DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues / Aplicação de abordagens de DDRT-PCR para aprimoramento de resultados no isolamento de DETs a partir de tecido cerebral de peixes

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Display Diferencial (DD) é uma técnica amplamente utilizada em estudos de expressão diferencial. A maioria desses estudos envolvendo espécies de peixes está restrita a espécies da América do Norte e Europa ou a espécies comerciais, como os salmoniformes. Estudos relacionados a peixes da América do Sul são ainda pouco explorados. Desse modo, o presente trabalho teve como objetivo descrever modificações na técnica de DD, a fim de melhorar os resultados relacionados ao isolamento de DETs (Transcritos Diferencialmente Expressos), utilizando Leporinus macrocephalus, peixe explorado comercialmente na América do Sul, como espécie para tal delineamento. Diferentes abordagens de DDRT-PCR foram desenvolvidas a partir de amostras de tecido cerebral e os produtos das reações foram analisados em gel de poliacrilamida 6% corados com 0,17% de nitrato de Prata (AgNO3). A utilização de reações de PCR sob condições de elevada estringência e oligonucleotídeos mais longos, com base em sequências cerne de VNTR (Número Variável de Repetições em Tandem), mostrou melhores resultados quando comparada a condições de baixa estringência e ao uso de oligonucleotídeos decâmeros. A estratégia empregada permitiu o isolamento de transcritos diferencialmente expressos em machos e fêmeas adultos de L. macrocephalus. Este estudo evidencia que modificações no método de DDRT-PCR garantem o melhor isolamento de DETs a partir de diferentes tecidos de peixes e asseguram a obtenção de dados mais sólidos relacionados a essa abordagem.

Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures: a review.

Structural modification of steroids through whole-cell biocatalysis is an invaluable procedure for the production of active pharmaceutical ingredients (APIs) and key intermediates. Modifications could be carried out with regio- and stereospecificity at positions hardly available for chemical agents. Much attention has been focused recently on the biotransformation of 17α-ethynyl substituted steroidal drugs using fungi, bacteria and plant cell cultures in order to obtained novel biologically active compounds with diverse structure features. Present article includes studies on biotransformation on 17α-ethynyl substituted steroidal drugs using microorganisms and plant cell cultures. Various experimental and structural elucidation methods used in biotransformational processes are also highlighted.

Stereoselective sorption by agricultural soils and liquid-liquid partitioning of trenbolone (17alpha and 17beta) and trendione.

Trenbolone acetate (TBA) is a synthetic anabolic hormone used for growth promotion in beef cattle, which excrete primarily 17alpha-trenbolone along with small amounts of 17beta-trenbolone and trendione. To aid in predicting transport of manure-borne TBA metabolites, multiconcentration sorption isotherms for 17alpha- and 17beta-trenbolone and trendione were generated with five autoclaved-sterilized soils that represented a range in soil properties. Hormone concentrations were measured independently in solution and soil phases, and quantified using liquid chromatography with electrospray mass spectrometry. In addition, partition coefficients between apolar hexane and water (K(hw)) and bipolar octanol and water (K(ow)) were measured for the three androgens to better ascertain the mechanisms that may be responsible for the sorption differences observed between isomers. In all five soils, trendione sorbed the most, and 17alpha- and 17beta-trenbolone isomers exhibited different sorption magnitudes. 17beta- trenbolone consistently sorbed a factor of 2 more than 17alpha-trenbolone. For all three androgens, sorption is proportional to the soil organic carbon (OC) content with average log OC-normalized distribution coefficients (log K(oc), L/kg OC) of 2.77 +/- 0.12 for 17alpha-trenbolone, 3.08 +/- 0.1 for 17beta-trenbolone and 3.38 +/- 0.19 for trendione, which suggests the dominance of hydrophobic partitioning. However, differences in K(hw) values between 17alpha- and 17beta-trenbolone were small indicating differences are not simply due to differences in aqueous activity. In contrast, similarly different K(ow) and K(oc) values for the two isomers indicate the likely contribution of H-bonding to stereoselective sorption.

Further constituents from the bark of Holarrhena pubescens.

Three compounds, pubadysone [11 alpha-hydroxy-18,20-oxido-3-oxo-pregna-1,4,17(20)-triene] (1), puboestrene [3-acetoxy-17-oxo-1,3,5(10)-estratriene] (2) and pubamide [3,18-dioxo-11 alpha-hydroxycona-1,4-diene] (3), have been isolated from the bark of Holarrhena pubescens. Their structures have been established through spectroscopic studies.