Crystal structure of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase complexed with an analogue of 1,3-bisphospho-d-glyceric acid.

We report here the first crystal structure of a stable isosteric analogue of 1,3-bisphospho-d-glyceric acid (1,3-BPGA) bound to the catalytic domain of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) in which the two phosphoryl moieties interact with Arg249. This complex possibly illustrates a step of the catalytic process by which Arg249 may induce compression of the product formed, allowing its expulsion from the active site. Structural modifications were introduced into this isosteric analogue and the respective inhibitory effects of the resulting diphosphorylated compounds on T. cruzi and Trypanosoma brucei gGAPDHs were investigated by enzymatic inhibition studies, fluorescence spectroscopy, site-directed mutagenesis, and molecular modelling. Despite the high homology between the two trypanomastid gGAPDHs (> 95%), we have identified specific interactions that could be used to design selective irreversible inhibitors against T. cruzi gGAPDH.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.     

T-cell restricted and unrestricted expression of transfected human interleukin-2 gene: phorbol ester- and calcium-inducible versus constitutive expression

Interleukin-2 (IL-2) gene expression is tightly controlled and generally limited to antigenic stimulation of T cells. To study the cell-specific expression of the IL-2 gene, we transfected the intact human IL-2 gene, including 2.0 kb of 5' and 0.3 kb of 3' flanking sequences, into mouse NIH-3T3 fibroblasts and BFS lymphoma T cells and into human epithelial HeLa cells. Stable transformants (NIH-3T3,HeLa and BFS cells) carried an intact transfected IL-2 gene and constitutively expressed cytoplasmic human IL-2 mRNA which was not detected in vector-transfected cells. Constitutive expression of IL-2 mRNA in human IL-2 gene-transfected NIH-3T3 and HeLa cells was associated to the secretion of bioactive IL-2 protein, while no IL-2 production was observed in untransfected or vector-transfected cells. Cytoplasmic IL-2 mRNA observed in transfectants was larger (1.4 kb) than endogenous IL-2 mRNA of human T cells, although smaller than RNA containing unspliced intact introns. No alternative promoters or polyadenylation signals were used by these cells, but some intronic sequences were present in the 1.4 kb mRNA. Phorbol ester and calcium ionophore did not modulate the expression of the transfected IL-2 gene in NIH-3T3 and HeLa cells, while these agents increased its expression in transfected BFS lymphoma T cells. We conclude that when transfected into lymphoid and non-lymphoid cells the intact human IL-2 gene is constitutively expressed, while its phorbol ester/calcium-mediated inducible expression is restricted to T cells. This suggests that the constitutive and inducible expression of the IL-2 gene can be dissociated and are presumably subjected to separate regulatory pathways.     

MCQTL: multi-allelic QTL mapping in multi-cross design

The aim of the MCQTL software package is to perform QTL mapping in multi-cross designs. It allows the analysis of the usual populations derived from inbred lines and can link the families by assuming that the QTL locations are the same in all of them. Moreover, a diallel modelling of the QTL genotypic effects is allowed in multiple related families. The implemented model is a linear regression model. A composite interval mapping and an iterative QTL mapping are implemented to deal with multiple QTL models. Marker cofactor selections by forward or backward stepwise methods are implemented as well as computation of threshold test value by permutation. AVAILABILITY: The program is available on request after signing a licence agreement; free of charge for academic and non-profit organizations at http://www.genoplante.org (Bioinformatics products).     

New insights into the retinal circulation: inflammatory lipid mediators in ischemic retinopathy

Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Consequently, the retina constitutes an excellent model for testing the functional consequences of membrane lipid peroxidation. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane (TXA2) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA2 and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.     

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

Unique properties of polyphenol stilbenes in the brain: more than direct antioxidant actions; gene/protein regulatory activity

The 'French Paradox' has been typically associated with moderate consumption of wine, especially red wine. A polyphenol 3,4',5-trihydroxy-trans-stilbene (a member of the non-flavonoids family), better known as resveratrol, has been purported to have many health benefits. A number of these valuable properties have been attributed to its intrinsic antioxidant capabilities, although the potential level of resveratrol in the circulation is likely not enough to neutralize free radical scavenging. The brain and the heart are uniquely vulnerable to hypoxic conditions and oxidative stress injuries. Recently, evidence suggests that resveratrol could act as a signaling molecule within tissues and cells to modulate the expression of genes and proteins. Stimulation of such proteins and enzymes could explain some the intracellular antioxidative properties. The modulation of genes could suffice as an explanation of some of resveratrol's cytoprotective actions, as well as its influence on blood flow, cell death, and inflammatory cascades. Resveratrol stimulation of the expression of heme oxygenase is one example. Increased heme oxygenase activity has led to significant protection against models of in vitro and in vivo oxidative stress injury. Resveratrol could provide cellular resistance against insults; although more work is necessary before it is prescribed as a potential prophylactic in models of either acute or chronic conditions, such as stroke, amyotrophic lateral sclerosis, Parkinson, Alzheimer, and a variety of age-related vascular disorders.     

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus).After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters.In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity.The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells.