Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.     

New exopolysaccharides produced by Aureobasidium pullulans grown on glucosamine

The polysaccharides produced by Aureobasidium pullulans, grown using glucosamine as the carbon source, were investigated by means of methylation analysis, affinity chromatography and NMR spectroscopy. The results indicated that, besides a small amount of pullulan, this micro-organism was capable of producing-in low yields-mixtures of at least two different complex polysaccharides containing mainly mannose and galactose. (1)H NMR spectra of two fractions obtained by lectin affinity chromatography indicated that one polymer was constituted exclusively of mannose residues while the other contained both galactofuranosyl and mannopyranosyl residues.     

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc–Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label‐free shotgun 2D‐nanoUPLC/MS^E to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc–susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large‐scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant–pathogen interaction in planta.     

Mechanical properties of physiological and pathological models of collagen peptides investigated via steered molecular dynamics simulations

In this work we used molecular simulations to investigate the elastic properties of collagen single chain and triple helix with the aim of understanding its features starting from first principles. We analysed ideal collagen peptides, homotrimeric and heterotrimeric collagen type I and pathological models of collagen. Triple helices were found much more rigid than single chains, thus enlightening the important role of interchain stabilizing forces, like hydrophobic interaction and hydrogen bonds. We obtained Young's moduli close to 4.5GPa for the ideal model of collagen and for the physiological heterotrimer, while the physiological homotrimer presented a Young's modulus of 2.51GPa, that can be related to a mild form of Osteogenesis Imperfecta in which only the homotrimeric form of collagen type I is produced. Otherwise, the pathological model (presenting a glycine to alanine substitution) showed an elastic modulus of 4.32GPa, thus only slightly lower than the ideal model. This suggests that this mutation only slightly affects the mechanical properties of the collagen molecule, but possibly acts on an higher scale, such as the packing of collagen fibrils.     

Collagen-specific T-cell repertoire in blood and synovial fluid varies with disease activity in early rheumatoid arthritis

Introduction  

Type II collagen is a DR4/DR1 restricted target of self-reactive T cells that sustain rheumatoid arthritis. The aim of the present study was to analyze the T-cell receptor repertoire at the onset of and at different phases in rheumatoid arthritis.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).     

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.     

Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Hypothyroidism provides resistance to kidney mitochondria against the injury induced by renal ischemia-reperfusion

Massive Ca(2+) accumulation in mitochondria, plus the stimulating effect of an inducing agent, i.e., oxidative stress, induces the so-called permeability transition, which is characterized by the opening of a nonspecific pore. This work was aimed at studying the influence of thyroid hormone on the opening of such a nonspecific pore in kidney mitochondria, as induced by an oxidative stress. To meet this objective, membrane permeability transition was examined in mitochondria isolated from kidney of euthyroid and hypothyroid rats, after a period of ischemia/reperfusion. It was found that mitochondria from hypothyroid rats were able to retain accumulated Ca(2+) to sustain a transmembrane potential after Ca(2+) addition, as well as to maintain matrix NAD(+) and membrane cytochrome c content. The protective effect of hypothyroidism was clearly opposed to that occurring in ischemic reperfused mitochondria from euthyroid rats. Our findings demonstrate that these mitochondria were unable to preserve selective membrane permeability, except when cyclosporin A was added. It is proposed that the protection is conferred by the low content of cardiolipin found in the inner membrane. This phospholipid is required to switch adenine nucleotide translocase from specific carrier to a non-specific pore.     

Synthesis,semipreparative HPLC separation,biological evaluation,and 3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase B inhibitors

The present study reports on synthesis in high yields (70–99%), HPLC enantioseparation, inhibitory activity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large series of (4-aryl-thiazol-2-yl)hydrazones (1–45). Most of the synthesized compounds proved to be potent and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors. Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chromatographic enantioseparation of these compounds obtained by a stereoconservative pattern. The separated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further structural modification of these lead compounds.