Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.     

Dynamical characterization of the heme NO oxygen binding (HNOX) domain. Insight into soluble guanylate cyclase allosteric transition

Since the discovery of soluble guanylate cyclase (sGC) as the mammalian receptor for nitric oxide (NO), numerous studies have been performed in order to understand how sGC transduces the NO signal. However, the structural basis of sGC activation is still not completely elucidated. Spectroscopic and kinetic studies showed that the key step in the activation mechanism was the NO-induced breaking of the iron proximal histidine bond in the so-called 6c-NO to 5c-NO transition. The main breakthrough in the understanding of sGC activation mechanism came, however, from the elucidation of crystal structures for two different prokaryotic heme NO oxygen (HNOX) domains, which are homologues to the sGC heme domain. In this work we present computer simulation results of Thermoanaerobacter tencogensis HNOX that complement these structural studies, yielding molecular explanations to several poorly understood properties of these proteins. Specifically, our results explain the differential ligand binding patterns of the HNOX domains according to the nature of proximal and distal residues. We also show that the natural dynamics of these proteins is intimately related with the proposed conformational dependent activation process, which involves mainly the alphaFbeta1 loop and the alphaA-alphaC distal subdomain. The results from the sGC models also support this view and suggest a key role for the alphaFbeta1 loop in the iron proximal histidine bond breaking process and, therefore, in the sGC activation mechanism.     

Metals contents in two fishes of different feeding behaviour in the Lower Paraná River and Río de la Plata Estuary

Cd, Cr, Cu and Pb concentrations were measured in the water and these plus Zn were measured in the diet, muscle and liver of the detritivorous fish Prochilodus lineatus and the omnivorous fish Pterodoras granulosus at two sites (Lower Paraná River and Río de la Plata right margin) located upstream and downstream, respectively, from Buenos Aires, an important source of xenobiotics. Cu and Cr concentrations in water were higher in the Río de la Plata coastal waters, while Cd and Pb were below detection limits. The stomach contents of P. lineatus showed a higher metal content at the estuary, attaining concentrations within the range reported for contaminated sediments. However, metals in fish tissue were not higher at the estuary and, except for Cd in liver, were lower than in the organic fraction of the stomach content. Thus, only Cd was accumulated. The P. granulosus diet was composed mainly of river vegetation debris and clams, Corbicula fluminea, at the estuary. Cd, Cu and Pb in liver were higher at the estuary while only Cu was higher in the diet at this site. The different nature of the diet seems associated with a differential bioavailability of its metal content. P. granulosus accumulates Cu at both sites and Cd at the estuary. Both fishes showed higher metal content in liver than in muscle. Cr in liver was below detection limits. No relation was found between metal content and fish size. Overall, metal content in fish tissues were low, except for Cu in P. granulosus and Cd in P. lineatus, similar to those of uncontaminated sites, suggesting that homeostatic mechanisms prevent metal accumulation.     

Motor unit recruitment strategies investigated by surface EMG variables.

During isometric contractions of increasing strength, motor units (MUs) are recruited by the central nervous system in an orderly manner starting with the smallest, with muscle fibers that usually show the lowest conduction velocity (CV). Theory predicts that the higher the velocity of propagation of the action potential, the higher the power at high frequencies of the detected surface signal. These considerations suggest that the power spectral density of the surface detected electromyogram (EMG) signal may give indications about the MU recruitment process. The purpose of this paper is to investigate the potential and limitations of spectral analysis of the surface EMG signal as a technique for the investigation of muscle force control. The study is based on a simulation approach and on an experimental investigation of the properties of surface EMG signals detected from the biceps brachii during isometric linearly increasing torque contractions. Both simulation and experimental data indicate that volume conductor properties play an important role as confounding factors that may mask any relation between EMG spectral variables and estimated CV as a size principle parameter during ramp contractions. The correlation between spectral variables and CV is thus significantly lower when the MU pool is not stable than during constant-torque isometric contractions. Our results do not support the establishment of a general relationship between spectral EMG variables and torque or recruitment strategy.     

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.     

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

Mode of amplification and reorganization of resistance genes during recent Arabidopsis thaliana evolution.

The NBS-LRR (nucleotide-binding site plus leucine-rich repeat) genes represent the major class of disease resistance genes in flowering plants and comprise 166 genes in the ecotype Col-0 of Arabidopsis thaliana. NBS-LRR genes are organized in single-gene loci, clusters, and superclusters. Phylogenetic analysis reveals nine monophyletic clades and a few phylogenetic orphans. Most clusters contain only genes from the same phylogenetic lineage, reflecting their origin from the exchange of sequence blocks as a result of intralocus recombination. Multiple duplications increased the number of NBS-LRR genes in the progenitors of Arabidopsis, suggesting that the present complexity in Col-0 may derive from as few as 17 progenitors. The combination of physical and phylogenetic analyses of the NBS-LRR genes makes it possible to detect relatively recent gene rearrangements, which increased the number of NBS-LRR genes by about 50, but which are almost never associated with large segmental duplications. The identification of 10 heterogeneous clusters containing members from different clades demonstrates that sequence sampling between different resistance gene loci and clades has occurred. Such events may have taken place early during flowering plant evolution, but they generated modules that have been duplicated and remobilized also more recently.