Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3% foram co-negativas, mas 27,6% foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.

Infestation of froghopper nymphs changes the amounts of total phenolics in sugarcane

The increased rate of sugarcane harvest without previous burn has provided a very favorable environment to the froghopper Mahanarva fimbriolota (Stal, 1854), with high Moisture and low temperature variation. Few works have Studied the response of sugarcane to this pest, so little is known about resistant cultivars. Plant phenolics are widely studied compounds because of their known antifierbivore effect. This research aims to determine if the attack of M.fimbriolata nymphs stimulates the acccumulation of total phenolics in sugarcane. The experiment was carried Out in greenhouse and arranged in completely randomized design, in a 3 x 2 x 4 factorial with three replications. Second instar nymphs of M. fimbriolota were infested at the following rates: control, 2-4 and 4-8 nymphs per pot (first-second infestations, respectively). Pots were covered with nylon net and monitored daily to isolate the effect of leaf Sucking adults. Leaf and root samples were collected and kept frozen in liquid nitrogen until analyses. Infested plants showed higher levels of phenolics in both root and leaf tissues. In roots, the cultivar SP80-1816 accumulated more phenolic compounds in response to the infestation of M. fimbriolata. on the other hand, higher levels were found in leaves and roots of control plants of SP86-42, which might be an indication of a non-preference mechanism. The increase of total phenolics in sugarcane infested with root-sucking froghopper nymphs does not seem to be useful to detect the resistance to this pest.

Ações de profissionais relativas ao banco de leite humano : uma perspectiva de mudança

This is an exploratory and descriptive study that aimed to investigate the actions of professionals in the context of breastfeeding, on the assumption that the actions taken by employees working together to postpartum and newborn are not competing to effect the distribution of pasteurized human milk so that it meets the needs of infants who depend on it. Thus, the study aimed to analyze the actions of medical and nursing staff of the distribution of pasteurized human milk to the newly born. The investigation was developed by action research in a federal hospital, located in the capital the state of Rio Grande do Norte, Brazil, reference assistance to women during pregnancy, childbirth and postpartum high risk in 2010. Study participants were fifty-five professionals chosen from the following inclusion criteria: to act in the NICU or rooming, being a pediatrician and / or neonatologists, nurses and technical nursing. According to the methodology of action research a questionnaire was applied, techniques in focus groups and courses were developed, and, finally, action evaluation. The project was submitted to the Ethics Committee at the Federal University of Rio Grande do Norte and approved with no protocol 448/2009. The problems identified in the responses issued by the social research were grouped into categories according to the similarity between them. The answer to the question of the survey - How is the need for pasteurized human milk for the newborns in neonatal intensive care unit and rooming identified? - Brought subsidies for action planning and implementation of strategies for change in the practice of professionals working in rooming and ICU. Thus, the study has relevance in social care and, when at the local level, will compete for the distribution pasteurized human milk to take effect as best as possible, as recommended by the Ministry of Health. It is also conceived that, in a macro view of society, it could contribute to minimizing the health problem that involves the child population

Temperature during soybean seed storage and the amount of electrolytes of soaked seeds solution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adição de areia para dispersão de solos na análise granulométrica

A acurácia da análise granulométrica depende da obtenção de suspensões de solo completamente dispersas e estáveis para possibilitar a separação das suas frações granulométricas. O objetivo do presente trabalho foi avaliar a eficácia da adição de quantidades e tamanhos de grãos de areia na fase de dispersão da análise granulométrica de solos, visando à maior acurácia na obtenção dos resultados da análise granulométrica. Os solos utilizados foram: Latossolo Vermelho eutroférrico (LVef), LatossoloVermelho acriférrico (LVwf), Latossolo Vermelho eutrófico (LVe), Argissolo Vermelho-Amarelo eutrófico (PVAe) e Nitossolo Vermelho eutroférrico (NVef). A dispersão das amostras dos solos foi realizada por meio da adição de hidróxido de sódio e agitação rotativa (60 rpm) por 16 h. O delineamento experimental adotado foi o inteiramente casualizado, com esquema fatorial 6 x 2, com três repetições. Os tratamentos foram constituídos por seis quantidades (0, 5, 10, 15, 20 e 25 g) e dois diâmetros (2,0-1,0 e 1,0-0,5 mm) de areia, adicionados na fase de dispersão da análise granulométrica dos solos. de acordo com as equações ajustadas, a adição de areia com diâmetro entre 1,0 e 0,5 mm nas quantidades de 21,4 g para LVef, 19,6 g para LVwf e 25,8 g para NVef proporciona, respectivamente para esses solos, aumentos de 50, 38 e 14,5 % nos teores de argila. No LVe e no PVAe não se justifica a adição de areia na análise granulométrica, pois esses solos não apresentaram problemas de dispersão. Os resultados demonstram que a adição de 25 g de areia, com diâmetro entre 1,0 e 0,5 mm, na fase de dispersão da análise granulométrica de solos argilosos com altos teores de óxidos de Fe e com dificuldades de dispersão, é eficiente para promover efetiva dispersão das partículas primárias do solo.

Características de latossolos roxos desenvolvidos de rochas alcalinas e básicas de Jaboticabal, SP

Comparou-se as características químicas, físicas e mineralógicas de Latossolos Roxos desenvolvidos de rocha alcalina (LRe) e de rocha eruptiva básica (LRd), da região de Jaboticabal, SP. Os resultados indicaram que o LRe apresenta-se num estádio de desenvolvimento, comparativamente, menos evoluído que o LRd, enquanto esse se apresenta com tendência para a maturidade. Os menores teores de argila e ferro total e maiores teores de saturação por bases, Ki e CTC da fração argila apresentados pelo LRe comprovam esta tendência. Conclui-se que a ocorrência de Latossolo Roxo eutrófico no campus da UNESP-Jaboticabal, pode estar associada a participação de rochas alcalinas na sua pedogênese.

Nutrição de micronutrientes em mudas de goiabeira em resposta ao uso de soluções nutritivas

The objective of this study was to evaluate the effect of different nutrient solutions oil the development and concentration of micronutrients ill guava cuttings. Two cultivars - Paluma and Seculo XXI - and four nutritional solutions were evaluated, using a factorial scheme (2x4) with three repetitions. At 90 days of hydroponic culture, dry mass and micronutrient nutrition in the cuttings were evaluated. The Paluma guava cuttings displayed greater development and nutritional requirements for boron, iron and manganese, as compared to cultivar Seculo M. The studied nutrient solutions did not affect accumulation, absorption or use efficiency of micronutrients in guava cuttings, except for copper and zinc, regardless of cultivar.

Liberação de micronutrientes de uma escória aplicada em um Argissolo Vermelho-Amarelo cultivado com mudas de goiabeira (Psidium guajava L.)

Os micronutrientes são importantes na nutrição das plantas, especialmente em solos tropicais com baixa concentração devido ao intemperismo. Como fonte alternativa de micronutrientes, tem-se a escória, resíduo da indústria de produção de ferro-gusa e aço. Assim, objetivou-se avaliar a escória como fonte de micronutrientes para mudas de goiabeira. O delineamento experimental foi em blocos ao acaso, com cinco repetições. As doses de escória foram aplicadas objetivando elevar em meia, uma vez, uma vez e meia, duas vezes e duas vezes e meia a saturação por bases do solo igual a 70%, correspondendo a 1,68; 3,36; 5,04; 6,72 e 8,40 g por vaso, além da testemunha sem aplicação. Após 90 dias de incubação da escória com o Argissolo Vermelho-Amarelo, cultivaram-se mudas de goiabeira (cv. Paluma) por 110 dias em vasos com 2,8 dm-3 de substrato, em viveiro telado, em Taquaritinga-SP, no período de outubro de 2000 a abril de 2001. A escória promoveu um efeito favorável na reação do solo e na disponibilidades de Zn, Cu, Mn e B do solo. Houve efeitos quadráticos nas concentrações de Zn, Cu e Mn do solo, que, por sua vez, estiveram associadas às doses de escória superiores a 5,8; 6,3 e 7,5 g por vaso, respectivamente, enquanto, para o B, esse efeito foi linear. A saturação por bases do solo, entre 51 e 55%, resultou em maior disponibilidade dos micronutrientes Zn, Cu e Mn no solo, ao passo que, para o B, esse valor foi de 65%. da mesma forma que ocorreu no solo, a aplicação da escória apresentou efeitos quadráticos nos teores de Zn, Cu e Mn da parte aérea e das raízes das mudas de goiabeira, enquanto, para o B, esse efeito foi linear. Concluiu-se, portanto, que a escória se comportou como material corretivo da acidez e como fonte de micronutrientes.

African-Derived mitochondria in South American native cattle breeds (Bos taurus): Evidence of a new taurine mitochondrial lineage

This article reports the nucleotide diversity within the control region of 42 mitochondrial chromosomes belonging to five South American native cattle breeds (Bos taurus). Analysis of these data in conjunction with B. taurus and B. indicus sequences from Africa, Europe, the Near East, India, and Japan allowed the recognition of eight new mitochondrial haplotypes and their relative positions in a phylogenetic network. The structure of genetic variation among different hypothetical groupings was tested through the molecular variance decomposition, which was best explained by haplotype group components. Haplotypes surveyed were classified as European-related and African-related. Unexpectedly, two haplotypes within the African cluster were more divergent from the African consensus than the latter from the European consensus. A neighbor-joining tree shows the position of two haplotypes compared to European/African mitochondrial lineage splitting. This different and putatively ancestral mitochondrial lineage (AA) is supported by the calibration of sequence divergence based on the Bos-Bison separation. The European/African mitochondria divergence might be subsequent (67,100 years before present) to that between AA and Africans (84,700 years before present), also preceding domestication times. These genetic data could reflect the haplotype distribution of Iberian cattle five centuries ago.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

Restriction fragment length polymorphism (RFLP) in Exon 2 of the BoLA-DRB3 gene in South American cattle

The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY, and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris

Although numerous studies have reported the production of skeletal muscle alpha -tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg2+-ATPase in the absence of troponin. on the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha -tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha -tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg2+-ATPase. (C) 2001 Academic Press.

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition. (C) 2003 Elsevier B.V. (USA). All rights reserved.

A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.