Specificity profile of NAT/NCS2 purine transporters in Sinorhizobium (Ensifer) meliloti

Sinorhizobium (Ensifer) meliloti is a model example of a soil alpha-proteobacterium which induces the formation of nitrogen-fixing symbiotic nodules on the legume roots. In contrast to all other rhizobacterial species, S. meliloti contains multiple homologs of nucleobase transporter genes that belong to NAT/NCS2 family (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2). We analyzed functionally all (six) relevant homologs of S. meliloti 1,021 using Escherichia coli K-12 as a host and found that five of them are high-affinity transporters for xanthine (SmLL9), uric acid (SmLL8, SmLL9, SmX28), adenine (SmVC3, SmYE1), guanine (SmVC3), or hypoxanthine (SmVC3). Detailed analysis of substrate profiles showed that two of these transporters display enlarged specificity (SmLL9, SmVC3). SmLL9 is closely related in sequence with the xanthine-specific XanQ of E. coli. We subjected SmLL9 to rationally designed site-directed mutagenesis and found that the role of key binding-site residues of XanQ is conserved in SmLL9, whereas a single amino-acid change (S93N) converts the xanthine/uric-acid transporter SmLL9 to a xanthine-preferring variant, due to disruption of an essential hydrogen bond with the C8 oxygen of uric acid. The results highlight the presence of several different purine nucleobase transporters in S. meliloti and imply that the purine transport might be important in the nodule symbiosis involving S. meliloti.     

Crystal structure of the BcZBP, a zinc-binding protein from Bacillus cereus

Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus thus provides a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The zinc-binding protein of B. cereus, BcZBP, is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity with the N-acetyl moiety of the N-acetylglucosamine and the diacetylchitobiose and triacetylchitotriose. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Here, we present the crystal structure of BcZBP at 1.8 A resolution. The N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas the C-terminal part consists of two beta-strands and two alpha-helices. In the crystal, the protein forms a compact hexamer, in agreement with solution data. A zinc binding site and a potential active site have been identified in each monomer. These sites have extensive similarities to those found in two known zinc-dependent hydrolases with deacetylase activity, MshB and LpxC, despite a low degree of amino acid sequence identity. The functional implications and a possible catalytic mechanism are discussed.     

An EG-VEGF-Dependent Decrease in Homeobox Gene NKX3.1 Contributes to Cytotrophoblast Dysfunction: A Possible Mechanism in Human Fetal Growth Restriction

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland–derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8–12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

Dynamics of Aerenchyma distribution in the cortex of sulfate-deprived adventitious roots of maize

BACKGROUND AND AIMS: Aerenchyma formation in maize adventitious roots is induced in nutrient solution by the deprivation of sulfate (S) under well-oxygenated conditions. The aim of this research was to examine the extent of aerenchyma formation in the cortex of sulfate-deprived adventitious roots along the root axis, in correlation with the presence of reactive oxygen species (ROS), calcium levels and pH of cortex cells and root lignification. METHODS: The morphometry of the second whorl of adventitious (W2) roots, subject to S-deprivation conditions throughout development, was recorded in terms of root length and lateral root length and distribution. W2 roots divided into sectors according to the mean length of lateral roots, and cross-sections of each were examined for aerenchyma. In-situ detection of alterations in ROS presence, calcium levels and pH were performed by means of fluorescence microscopy using H(2)DCF-DA, fluo-3AM and BCECF, respectively. Lignification was detected using the Wiesner test. KEY RESULTS: S-deprivation reduced shoot growth and enhanced root proliferation. Aerenchyma was found in the cortex of 77 % of the root length, particularly in the region of emerging or developing lateral roots. The basal and apical sectors had no aerenchyma and no aerenchyma connection was found with the shoot. S-deprivation resulted in alterations of ROS, calcium levels and pH in aerenchymatous sectors compared with the basal non-aerenchymatous region. Lignified epidermal layers were located at the basal and the proximal sectors. S-deprivation resulted in shorter lateral roots in the upper sectors and in a limited extension of the lignified layers towards the next lateral root carrying sector. CONCLUSIONS: Lateral root proliferation is accompanied by spatially localized induced cell death in the cortex of developing young maize adventitious roots during S-deprivation.     

Search for an Association between V249I and T280M CX3CR1 Genetic Polymorphisms,Endothelial Injury and Preeclampsia: The ECLAXIR Study

Background

Preeclampsia and coronary-artery disease share risk factors, suggesting common pathophysiological mechanisms. CX3CR1/CX3CL1 mediates leukocyte migration and adhesion and has been implicated in the pathophysiology of several inflammatory diseases. M280/I249 variants of CX3CR1 are associated with an atheroprotective effect and reduced endothelial dysfunction. The aim of this study was to search for an association between V249I and T280M polymorphisms of CX3CR1, preeclampsia and endothelial dysfunction.

Methodology/Principal Findings

We explored these polymorphisms with real-time polymerase chain reaction in a case-control study (184 white women with preeclampsia and 184 matched normotensive pregnant women). Endothelial dysfunction biomarkers including von Willebrand factor, VCAM-1 and thrombomodulin, as well as the soluble form of CX3CL1 were measured by enzyme-linked immunosorbent assays (ELISA). The I249 and M280 alleles were associated neither with preeclampsia, nor with its more severe form or with endothelial injury. In contrast, we found a trend toward increased CX3CL1 levels in preeclampsia patients, especially in early-onset- preeclampsia as compared to its level in later-onset- preeclampsia.

Conclusions/Significance

This is the first study to characterize the CX3CR1 gene polymorphisms in patients with preeclampsia. We found no differences in genotype or haplotype frequencies between patients with PE and normal pregnancies, suggesting that maternal CX3CR1 V249I and T280M polymorphisms do not increase susceptibility to preeclampsia. Further studies should be performed to directly evaluate the pathophysiological role of CX3CL1, a molecule abundantly expressed in endometrium, which has been shown to stimulate human trophoblast migration.     

Analysis of gene expression in operons of Streptomyces coelicolor

Background  

Recent studies have shown that microarray-derived gene-expression data are useful for operon prediction. However, it is apparent that genes within an operon do not conform to the simple notion that they have equal levels of expression.