Sequencing <Emphasis Type="Italic">EVC</Emphasis> and <Emphasis Type="Italic">EVC2</Emphasis> identifies mutations in two-thirds of Ellis–van Creveld syndrome patients

Ellis–van Creveld syndrome (EvC) is caused by mutations in EVC and EVC2, genes in a divergent orientation separated by only 2.6 kb. We systematically sought mutations in both genes in a panel of 65 affected individuals to assess the proportion of cases resulting from mutations in each gene. We PCR amplified and sequenced the coding exons of both genes. We investigated mutations that could affect splicing by in vitro splicing assays and cDNA analysis. We have identified EVC mutations in 20 cases (31%); in all of these we have detected the mutation on each allele. We have identified EVC2 mutations in 25 cases (38%); in 22 of these we have isolated a mutation on each allele. The majority of the mutations introduce a premature termination codon. We sequenced the region between the two genes in 10 of the 20 cases in which we had not identified a mutation in either gene, revealing only one SNP that was not a common polymorphism. As we have not identified mutations in either gene in 20 cases (31%) it is possible that there is further genetic heterogeneity. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Structural framework for DNA translocation via the viral portal protein

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal-DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a 'mexican wave' of positional and conformational changes propagating sequentially along this belt.     

Is cell culture stressful? Effects of degradable and nondegradable culture surfaces on U937 cell function

In vitro cell culture has become one of the most widely used techniques in biological and health sciences research, with the most common culture supports being either tissue culture grade polystyrene (TCPS) or polydimethylsiloxane (PDMS). It has previously been shown that monocyte-derived macrophages (MDMs) respond to material surface chemistry, synthesizing and releasing degradative activities that could produce products, which alter the cell's response. In this study, functional parameters of differentiated U937 macrophage-like cells were compared when cultured on nondegradable standard control surfaces versus models of biomaterials (polycarbonate-based polyurethanes) used in the manufacture of medical devices previously shown to degrade and/or elicit pathways of inflammation. Although the influence of PDMS and TCPS on cell function is often underappreciated by investigators, both surfaces elicited enzyme markers of inflammation. Cells on TCPS had the highest intracellular and released esterase activities and protein levels. Cells on PDMS had the most released acid phosphatase activity and protein (P < 0.001), as well as de novo 57- and 59-kDa released proteins. The criteria for defining an activated cell phenotype become critically important when materials such as PDMS and TCPS are used as standard control surfaces whether in experiments for research in elucidating metabolic pathways or in screening drugs and materials for therapeutic uses.     

SHIP2 controls PtdIns(3,4,5)P(3) levels and PKB activity in response to oxidative stress

Reactive oxygen species (ROS) are known to be involved in redox signalling pathways that may contribute to normal cell function as well as disease progression. The tumour suppressor PTEN and the inositol 5-phosphatase SHIP2 are critical enzymes in the control of PtdIns(3,4,5)P(3) level. It has been reported that oxidants, including those produced in cells such as macrophages, can activate downstream signalling via the inactivation of PTEN. The present study evaluates the potential impact of SHIP2 on phosphoinositides in cells exposed to sodium peroxide. We used a model of SHIP2 deficient mouse embryonic fibroblasts (MEFs) stimulated by H(2)O(2): at 15 min, PtdIns(3,4,5)P(3) was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P(2) could be detected at 15 or 120 min incubation of the cells with H(2)O(2) (0.6 mM). PKB activity was also upregulated in SHIP2 -/- cells as compared to +/+ cells in response to H(2)O(2). SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role as a lipid phosphatase in the control of PtdIns(3,4,5)P(3) level in response to H(2)O(2). We conclude that SHIP2 lipid phosphatase activity plays an important role in the metabolism PtdIns(3,4,5)P(3) which is demonstrated in oxygen stressed cells.     

DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

Novel nucleotide triphosphates as potent P2Y2 agonists with enhanced stability over UTP

The synthesis of a series of novel C-linked nucleotide triphosphates is reported. These exhibit excellent agonist potency and selectivity for the P2Y2 receptor with a number of examples having EC50 values below 10 nM. Representative compounds from the N-linked and C-linked series showed enhanced metabolic stability compared with that of the natural ligand UTP.     

Novel 2-aminobenzothiazoles as selective neuronal nitric oxide synthase inhibitors

A series of substituted 2-aminobenzothiazole compounds have been synthesized and evaluated as nitric oxide synthase (NOS) inhibitors. Compound 14 shows activity in the nM range and is selective for the human neuronal NOS isoform. We have also evaluated the compounds against the rat NOS isoforms. For some of the compounds, there are significant differences in NOS inhibitory activities between the human and rat enzymes. For example, compound 10b has nM activity against the rat nNOS while low microM activity against the human nNOS.     

Glycoprotein structural genomics: solving the glycosylation problem

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.