Characterization of the promoter activity of a poxvirus conserved element

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.     

Central distribution of nociceptive intradental afferent nerve fibers in the rat

The central distribution of intradental afferent nerve fibers was investigated by combining electron microscopic observations with a selective method for inducing degeneration of the A delta- and C-type afferent fibers. Degenerating terminals were found on the proprioceptive mesencephalic trigeminal neurons and on dendrites in the neuropil of the trigeminal motor nucleus after application of capsaicin to the rat's lower incisor tooth pulp. The results give anatomical evidence of new sites of central projection of intradental A delta- and C-type fibers whereby the nociceptive information from the tooth pulp can affect jaw muscle activity.     

Solubility improvement of an anthrax toxin peptide inhibitor by rational amino acid randomization

We previously described a potent anthrax toxin inhibitor, based on a phage-library-selected peptide sequence, synthesized as a tetra-branched molecule on a lysine core and further modified for improvement of activity [Pini et al., Biochem. J., 2006, 395, 157]. This branched peptide had very low solubility because of several hydrophobic residues in the peptide sequence. This complicated molecule purification and manufacturing. Here we report a rational modification of the peptide sequence, obtained by construction and selection of several mini libraries of branched peptides, containing sequences randomized in non crucial positions of the original peptide. Mini libraries were screened for solubility and inhibitory activity. This procedure enabled us to obtain a new peptide with a better solubility and identical inhibitory activity.     

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.     

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50–50 nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC₅₀ values of 1.22 ± 0.01, 3.75 ± 0.02 and 3.60 ± 0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC₅₀ 10.3 ± 0.03) when compared with known flavonols 5–7, and interestingly increased when tested against ABTS scavenging activity.     

ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development

Arabidopsis abcb1 abcb19 double mutants defective in the auxin transporters ABCB1/PGP1 and ABCB19/PGP19 are altered in stamen elongation, anther dehiscence and pollen maturation. To assess the contribution of these transporters to stamen development we performed phenotypic, histological analyses, and in situ hybridizations on abcb1 and abcb19 single mutant fl owers. We found that pollen maturation and anther dehiscence are precocious in the abcb1 but not in the abcb19 mutant. Accordingly, endothecium ligni fication is altered only in abcb1 anthers. Both abcb1 and abcb1 abcb19 stamens also show altered early development, with asynchronous anther locules and a multilayer tapetum. DAPI staining showed that the timing of meiosis is asynchronous in abcb1 abcb19 anther locules, while only a small percentage of pollen grains are nonviable according to Alexander's staining. In agreement, TAM(TARDY ASYNCHRONOUS MEIOSIS), as well as BAM2(BARELY ANY MERISTEM)—involved in tapetal cell development—are overexpressed in abcb1 abcb19 young fl ower buds. Corre spondingly, ABCB1 and ABCB19 mRNA localization supports the observed phenotypes of abcb1 and abcb1 abcb19 mutant anthers. In conclusion, we provide evidence that auxin transport plays a signi ficant role both in early and late stamen development: ABCB1 plays a major role during anther development, while ABCB19 has a synergistic role.     

Alkaline cooking and tortilla quality in maize grains from the humid,tropical lands of Mexico

Maize (Zea mays L.) tortilla is the major staple food for the Mexican population. Nine tropical maize genotypes were evaluated. All samples had white grains, a common characteristic in tropical maize, and therefore they were appropriate for nixtamalized flour industry. Grain, flour, masa and tortilla characteristics of each maize genotype were evaluated. Length, width, thickness, weight of 1000 grains and hardness of grain were determined. Moisture content, proteins, fat, ash, mean particle size, water absorption index, enthalpy, and flour temperature were also evaluated. Adhesiveness and cohesiveness were evaluated in masa. Moisture content, protein, capacity to puff up, roll making, tension and cutting strength were determined in tortillas. There were significant differences (p≤0.05) in most of the evaluated characteristics. Grain length values varied between 9.26 and 11.02 mm for populations 23 and 22, respectively. Grain hardness oscillated between 11.17 (population 32) and 14.75 (landrace Mejen). According to the weight of 1000 grains most genotypes had small grains. The minimum and maximum moisture values of flour and tortillas were 8.33-9.99% and 46.20-50.36%, respectively. The texture of tortillas elaborated from population 32 and landrace Mejen had the lowest tension and cutting strength, resulting the best genotypes for making tortilla.