Inhibition of chloroplast coupling factor by naphthylglyoxal

The trypsin-activated Ca²⁺ -ATPase of spinach chloroplast membranes was completely inhibited by treatment with naphthylglyoxal, a fluorescent compound that should bind covalently to arginine residues. The inhibition followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentration gave values near unity, suggesting that inactivation is a consequence of modifying one arginine residue per active site. Partial protection against naphthylglyoxal was afforded by ADP and ATP, with either less or no protection by other nucleotide bases. At inhibition levels less than complete, the K_m for ATP was not affected but the V_max of the enzyme was diminished. The light-dependent exchange of tightly bound nucleotides on the membrane-bound enzyme was not inhibited by naphthylglyoxal treatment, indicating significant retention of the conformational response of the enzyme to the membrane high-energy state. Using [³H]naphthylglyoxal, the extent of inhibition was a linear function of the amount of naphthylglyoxal bound up to 60% inhibition. The curves extrapolated to 2 mol naphthylglyoxal bound, associated with complete inhibition of ATPase. The radioactive naphthylglyoxal was distributed equally between α- and β-subunits.     

Encapsulation of micropropagated buds of six woody species

Regrowth after encapsulation in a sodium alginate matrix of micropropagated buds from six different in vitro proliferated woody species was evaluated. Actinidia deliciosa Liang & Ferguson (kiwifruit), Betula pendula Roth (birch), Crataegus oxyacantha L. (hawthorn), Malus spp. (apple), Rubus spp. (blackberry) and Rubus idaeus L. (raspberry) propagated in vitro were used as bud sources. Encapsulation with sodium alginate and subsequent regrowth on nutrient rich medium was compared to encapsulation with nutrient-enriched alginate capsules followed by regrowth on nutrientless medium. Apical and sub-apical buds of Malus (rootstock M. 27 and cultivar Starkspur Red) were also compared for encapsulation and regrowth ability. All species showed a regrowth after encapsulation, but only if cultured on enriched media. M.27 apical and sub-apical buds showed different regrowth ability after encapsulation with sodium alginate. Applicability of encapsulation of single micropropagated tree buds is discussed.     

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.     

Alpha-tocopherol pretreatment improves endothelium-dependent vasodilation in aortic strips of young and aging rats exposed to oxidative stress

Acetylcholine-induced, endothelium-dependent relaxation of norepinephrine-precontracted aortic strips, was severely impaired after exposure to a hypoxanthine/xanthine oxidase reaction generating oxygen radicals. This effect was more evident in aortic strips of aging rats (24 months old) in comparison to young rats (3 months old). The addition of authentic ·NO (1 M) completely relaxed aortic strips exposed to oxidative stress both in young and aging rats. In vitro EPR measurements showed that the ·NO signal was reduced by enzymatic O₂-generating reaction.The activity of a partial purified preparation of constitutive NO synthase from rat cerebellum was significantly decreased after exposure to exogenous oxygen radicals. Pretreatment of aortic strips with 100 M alpha-tocopherol-phosphate, produced a significant improvement of acetylcholine-dependent relaxation in the aortic strips exposed to oxidative stress, particularly in the aged vessel. The content of malondialdehyde in aortic tissue did not change after oxidative stress or alpha-tocopherol pretreatment. Alpha-tocopherol was unable to recover the NO synthase activity depressed in vitro by hypoxanthine/xanthine oxidase reaction. This study confirms that an oxidative stress impairs the endothelium-mediated vasodilation. Alpha-tocopherol pretreatment protects the vessel against this damage. The mechanism of action of alpha-tocopherol is unknown, but seems unrelated to an antioxidant activity.Abbreviations ACh acethylcholine - EPR electron paramagnetic resonance - ROS reactive oxygen species - MDA malondialdehyde - NE norepinephrine - cNOS constitutive nitric oxide synthase     

Self-assembly of biopolymeric structures below the threshold of random cross-link percolation.

Self-assembly of extended structures via cross-linking of individual biomolecules often occurs in solutions at concentrations well below the estimated threshold for random cross-link percolation. This requires solute-solute correlations. Here we study bovine serum albumin. Its unfolding causes the appearance of an instability region of the sol, not observed for native bovine serum albumin. As a consequence, spinodal demixing of the sol is observed. The thermodynamic phase transition corresponding to this demixing is the determinative symmetry-breaking step allowing the subsequent occurrence of (correlated) cross-linking and its progress up to the topological phase transition of gelation. The occurrence of this sequence is of marked interest to theories of spontaneous symmetry-breaking leading to morphogenesis, as well as to percolation theories. The present results extend the validity of conclusions drawn from our previous studies of other systems, by showing in one single case, system features that we have hitherto observed separately in different systems. Time-resolved experimental observations of the present type also bring kinetic and diffusional processes and solute-solvent interactions into the picture of cross-link percolation.     

Metabolism and Trafficking of N-Type Voltage-Operated Calcium Channels in Neurosecretory Cells

The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity -conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. -conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis.     

Town and Gender Effects on Hair Lead Levels in Children from Three Sardinian Towns (Italy) with Different Environmental Backgrounds

This study reports hair lead (PbH) levels measured in 2002 in 193 children from three Sardinian towns: Carbonia, Gonnesa, and Sinnai. Carbonia and Gonnesa are in a polluted area of Sardinia due to their vicinity to the industrial zone of Portovesme. As a consequence of its economy and location, Sinnai is not exposed to lead pollution. PbH concentrations were determined by inductively coupled plasma atomic absorption spectrometry. The aim of this study was to evaluate if hair is a reliable biomarker to determine different degrees of exposure of populations to lead pollution and if there is a tendency to higher accumulation by males or females. The girls of Carbonia had the highest mean PbH value (2.21 microg/g), followed by the Gonnesa girls (2.03 microg/g), Carbonia boys (1.86 microg/g), Gonnesa boys (0.91 microg/g), and finally the Sinnai boys (0.68 microg/g) and girls (0.50 microg/g). Two-way analysis of covariance, with age as covariate, revealed a significant effect of town and sex on log PbH. Spearman's rank correlation coefficient indicated a significant positive concordance between PbH levels and gender (score for males=1, females=2). The results suggest that hair is a reliable biomarker to determine different levels of exposure of populations to lead pollution, and they indicate that females tend to accumulate lead in the hair more than males of the same age.     

Allosteric regulation of histidine kinases by their cognate response regulator determines cell fate

The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.     

The pathological splicing mutation c.6792C>G in NF1 exon 37 causes a change of tenancy between antagonistic splicing factors

We have previously identified an ESE in NF1 exon 37 whose disruption by the pathological mutation c.6792C>G caused aberrant splicing. We now investigate the RNA-protein complexes affected by the c.6792C>G mutation observing that this concurrently decreases the affinity for the positive splicing factor YB-1 and increases the affinity for the negative splicing factors, hnRNPA1, hnRNPA2 and a new player in these type of complexes, DAZAP1. Our findings highlight the complexity of the interplay between positive and negative factors in the exon inclusion/skipping outcome. Furthermore, our observations stress the role of a wide genomic context in NF1 exon 37 definition.