EPR studies of chlorocatechol 1,2-dioxygenase: evidences of iron reduction during catalysis and of the binding of amphipatic molecules

Chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is a dioxygenase responsible for ring cleavage during the degradation of recalcitrant aromatic compounds. We determined the zero-field splitting of the Fe(III) cofactor (|D| = 1.3 +/- 0.2 cm(-1)) by electron paramagnetic resonance (EPR) experiments that along with other structural data allowed us to infer the Fe(III) coordination environment. The EPR spectrum of the ion shows a significantly decrease of the g = 4.3 resonance upon substrate binding. This result is rationalized in terms of a mechanism previously proposed, where catechol substrate is activated by Fe(III), yielding an exchange-coupled Fe(II)-semiquinone (pair). The Pp 1,2-CCD capacity of binding amphipatic molecules and the effects of such binding on protein activity are also investigated. EPR spectra of spin labels show a protein-bound component, which was characterized by means of spectral simulations. Our results indicate that Pp 1,2-CCD is able to bind amphipatic molecules in a channel with the headgroup pointing outwards into the solvent, whereas the carbon chain is held inside the tunnel. Protein assays show that the enzyme activity is significantly lowered in the presence of stearic-acid molecules. The role of the binding of those molecules as an enzyme activity modulator is discussed.     

Conformationally sensitive residues in extracellular loop 5 of the Na+/dicarboxylate co-transporter

The Na+/dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate2-. A previous study (Pajor, A. M. (2001) J. Biol. Chem. 276, 29961-29968), identified four amino acids, Ser-478, Ala-480, Ala-481, and Thr-482, near the extracellular end of transmembrane helix (TM) 9 that are likely to form part of the permeation pathway of the transporter. All four cysteine-substituted mutants were sensitive to inhibition by the membrane-impermeant reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) and protected by substrate. In the present study, we continued the cysteine scan through extracellular loop 5 and TM10, from Thr-483 to Val-528. Most cysteine substitutions were well tolerated, although cysteine mutations of some residues, particularly within the TM, produced proteins that were not expressed on the plasma membrane. Six residues in the extracellular loop (Thr-483, Thr-484, Leu-485, Leu-487, Ile-489, and Met-493) were sensitive to chemical labeling by MTSET, depending on the conformational state of the protein. Transport inhibition by MTSET could be prevented by substrate regardless of temperature, suggesting that the likely mechanism of substrate protection is steric hindrance rather than large-scale conformational changes associated with translocation. We conclude that extracellular loop 5 in NaDC-1 appears to have a functional role, and it is likely to be located in or near the substrate translocation pore in the protein. Conformational changes in the protein affect the accessibility of the residues in extracellular loop 5 and provide further evidence of large-scale changes in the structure of NaDC-1 during the transport cycle.     

In Situ Investigation of Leaf Water Status by Portable Unilateral Nuclear Magnetic Resonance

A portable unilateral nuclear magnetic resonance (NMR) instrument was used to detect in field conditions the water status of leaves of herbaceous crops (Zea mays, Phaseolus vulgaris), mesophyllous trees (Populus nigra), and natural Mediterranean vegetation characterized by water-spending shrubs (Cistus incanus) and water-saving sclerophyllous trees (Quercus ilex). A good relationship was observed between NMR signal, leaf relative water content, and leaf transpiration in herbaceous leaves undergoing fast dehydration or slowly developing a drought stress. A relationship was also observed between NMR signal and water potential of Populus leaves during the development of a water stress and when leaves recovered from the stress. In the natural vegetation, the relationship between NMR signal and water status was found in Cistus, the species characterized by high transpiration rates, when measured during a drought stress period and after a rainfall. In the case of the sclerophyllous Quercus, the NMR signal, the relative water content, and the transpiration rate did not change at different leaf water status, possibly because a large amount of water is compartmentalized in cellular structures and macromolecules. The good association between NMR signal and relative water content was lost in leaves exposed for 24 h to dehydration or to an osmotic stress caused by polyethylene glycol feeding. At this time, the transverse relaxation time became longer than in leaves maintained under optimal water conditions, and two indicators of membrane damage, the ion leakage and the emission of products of membrane lipoxygenation [(Z)-3-hexenal, (Z)-3-hexenol, and (E)-2-hexenol], increased. These results taken all together give information on the physiological state of a leaf under a developing stress and show the usefulness of the NMR instrumentation for screening vegetation health and fitness in natural and cultivated conditions. It is concluded that the portable unilateral NMR instrument may be usefully employed in field conditions to monitor nondestructively the water status of plants and to assist agricultural practices, such as irrigation scheduling, to minimize stomatal closure and the consequent limitation to plant production.     

The delta e13 isoform of the calcitonin receptor forms a six-transmembrane domain receptor with dominant-negative effects on receptor surface expression and signaling

The CTRdelta e13 splice variant of the rabbit calcitonin receptor, which lacks the 14 amino acids of the seventh transmembrane domain (TMD) that are encoded by exon 13, is poorly expressed on the cell surface, fails to mobilize intracellular calcium or activate Erk, and inhibits the cell surface expression of the full-length C1a isoform. Nuclear magnetic resonance- and fluorescence-activated cell sorter-based experiments showed that the residual seventh TMD of CTRdelta e13 fails to partition into the lipid bilayer, resulting in an extracellular C terminus. Truncating the receptor after residue 397 to delete the cytoplasmic tail resulted in reduced cell surface expression and an inability to mobilize intracellular calcium or activate Erk, but the truncated receptor did not inhibit C1a cell surface expression. In contrast, when the receptor was truncated after residue 374 to eliminate the entire seventh TMD domain and the C-terminal domain, the resulting receptor reduced the cell surface expression of C1a in a manner similar to that of CTRdelta e13. Thus, normal cell surface expression, mobilization of intracellular calcium, and Erk activation requires the cytoplasmic C-terminal tail of the CTR, whereas the absence of the seventh TMD in the transmembrane helical bundle causes the dominant-negative effect on the surface expression of C1a.     

Linking two immuno-suppressive molecules: indoleamine 2,3 dioxygenase can modify HLA-G cell-surface expression

Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1-negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potential implications in fetal-maternal tolerance are discussed.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

Obesity--new threat to Croatian longevity

The aim of this study was to examine the association of weight gain and life expectancy at birth in Croatia. Mean body mass index was based on the data from the Croatian Adult Health Survey 2003. Birth rate and mortality data needed for life expectancy calculation were supplied by the Central Bureau of Statistics. The results suggest that the increase in mean body mass index value (1.31 kg m(-2) for women and 1.41 kg m(-2) for men) will shorten life expectancy at birth for one year. Obesity, if unchecked, might have a negative effect on life expectancy in Croatia. Despite widespread knowledge about how to reduce the severity of the problem, observed trends in obesity in Croatia continue to worsen. These trends threaten to diminish the health and life expectancy of current and future generations.     

Modeling trap-awareness and related phenomena in capture-recapture studies

Trap-awareness and related phenomena whereby successive capture events are not independent is a feature of the majority of capture-recapture studies. This phenomenon was up to now difficult to incorporate in open population models and most authors have chosen to neglect it although this may have damaging consequences. Focusing on the situation where animals exhibit a trap response at the occasion immediately following one where they have been trapped but revert to their original naïve state if they are missed once, we show that trap-dependence is more naturally viewed as a state transition and is amenable to the current models of capture-recapture. This approach has the potential to accommodate lasting or progressively waning trap effects.     

Influenza a viruses from wild birds in Guatemala belong to the North American lineage

The role wild bird species play in the transmission and ecology of avian influenza virus (AIV) is well established; however, there are significant gaps in our understanding of the worldwide distribution of these viruses, specifically about the prevalence and/or significance of AIV in Central and South America. As part of an assessment of the ecology of AIV in Guatemala, we conducted active surveillance in wild birds on the Pacific and Atlantic coasts. Cloacal and tracheal swab samples taken from resident and migratory wild birds were collected from February 2007 to January 2010.1913 samples were collected and virus was detected by real time RT-PCR (rRT-PCR) in 28 swab samples from ducks (Anas discors). Virus isolation was attempted for these positive samples, and 15 isolates were obtained from the migratory duck species Blue-winged teal. The subtypes identified included H7N9, H11N2, H3N8, H5N3, H8N4, and H5N4. Phylogenetic analysis of the viral sequences revealed that AIV isolates are highly similar to viruses from the North American lineage suggesting that bird migration dictates the ecology of these viruses in the Guatemalan bird population.