Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

The effect of interval training in children with exercise-induced asthma competing in soccer

A lot of emphasis has been placed in screening individuals with exercise-induced bronchospasm in order to avoid persistence bronchial hyperactivity and consequent chronic silent inflammation of the respiratory tract. The purpose of this study was to evaluate the effect of interval training on the respiratory function and endurance in children with exercise-induced asthma (EIA) participating in the sport of soccer. Twenty-nine boys ages 10-14, who developed EIA after a 6-minute free running test (decline in forced expiratory volume in 1 second: FEV(1)10%), participated in the study. They were divided into 2 groups (experimental: n = 18, and control: n = 11), fulfilling the same criteria (i.e., age, body height and weight, and severity of asthma). The experimental group exercised with the interval training method for a period of 8 weeks, (3 sessions per week), whereas the control group exercised with the usual football program. Measurements were made for FEV(1) and endurance in both groups, before and after the application of training (8 weeks). Following the implementation of the training program, a significant improvement in FEV(1) and endurance was documented in the experimental group, as well as significant differences between the 2 groups. In conclusion, duration and aerobic training via the interval method seems to be beneficial to soccer players with EIA.     

Oxidative stress biomarkers responses to physical overtraining: implications for diagnosis

Overtraining syndrome is characterized by declining performance and transient inflammation following periods of severe training with major health implications for the athletes. Currently, there is no single diagnostic marker for overtraining. The present investigation examined the responses of oxidative stress biomarkers to a resistance training protocol of progressively increased and decreased volume/intensity. Twelve males (21.3+/-2.3 years) participated in a 12-week resistance training consisting of five 3-week periods (T1, 2 tones/week; T2, 8 tones/week; T3, 14 tones/week; T4, 2 tones/week), followed by a 3-week period of complete rest. Blood/urine samples were collected at baseline and 96 h following the last training session of each period. Performance (strength, power, jumping ability) increased after T2 and declined thereafter, indicating an overtraining response. Overtraining (T3) induced sustained leukocytosis, an increase of urinary isoprostanes (7-fold), TBARS (56%), protein carbonyls (73%), catalase (96%), glutathione peroxidase, and oxidized glutathione (GSSG) (25%) and a decline of reduced glutathione (GSH) (31%), GSH/GSSG (56%), and total antioxidant capacity. Isoprostanes and GSH/GSSG were highly (r=0.764-0.911) correlated with performance drop and training volume increase. In conclusion, overtraining induces a marked response of oxidative stress biomarkers which, in some cases, was proportional to training load, suggesting that they may serve as a tool for overtraining diagnosis.     

The molecular basis of selective promoter activation by the sigmaS subunit of RNA polymerase

Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, sigmaS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, sigma70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing sigmaS or sigma70, or by both holoenzymes. How these promoter features impose sigmaS selectivity is further discussed. Moreover, additional pathways allow sigmaS to compete more efficiently than sigma70 for limiting amounts of core RNAP (E) and thereby enhance EsigmaS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes.     

Expression of the yeast cpd1 gene in tobacco confers resistance to the fungal toxin cercosporin

Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.     

Proteomic analysis of childhood de novo acute myeloid leukemia and myelodysplastic syndrome/AML: correlation to molecular and cytogenetic analyses

The aim of this study was to investigate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) and to provide additional data regarding the proteomic analysis of AML. The protein profiles obtained were correlated to cytogenetic and molecular analyses. Bone marrow (BM) and peripheral blood (PB) samples were obtained during MDS diagnosis, at MDS transformation to AML, at de novo AML diagnosis and 3 months following treatment. As controls, non-leukemic pediatric patients were studied. Cytogenetic and molecular analyses were carried out by G banding and polymerase chain reaction followed by sequencing, respectively. Differential proteomic analysis was performed by two-dimensional gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. No significant correlations were noted between protein patterns and cytogenetic or molecular analyses. Certain suppressor genes, metabolic enzymes, immunoglobulins and actin-binding proteins were differentially expressed by BM or PB plasma and cell lysates compared to controls. The obtained data showed that vitamin D and gelsolin played contradicting roles in contributing and restraining leukemogenesis, while MOES, EZRI and AIFM1 could be considered as biomarkers for AML.