimental conditions. However, when the intracellular Ca2+ concentration increased, AVEC displayed apoptotic features upon exposure to NO. This apoptosis was confirmed by the release of oligonucleosomes to the cytosol and by the characteristic DNA laddering observed after electrophoresis in agarose gels. Ca2+‐mobilizing agents and interleukin‐1β (IL‐1β) also elicited an apoptotic response in these cells through a mechanism that required NO synthesis. The ability of NO and intracellular Ca2+ to promote apoptosis was dependent on the number of passages of the cells in culture, suggesting the loss of protective factors in the course of ex vivo cell culture. Because AVEC exhibit an important capacity to increase the intracellular Ca2+ concentration in response to a wide array of agonists, this condition might affect the integrity of the vascular system under pathological circumstances such as those prevailing in the course of septic shock.
2,7‐Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation
Authors: Possel Heiko , Noack Heiko , Augustin Wolfgang , Keilhoff Gerburg , Wolf Gerald
Published: July 2002

Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage, including apoptosis and necrosis. Their proposed actions comprise lipid peroxidation, DNA damage, destruction of the mitochondrial respiratory chain and protein modifications. Recent experiments underline the importance of peroxynitrite, the reaction product of the two potent reactive species nitric oxide and superoxide. Several fluorogenic compounds have been used in order to determine ROS formation in living cells. Besides dihydrorhodamine‐123 (DHR‐123), at present mostly applied to monitor peroxynitrite, 2,7‐dihydrodichlorofluorescein (DCF‐H) is used for detection of hydrogen peroxide and nitric oxide. We employed a cell free approach to evaluate the specificity and sensitivity of DCF‐H to various oxidizing compounds. Our studies imply that DCF‐H is much more sensitive to peroxynitrite oxidation than any other compound tested. In order to study peroxynitrite generation within individual cells, primary glial cultures loaded with DCF‐H were monitored with a laser scanning microscope. Microglia, stimulated to simultaneously produce the peroxynitrite precursors nitric oxide and superoxide, displayed the greatest increase in DCF fluorescence, whereas microglia producing either nitric oxide or superoxide alone showed a relatively small increase in DCF fluorescence. In conclusion, DCF‐H was demonstrated to be an excellent peroxynitrite marker with the potential to detect peroxynitrite formation in living cells.