Synthesis and molecular modeling of a lisinopril-tryptophan analogue inhibitor of angiotensin I-converting enzyme

With a view to developing a more C-domain-selective angiotensin I-converting enzyme (ACE)-inhibitor, a novel analogue of lisinopril has been synthesized which incorporates a bulky P(2)(') tryptophan functionality. This inhibitor demonstrated a significantly increased specificity for the C-domain as compared with lisinopril. Molecular docking revealed hydrophobic and hydrogen-bonding interactions with residues of the C-domain S(2)(') subsite.     

Tagging of Escherichia coli proteins with new cassettes allowing in vivo systematic fluorescent and luminescent detection,and purification from physiological expression levels

We designed cassettes allowing the systematic fusion of fluorescent or luminescent proteins preceded by the calmodulin binding peptide tag to the C–terminus of Escherichia coli proteins. The chromosomal insertion, and thus physiological expression level of these fusions, permits the study of protein localization by fluorescent microscopy and protein quantification, in vivo and dynamically in diverse conditions. Furthermore, the calmodulin binding peptide tag allows standard detection, affinity purification, and co–purification experiments. These cassettes are therefore very valuable for the versatility of experiments they make available for a given strain, from biochemistry to dynamic and in vivo studies.     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.     

Composition, and antimicrobial and remarkable antiprotozoal activities of the essential oil of rhizomes of Aframomum sceptrum K. Schum. (Zingiberaceae)

The essential oil from the rhizomes of Aframomum sceptrum (Zingiberaceae) was analyzed by GC/MS, and its major constituents were found to be β‐pinene (12.7%), caryophyllene oxide (10.0%), and cyperene (6.0%). The oil was also evaluated for antimicrobial activities, in comparison with β‐pinene, caryophyllene oxide, and the leaf essential oil of Melaleuca alternifolia (Myrtaceae). The A. sceptrum essential oil exhibited bacteriostatic activity against the Gram‐positive bacteria Bacillus subtilis, Staphylococcus epidermidis, and S. aureus, but not against Gram‐negative bacteria. Moreover, it showed mild fungicidal activity against Candida albicans and Aspergillus fumigates, and remarkable antiprotozoal activity against Trypanosoma brucei brucei (MLC of 1.51 μl/ml) and Trichomonas vaginalis (IC₅₀ of 0.12±0.02 and MLC of 1.72 μl/ml).