Protein interactions within the TcZFP zinc finger family members of Trypanosoma cruzi: implications for their functions

The small zinc finger proteins tbZFP1 and tbZFP2 have been implicated in the control of Trypanosoma brucei differentiation to the procyclic form. Here, we report that the complete ZFP family in Trypanosoma cruzi is composed by four members, ZFP1A and B, and ZFP2A and B. ZFP1B is a paralog specific gene restricted to T. cruzi, while the ZFP2A and B paralogs diverged prior to the trypanosomatid lineage separation. Moreover, we demonstrate that TcZFP1 and TcZFP2 members interact with each other and that this interaction is mediated by a WW domain in TcZFP2. Also, TcZFP2B strongly homodimerizes by a glycine rich region absent in TcZFP2A. We propose a model to discuss the relevance of these protein-protein interactions in terms of the functions of these proteins.     

Long-distance magnetic coupling in dinuclear copper(II) complexes with oligo-para-phenylenediamine bridging ligands

Two novel dinuclear copper(II) complexes of formulae [Cu₂(tren)₂(bpda)](ClO₄)₄ (2) and [Cu₂(tren)₂(tpda)](ClO₄)₄ (3) containing the tripodal tris(2-aminoethyl)amine (tren) terminal ligand and the 4,4′-biphenylenediamine (bpda) and 4,4″-p-terphenylenediamine (tpda) bridging ligands have been synthesized and structurally, spectroscopically, and magnetically characterized. Their experimentally available electronic spectroscopic and magnetic properties have been reasonably reproduced by DFT and TDDFT calculations. Single crystal X-ray diffraction analysis of 2 shows the presence of dicopper(II) cations where the bpda bridging ligand adopts a bismonodentate coordination mode toward two [Cu(tren)]²⁺ units with an overall non-planar, orthogonal anti configuration of the N-Cu-N threefold axis of the trigonal bipyramidal Cu^II ions and the biphenylene group. The electronic absorption spectra of 2 and 3 in acetonitrile reveal the presence of four moderately weak d-d transitions characteristic of a slightly distorted trigonal bipyramid stereochemistry of the Cu^II ions. TDDFT calculations on 2 identify these transitions as those taking place between the four lower-lying, doubly occupied a₂ (d_yz)², b₂ (d_xz)², b₁ (d_xy)², and a₁ (d_x²-y²)² orbitals and the upper, singly occupied a₁ (d_z²)¹ orbital of each trigonal bipyramidal Cu^II ion. Variable-temperature magnetic susceptibility measurements of 2 and 3 show the occurrence of moderate (J = −8.5 cm⁻¹) to weak intramolecular antiferromagnetic couplings (J = −2.0 cm^-1) [H = −JS₁·S₂ with S₁ = S₂ = S_Cu = ½] inspite of the relatively large copper-copper separation across the para-substituted biphenylene- (r = 12.3 Å) and terphenylenediamine (r = 16.4 Å) bridges, respectively. DFT calculations on 2 and 3 support the occurrence of a spin polarization mechanism for the propagation of the exchange interaction between the two unpaired electrons occupying the d_z² orbital of each trigonal bipyramidal Cu^II ion through the predominantly π-type orbital pathway of the oligo-p-phenylenediamine bridges, as reported earlier for the parent compound [Cu₂(tren)₂(ppda)](ClO₄)₄·2H₂O (1) with the 1,4-phenylenediamine (ppda) bridging ligand. Finally, a rather slow exponential decay of the antiferromagnetic coupling (-J) with the number of phenylene repeat units, -(C₆H₄)_n- (n = 1-3), has been found both experimentally and theoretically along this series of oligo-p-phenylenediamine-bridged dicopper(II) complexes. These results further support the ability of linear π-conjugated oligo-p-phenylene spacers to transmit the exchange interaction between the unpaired electrons of the two Cu^II centers with intermetallic distances in the range of 7.5-16.4 Å.     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Event-driven time-optimal control for a class of discontinuous bioreactors

Discontinuous bioreactors may be further optimized for processing inhibitory substrates using a convenient fed-batch mode. To do so the filling rate must be controlled in such a way as to push the reaction rate to its maximum value, by increasing the substrate concentration just up to the point where inhibition begins. However, an exact optimal controller requires measuring several variables (e.g., substrate concentrations in the feed and in the tank) and also good model knowledge (e.g., yield and kinetic parameters), requirements rarely satisfied in real applications. An environmentally important case, that exemplifies all these handicaps, is toxicant wastewater treatment. There the lack of online practical pollutant sensors may allow unforeseen high shock loads to be fed to the bioreactor, causing biomass inhibition that slows down the treatment process and, in extreme cases, even renders the biological process useless. In this work an event-driven time-optimal control (ED-TOC) is proposed to circumvent these limitations. We show how to detect a "there is inhibition" event by using some computable function of the available measurements. This event drives the ED-TOC to stop the filling. Later, by detecting the symmetric event, "there is no inhibition," the ED-TOC may restart the filling. A fill-react cycling then maintains the process safely hovering near its maximum reaction rate, allowing a robust and practically time-optimal operation of the bioreactor. An experimental study case of a wastewater treatment process application is presented. There the dissolved oxygen concentration was used to detect the events needed to drive the controller.     

Mutation of Membrane Type-1 Metalloproteinase, MT1-MMP, Causes the Multicentric Osteolysis and Arthritis Disease Winchester Syndrome

The "vanishing bone" syndromes represent a group of rare skeletal disorders characterized by osteolysis and joint destruction, which can mimic severe rheumatoid arthritis. Winchester syndrome was one of the first recognized autosomal-recessive, multicentric forms of the disorder. It was originally described nearly 50 years ago in two sisters with a severe crippling osteolysis. Using cultured fibroblasts from the proband, we have now identified homozygous mutations in membrane type-1 metalloproteinase (MT1-MMP or MMP14). We demonstrate that the resulting hydrophobic-region signal-peptide substitution (p.Thr17Arg) decreases MT1-MMP membrane localization with consequent impairment of pro-MMP2 activation, and we propose a structure-based mechanism for this effect.     

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.