Exogenous cadaverine induces oxidative burst and reduces cadaverine conjugate content in the common ice plant

The effect of free cadaverine (Cad) on its conjugates formation was analyzed in roots of the common ice plants (Mesembryanthemum crystallinum L.). It was found for the first time that Cad could induce oxidative burst in the roots of adult plants, as was evident from the sharp decrease in the content of Cad soluble or insoluble conjugates. This unusual effect was associated with the increased oxidative degradation of exogenous Cad (1mM, 1.5h) and intense H(2)O(2) production in the root cells of adult plants. Root treatment of both juvenile and adult plants with H(2)O(2) (1mM, 1.5h) reduced the content of soluble Cad conjugates and increased the content of their components, free Cad and phenols. We also found that one of the possible reasons of the negative effect of exogenous diamine on the formation of conjugated forms in adult roots was alkalization of the root apoplast at Cad addition to nutrient medium and the unusual O(2)(-) synthase function as a pH-dependent guaiacol peroxidase in the presence of a high content of H(2)O(2). This was confirmed by the data on the accumulation of O(2)(-) and enhanced superoxide dismutase activity in adult roots under treatment with Cad. It is possible that the accumulation of O(2)(-) together with H(2)O(2) was also responsible for oxidative burst, which induced a decrease in the content of Cad conjugates in adult roots of the common ice plants.     

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X‐ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate‐binding site. ROP kinases contain a typical bilobate kinase fold and a novel N‐terminal extension that both stabilizes the N‐lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N‐terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N‐terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP‐binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.     

Metal-controlled interdomain cooperativity in parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca²⁺ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg²⁺- and Na⁺-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg²⁺- and Na⁺-loaded forms of pike α-PA), to the more complex mechanisms (Ca²⁺-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca²⁺-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na⁺-bound state), thermophilic (Mg²⁺-form), or even of the hyperthermophilic origin (Ca²⁺-form).     

Edraianthus × lakusicii (Campanulaceae) a new intersectional natural hybrid: morphological and molecular evidence

The genus Edraianthus A.DC. has a center of distribution in the Balkans and it is one of the most prominent groups of endemic plants in this region. During our recent fieldtrip to Mt. Lov?en (Montenegro), putative hybrid individuals were encountered, morphologically intermediate between two sympatric taxa, E. tenuifolius (E. sect. Edraianthus) and E. wettsteinii subsp. lovcenicus (E. sect. Uniflori). Multivariate morphometric and molecular analyses were carried out to investigate the occurrence of hybridization between these two species. As a result, a new nothospecies is described here, Edraianthus × lakusicii V. Stevanovi? & D. Laku?i?, a natural hybrid between E. tenuifolius and E. wettsteinii subsp. lovcenicus. At present, this hybrid is known only from the single locality of Mt. Lov?en. Its population size is estimated to be <50 mature individuals and the estimated “area of occupancy” is smaller than 1 km².     

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca²⁺ ([Ca²⁺]_i), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC₂₀) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF_2α-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF₂α-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca²⁺]_i, but caused a Y27632-sensitive constriction in α-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC₂₀ at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca²⁺ sensitization and vasoconstriction independent of hydrogen peroxide.     

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c’s conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria—its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition—are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.     

On projection-reconstruction NMR

Three most simple Projection-Reconstruction algorithms, namely, the Lowest-Value, Additive Back-Projection and Hybrid Back-Projection/Lowest-Value algorithms, are analyzed. A new, also simple, algorithm that reconstructs the spectrum by utilizing the amplitude histogram at each reconstruction point, is explored. The algorithms are tested using simulated spectra. While all the algorithms considered can potentially result in substantial reduction of the amount of data needed for reconstruction, they can suffer from a number of drawbacks. In particular, they often fail when the spectra are noisy and/or contain overlapping peaks. When compared to the existing algorithms, the new, histogram-based algorithm has the potential advantage of being able to deal with spectra containing peaks of opposite phase.