Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII’s preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.     

Weak population spatial genetic structure and low infraspecific specificity for fungal partners in the rare mycoheterotrophic orchid Epipogium aphyllum

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (F_ST = 0.106 for nuclear microsatellites and F_ST = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (R_r = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism.

     

Soil protist function varies with elevation in the Swiss Alps

Protists are abundant and play key trophic functions in soil. Documenting how their trophic contributions vary across large environmental gradients is essential to understand and predict how biogeochemical cycles will be impacted by global changes. Here, using amplicon sequencing of environmental DNA in open habitat soil from 161 locations spanning 2600 m of elevation in the Swiss Alps (from 400 to 3000 m), we found that, over the whole study area, soils are dominated by consumers, followed by parasites and phototrophs. In contrast, the proportion of these groups in local communities shows large variations in relation to elevation. While there is, on average, three times more consumers than parasites at low elevation (400–1000 m), this ratio increases to 12 at high elevation (2000–3000 m). This suggests that the decrease in protist host biomass and diversity toward mountains tops impact protist functional composition. Furthermore, the taxonomic composition of protists that infect animals was related to elevation while that of protists that infect plants or of protist consumers was related to soil pH. This study provides a first step to document and understand how soil protist functions vary along the elevational gradient.     

Sucrose feeding during pregnancy and lactation elicits distinct metabolic response in offspring of an inbred genetic model of metabolic syndrome

The importance of early environment, including maternal diet during pregnancy, is suspected to play a major role in pathogenesis of metabolic syndrome and related conditions. One of the proposed mechanisms is a mismatch between the prenatal and postnatal environments, leading to misprogramming of the metabolic and signaling pathways of the developing fetus. We assessed whether the exposure to high-sucrose diet (HSD) alleviates the detrimental effects of sucrose feeding in later life (predictive adaptive hypothesis) in a highly inbred model of metabolic syndrome, the PD/Cub rat. Rat dams were continuously fed either standard or HSD (70% calories as sucrose) starting 1 wk before breeding, throughout pregnancy, at birth, and until weaning of the offspring. After weaning, all male offspring were fed HSD until the age of 20 wk, when detailed metabolic and morphometric profiles were ascertained. The early life exposure to a sucrose-rich diet resulted in distinct responses to longtime postnatal HSD feeding. Offspring of the sucrose-fed mothers displayed higher adiposity and substantial increases in triglyceride liver content together with unfavorable distribution of cholesterol into lipoprotein subfractions. On the other hand, their adiponectin concentrations were significantly higher, and insulin sensitivity of skeletal muscle was enhanced compared with the offspring of standard diet-fed mothers. Triglycerides, free fatty acids, overall glucose tolerance, and the insulin sensitivity of adipose tissue were comparable in both groups. In the genetically identical animals, maternal HSD feeding elicited a variety of subtle effects but did not lead to predictive adaptive protection from most HSD-induced metabolic derangements.     

Modulation of expression of the sigma receptors in the heart of rat and mouse in normal and pathological conditions

The aim of the present work was to study the effect of various stressors (hypoxia, cold, immobilization) on the gene expression of sigma receptors in the left ventricles of rat heart. We have clearly shown that gene expression of sigma receptors is upregulated by strong stress stimuli, such as immobilization and/or hypoxia. Nevertheless, cold as a milder stressor has no effect on sigma receptor's mRNA levels. Signalling cascade of sigma receptors is dependent on IP(3) receptors, since silencing of both, type 1 and 2 IP(3) receptors resulted in decreased mRNA levels of sigma receptors. Physiological relevance of sigma receptors in the heart is not clear yet. Nevertheless, based on the already published data we can assume that sigma receptors might participate in contractile responses in cardiomyocytes.     

Schizophrenia and family

Family represents an important supportive social network for most patients with schizophrenia. In order to provide safe and low-stress environment, necessary for the successful long-term treatment of schizophrenia the family must be helped. Family members suffer both emotionally and financially. Their burden is high and quality of life is low. Relatives change their life values and preferences when a severe mental illness occurs in the family and are ready to cooperate. Family interventions constitute an integral part of complex schizophrenia treatment. To be successful interventions must take into account relatives beliefs, perspectives and issues of concern. The goal of this paper is to 1) overview studies on family burden, 2) provide theoretical background for family interventions, 3) describe a family psychoeducational approach as an optimal treatment modification.     

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder,solution, DPC micelles and at the air–water interface

Cateslytin (bCGA ³⁴⁴RSMRLSFRARGYGFR³⁵⁸), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Conformation of DNA GG intrastrand cross-link of antitumor oxaliplatin and its enantiomeric analog

Downstream processes that discriminate between DNA adducts of a third generation platinum antitumor drug oxaliplatin and conventional cisplatin are believed to be responsible for the differences in their biological effects. These different biological effects are explained by the ability of oxaliplatin to form DNA adducts more efficient in their biological effects. In this work conformation, recognition by HMG domain protein and DNA polymerization across the major 1,2-GG intrastrand cross-link formed by cisplatin and oxaliplatin in three sequence contexts were compared with the aid of biophysical and biochemical methods. The following major differences in the properties of the cross-links of oxaliplatin and cisplatin were found: i), the formation of the cross-link by oxaliplatin is more deleterious energetically in all three sequence contexts; ii), the cross-link of oxaliplatin bends DNA slightly but systematically less in all sequence contexts tested; iii), the affinity of HMG domain protein to the cross-link of oxaliplatin is considerably lower independent of the sequence context; and iv), the Klenow fragment of DNA polymerase I pauses considerably more at the cross-link of oxaliplatin in all sequence contexts tested. We have also demonstrated that the chirality at the carrier ligand of oxaliplatin can affect its biological effects.     

Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study

Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 x LD(50)). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a "mean" of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.     

Recruitment of Cdc42 through the GAP domain of RLIP participates in remodeling of the actin cytoskeleton and is involved in Xenopus gastrulation

The transduction pathways that branch out of fibroblast growth factor signaling are essential for the induction of the mesoderm and the specification of the vertebrate body plan. One of these pathways is thought to control remodeling of the actin cytoskeleton through the Ral binding protein (RLIP also known as RalBP1), an effector of the small G protein Ral. RLIP contains a region of homology with the GTPase-activating protein (GAP) domain involved in the regulation of GTPases of the Rho family. We demonstrate here that the GAP domain of RLIP is responsible for the stability of the actin cytoskeleton in Xenopus laevis embryos. We also demonstrate that the complete N-terminal domain of RLIP containing the μ2 binding domain (μ2BD) and the GAP domain induces disruption of the actin cytoskeleton when targeted to the plasma membrane. Neither domain, however, has any effect on the actin cytoskeleton when individually targeted to the plasma membrane. We also determined that Cdc42-GDP, but neither Rac-GDP nor Rho-GDP, rescues the effect of expression of the membrane-localized Xenopus RLIP on the actin cytoskeleton. We show that the GAP domain of RLIP interacts in vivo with Cdc42-GTP and Cdc42-GDP. Finally, a single mutation (K244A) in the GAP sequence prevented embryos from gastrulating. These results demonstrate that to participate in the control of the actin cytoskeleton, RLIP needs its complete N-terminal region coding for the μ2BD and the GAP domain. We suggest that RLIP, by coordinating two complementary mechanisms, the endocytosis of clathrin-coated pits and the remodeling of cortical actin, participates in the gastrulation process.