High-Yield Expression in Escherichia coli and Purification of Mouse Ubiquitin-Activating Enzyme E1

Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.     

Multivariate analysis to determine the genetic distance among backcross papaya (Carica papaya) progenies

Morpho-agronomic and molecular (RAPD and ISSR markers) data were used to evaluate genetic distances between papaya backcross progenies in order to help identify agronomically superior genotypes. Thirty-two papaya progenies were evaluated based on 15 morpho-agronomic characteristics, 20 ISSR and 19 RAPD primers. Manhattan, Jaccard and Gower distances were used to estimate differences based on continuous and binary data and combined analyses, respectively. Except for production, there were significant differences in the continuous variables among the genotypes. The molecular analysis revealed 193 dominant markers (ISSR and RAPD), being 53 polymorphic loci. Among the various clusters that were generated, the one based on a combined analysis of morpho-agronomic and molecular data gave the highest cophenetic correlation (0.72) compared to individual analysis, consistently allocating the progenies into six groups. We found that the Gower algorithm was more coherent in the discrimination of the genotypes, demonstrating that a combination of molecular and agronomic data is valuable for studies of genetic dissimilarity in papaya.     

Purification and Biological Activities of Abelmoschus esculentus Seed Lectin

The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0?kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1?mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44?%, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10?mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69?%, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0?kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.     

Synthesis of a Natural Chalcone and Its Prenyl Analogs – Evaluation of Tumor Cell Growth‐Inhibitory Activities,and Effects on Cell Cycle and Apoptosis

Six prenyl (=3‐methylbut‐2‐en‐1‐yl) chalcones (=1,3‐diphenylprop‐2‐en‐1‐ones), 2 – 7 , and one natural non‐prenylated chalcone, 1 , have been synthesized and evaluated for their in vitro growth‐inhibitory activity against three human tumor cell lines. A pronounced dose‐dependent growth‐inhibitory effect was observed for all prenylated derivatives, except for 7 . The chalcone possessing one prenyloxy group at C(2′), i.e., 2 , was the most active derivative against the three human tumor cell lines (5.9<GI₅₀<7.7 μM ). The majority of compounds caused an increase in percentage of apoptotic cells and/or they interfered with cell cycle distribution in the MCF‐7 cell line.     

Isoxazolines and isoxazoles as factor Xa inhibitors

3,4,5-Trisubstituted isoxazolines (2) and isoxazoles (3) were prepared and evaluated for their in vitro and in vivo antithrombotic efficacy. They were compared to 3,5,5-trisubstituted isoxazolines (1) for Factor Xa selectivity and potency. They were also compared in an arterio-venous (A-V) shunt model of thrombosis.     

Synthesis of 2-deoxy-2-fluoro and 1,2-ene derivatives of the naturally occurring glycosidase inhibitor, salacinol, and their inhibitory activities against recombinant human maltase glucoamylase

2-Deoxy-2-fluorosalacinol and a 1,2-ene derivative of the naturally occurring glycosidase inhibitor salacinol were synthesized for structure activity studies with human maltase glucoamylase (MGA). 2-Deoxy-2-fluorosalacinol was synthesized through the coupling reaction of 2-deoxy-2-fluoro-3,5-di-O-p-methoxybenzyl-1,4-anhydro-4-thio-D-arabinitol with 2,4-O-benzylidene-l-erythritol-1,3-cyclic sulfate in hexafluoroisopropanol (HFIP) containing 0.3 equiv of K(2)CO(3). Excess of K(2)CO(3) resulted in the elimination of HF from the coupled product, and the formation of an alkene derivative of salacinol. Nucleophilic attack of the 1,4-anhydro-4-thio-D-arabinitol moiety on the cyclic sulfate did not proceed in the absence of K(2)CO(3). No reaction was observed in acetonitrile containing K(2)CO(3). The target compounds were obtained by deprotection with TFA. The 2-deoxy-1-ene derivative of salacinol and 2-deoxy-2-fluorosalacinol inhibited recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose, with an IC(50) value of 150 microM and a K(i) value of 6+/-1 microM, respectively.     

Tissue-specific defects are caused by loss of the Drosophila MAN1 LEM domain protein

The nuclear lamina represents a protein network required for nuclear structure and function. One family of lamina proteins is defined by an approximately 40-aa LAP2, Emerin, and MAN1 (LEM) domain (LEM-D) that binds the nonspecific DNA-binding protein, barrier-to-autointegration factor (BAF). Through interactions with BAF, LEM-D proteins serve as a bridge between chromosomes and the nuclear envelope. Mutations in genes encoding LEM-D proteins cause human laminopathies that are associated with tissue-restricted pathologies. Drosophila has five genes that encode proteins with LEM homology. Using yeast two-hybrid analyses, we demonstrate that four encode proteins that bind Drosophila (d)BAF. In addition to dBAF, dMAN1 associates with lamins, the LEM-D protein Bocksbeutel, and the receptor-regulated Smads, demonstrating parallel protein interactions with vertebrate homologs. P-element mobilization was used to generate null dMAN1 alleles. These mutants showed decreased viability, with surviving adults displaying male sterility, decreased female fertility, wing patterning and positioning defects, flightlessness, and locomotion difficulties that became more severe with age. Increased phospho-Smad staining in dMAN1 mutant wing discs is consistent with a role in transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signaling. The tissue-specific, age-enhanced dMAN1 mutant phenotypes are reminiscent of human laminopathies, suggesting that studies in Drosophila will provide insights into lamina dysfunction associated with disease.