Circular dichroism of pig and bovine lactadherins and their affinity for the pig zona pellucida

We have purified and characterized pig and bovine milk lactadherins. Studies by circular dichroism spectroscopy indicate that the two proteins present a similar folding pattern. Results have been discussed in terms of their affinity for pig zona pellucida in order to use these proteins as analogs of pig sperm lactadherin in gamete studies.     

Thalamic development induced by Shh in the chick embryo

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.     

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

Background  

Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein.     

Sequence determinants of protein aggregation: tools to increase protein solubility

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, very often the target protein accumulates into insoluble aggregates in a misfolded and biologically inactive form. Bacterial inclusion bodies are major bottlenecks in protein production and are hampering the development of top priority research areas such structural genomics. Inclusion body formation was formerly considered to occur via non-specific association of hydrophobic surfaces in folding intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria resembles to the well-studied process of amyloid fibril formation. Both processes appear to rely on the formation of specific, sequence-dependent, intermolecular interactions driving the formation of structured protein aggregates. This similarity in the mechanisms of aggregation will probably allow applying anti-aggregational strategies already tested in the amyloid context to the less explored area of protein aggregation inside bacteria. Specifically, new sequence-based approaches appear as promising tools to tune protein aggregation in biotechnological processes.     

Chromatographic evaluation of the toxicity in fish of pesticides

Ecotoxicity assessment is essential before placing new chemical substances on the market. An investigation of the use of the chromatographic retention (log k) in biopartitioning micellar chromatography (BMC) as an in vitro approach to evaluate the toxicity in fish of pesticides (acute toxicity levels as pLC(50)) is proposed. A heterogeneous data set of 85 pesticides from six chemical families with available experimental fish toxicity data (ECOTOX database from U.S. Environmental Protection Agency (EPA)) was used. For pesticides exhibiting non-polar narcosis mechanism in fish (non-specific toxicity), more reliable models and precise pLC(50) estimations are obtained from log k (quantitative retention-activity relationships, QRAR) than from log P (quantitative structure-activity relationships, QSAR) or ECOSAR (ECOSAR program from U.S. EPA).     

Pentamidine is an antiparasitic and apoptotic drug that selectively modifies ubiquitin

We have determined the cytotoxic properties of pentamidine isethionate (2) towards the promastigotes of the protozoan parasite Leishmania infantum. The leishmanicidal activity of 2 was 60 times higher after 72 h of incubation than that of cisplatin (4). The pentamidine salt 2 induced a higher amount of programmed cell death (PCD) than cisplatin, which is associated with inhibition of DNA synthesis and cell-cycle arrest in the G2/M phase. Circular dichroism (CD) data indicate that binding of 2 to calf-thymus DNA (CT-DNA) induces conformational changes in the DNA double helix, consistent with a B-->A transition. Moreover, the interaction of 2 with ubiquitin led to a 6% increase in the beta-sheet content of the protein as observed by CD spectroscopy. Fluorescence-spectroscopy studies agreed with the CD data, showing that the pentamidine portion of 2 induces a significant decrease in the fluorescence of the Ub residues Phe4 and Phe45 located on the beta-cluster of the molecule, but not of Tyr59 on the alpha-cluster. These data indicate that pentamidine specifically modifies the beta-cluster, i.e., the 'basic face' of ubiquitin. Our results suggest that the biochemical mechanism of action of pentamidine may be a consequence of its dual binding to DNA and proteins.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Expression of canonical SOS genes is not under LexA repression in Bdellovibrio bacteriovorus

The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the delta-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other delta-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the delta-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.     

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.     

Identification and characterization of an interspersed repetitive DNA fragment in Plasmodium vivax with potential use for specific parasite detection

We cloned and characterized a Plasmodium vivax repeat element of 7872bp named PvRE7.8. Several internal tandem repeats were found along the sequence. The repetitive nature of the PvRE7.8 element was confirmed by hybridization of a P. vivax YAC library. Based on the data bank analysis and the presence of two contiguous putative genes that may encode proteins related to DNA metabolism, PvRE7.8 could be considered an inactivated transposon-LINE element. By using Pv79 as probe or primers derived from Pv79-flanking sequences, P. vivax DNA Could be detected from whole blood and mosquito samples. We consider that the repeat element described here has potential for P. vivax malaria diagnosis and for epidemiological analysis of P. vivax transmission areas.