Biglycan: an emerging small leucine-rich proteoglycan (SLRP) marker and its clinicopathological significance

Molecular and Cellular Biochemistry - Extracellular matrix (ECM) plays an important role in the structural organization of tissue and delivery of external cues to the cell. Biglycan, a class I...     

Variation in fructooligosaccharide contents during plant development and in different cultivars of durum wheat

Abstract

Fructooligosaccharides (FOS) have received a great deal of attention for their properties as prebiotic components. Several plants store FOS in their tissues as carbohydrate source or as osmoregulators. In comparison with the other natural FOS sources, cereals, in particular durum wheat, store a significant amount of these oligosaccharides during their growth. To gain a deeper insight on the relationships between FOS and plant/kernel development, FOS levels were analysed in the grains and stems of a widely cultivated durum wheat cultivar (Simeto), during the whole period of grain filling. In addition, a broad analysis of FOS contents was carried out by comparing their levels in the stems and grains of 45 cultivars of durum wheat harvested at two development stages. Results show a large variation among cultivars in their capability to metabolise FOS.     

Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage

The development of an appropriate technique for the identification of autolysin-defective mutants of pneumococcus has been a fundamental step to carry out studies on the molecular characteristics of the lytic enzymes of Streptococcus pneumoniae and its bacteriophage. Our results show that the principal pneumococcal autolysin (an amidase) is responsible for the separation of the daughter cells at the end of the cell division. On the other hand, this system provides a reliable experimental model to support the extended idea concerning the modular organization of most proteins. The comparative analyses of the deduced amino acid sequences of these enzymes, as well as the construction of functional chimeric phage-bacterial enzymes, demonstrate that the C-terminal domain, which contains a large number of repeated amino acid motifs, is the substrate-binding domain, whereas the N-terminal domain provides enzymatic specificity. We propose that the pneumococcal lytic enzymes have evolved by modular exchange providing examples of the types of novel genes that the bacteria or the phage might create to allow them to become adapted to new environmental situations.     

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N -acetyl-muramoyl- L -alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis , whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.     

Wordom: a program for efficient analysis of molecular dynamics simulations

Wordom is a versatile program for manipulation of molecular dynamics trajectories and efficient analysis of simulations. Original tools in Wordom include a procedure to evaluate significance of sampling for principal component analysis as well as modules for clustering multiple conformations and evaluation of order parameters for folding and aggregation. The program was developed with special emphasis on user-friendliness, effortless addition of new modules and efficient handling of large sets of trajectories.     

Trypanosoma cruzi: in vivo evaluation of iron in skin employing X-ray fluorescence (XRF) in mouse strains that differ in their susceptibility to infection

Trypanosoma cruzi, the causative agent of Chagas' disease (CD), is a substantial public health concern in Latin America. Laboratory mice inoculated with T. cruzi have served as important animal models of acute CD. Host hypoferremic responses occur during T. cruzi infection; therefore, it has been hypothesized that T. cruzi requires iron for optimal growth in host cells and, unlike extracellular pathogens, may benefit from host hypoferremic responses. Recent technological improvements of X-ray fluorescence are useful for diagnostics or monitoring in biomedical applications. The goal of our study was to determine whether the iron availabilities in Swiss and C57BL/6 mice differ during the acute phase of T. cruzi infection and whether the availability correlates with oxidative stress in the susceptible and resistant phenotypes identified in these mice. Our results showed that the decrease in iron levels in the skin of resistant infected mice correlated with the increase in oxidative stress associated with anemia and the reduction in parasite burden.     

Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction

The transfer of electrons and protons between membrane-bound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH(2)). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q(1)-site and Q(2)-site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q(2)-site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.     

Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity

The tocopherol transfer protein (TTP) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[1,2,5]oxadiazol-4-ylamino)nonyl]chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue shifted, and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analogue binds to TTP reversibly and with high affinity (K(d) = 8.5 +/- 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the ataxia with vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (K(d) = 71 +/- 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high-resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction.