High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T‐cell‐mediated immune responses in HIV‐positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non‐food crop, and tomato, a food crop with an edible fruit. Optimized p24‐Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.     

Plastid transformation of high‐biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV‐1) p24 antigen

Chloroplast transformation of the high‐biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV‐1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast‐codon‐optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 µg/g fresh weight or ~2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5‐yellow) with detectable p24 accumulation (up to approximately 450 µg/g fresh weight or ~4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5‐yellow leaves was reduced by ~40%. The pZF5‐yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3′ untransformed region in the plastid genome. Chloroplast‐expressed p24 was recognized by a conformation‐dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5‐yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion‐exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N‐terminal sequencing and mass spectrometry.     

Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes

Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.     

PTP1B inhibitors: synthesis and evaluation of difluoro-methylenephosphonate bioisosteres on a sulfonamide scaffold

We have synthesized and evaluated a series of triaryl sulfonamide-based PTP1B inhibitors in which a difluoro-methylenephosphonate group of a potent lead has been replaced by potential bioisosteric replacements. Several mono- or di-charged compounds (8a, 8b, and 15a) were shown exhibit inhibitory activity in the low micromolar range, demonstrating the feasibility of using this approach in identifying non-phosphonate pTyr mimetics in a small molecular scaffold. These results also provide a useful indication of the relative effectiveness of these pTyr mimetics.     

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.     

Chemical, structural and biological studies of cis-[Pt(3-Acpy)2Cl2]

Recent developments in the field of platinum anticancer drugs have revealed that compounds containing derivates of pyridine may exhibit highly cytotoxic activity against a variety of tumor cells, with AMD473 (cis-PtCl₂(NH₃)(2-methylpyridine)) as one of the most relevant examples. Following these advances, this paper describes the synthesis, characterization and X-ray structure of the square-planar compound cis-[Pt(3-Acpy)₂Cl₂] (1, Acpy stands for acetylpyridine), where the coordination of 3-acetylpyridine takes places through the pyridine nitrogen of the ligand. The structural arrangement of this compound is highly peculiar and it is the first example with two of these 3-acetylpyridine molecules in a cis disposition. In addition, the anticancer and antibacterial activities of this compound together with studies of DNA binding are also described in detail, with selective activity of compound 1 against A2780R cells. cis-[Pt(3-Acpy)₂Cl₂] apparently coordinates to the DNA double helix upon exchange of at least one of the Cl⁻ ions with the media and shows very interesting bacteriolytic and bacteriostatic activity against Escherichia coli and Streptomyces, respectively.     

Peptide-mediated DNA condensation for non-viral gene therapy

The construction of non-viral, virus-like vehicles for gene therapy involves the functionalization of multipartite constructs with nucleic acid-binding, cationic agents. Short basic peptides, alone or as fusion proteins, are appropriate DNA binding and condensing elements, whose incorporation into gene delivery vehicles results in the formation of protein–DNA complexes of appropriate size for cell internalization and intracellular trafficking. We review here the most used cationic peptides for artificial virus construction as well as the recently implemented strategies to control the architecture and biological activities of the resulting nanosized particles.     

Scale-sensitive landscape complementation determines habitat suitability for a territorial generalist

Evaluating habitat suitability is often complex, as habitat effects may be scale‐dependent, critical resources may be spatially segregated, and resource availability may also depend on intra‐ and inter‐specific interactions. Using analyses that spanned multiple years and spatial scales, we investigated habitat requirements of a territorial generalist, the common raven Corvus corax, in a relatively pristine woodland, Bia?owie?a Forest (E Poland). We compiled data from multiple raven surveys conducted between 1985 and 2001. Ravens were regularly distributed over the entire study area but declined in density over 50% within the 16 yr interval. In the same period game and forest management significantly reduced ungulate densities and likely diminished the habitat quality with regard to food supply, especially carrion. To better understand habitat requirements of ravens we studied breeding performance in relation to three different habitat types across multiple scales: open areas, coniferous‐dominated forest, and deciduous‐dominated forest. We found a prominent dissimilarity between raven nesting and foraging habitats highlighting the importance of resource complementation for ravens. On a fine scale, large old pines were exclusively selected as nesting trees and nesting areas were generally coniferous‐dominated. However, at increasingly broader scales, coniferous habitats were negatively associated with raven reproductive success as those habitats likely provide a lower food supply. Only where the coniferous nesting areas at smaller scales were complemented with high percentages of deciduous forests and open areas at broader scales did the breeding performance increase. In addition to habitat composition, intra‐specific interactions were important determinants of reproductive performance and very successful neighbors decreased reproductive performance of a focal pair. Most of previous studies have investigated resource complementation in terms of habitat edges or proximity of complementing resource patches. Our study demonstrates that the concept of landscape complementation also applies to gradients in landscape composition and emphasizes the importance of scales and intraspecific interactions in habitat analyses.