Autosomal dominant hereditary spastic paraplegia: Novel mutations in the REEP1 gene (SPG31)

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>SPG4 </it>gene (spastin) and in the <it>SPG3A </it>gene (atlastin) account for the majority of 'pure' autosomal dominant form of hereditary spastic paraplegia (HSP). Recently, mutations in the <it>REEP1 </it>gene were identified to cause autosomal dominant HSP type SPG31. The purpose of this study was to determine the prevalence of <it>REEP1 </it>mutations in a cohort of 162 unrelated Caucasian index patients with 'pure' HSP and a positive family history (at least two persons per family presented symptoms).</p> <p>Methods</p> <p>162 patients were screened for mutations by, both, DHPLC and direct sequencing.</p> <p>Results</p> <p>Ten mutations were identified in the <it>REEP1 </it>gene, these included eight novel mutations comprising small insertions/deletions causing frame shifts and subsequently premature stop codons, one nonsense mutation and one splice site mutation as well as two missense mutations. Both missense mutations and the splice site mutation were not identified in 170 control subjects.</p> <p>Conclusion</p> <p>In our HSP cohort we found pathogenic mutations in 4.3% of cases with autosomal dominant inheritance. Our results confirm the previously observed mutation range of 3% to 6.5%, respectively, and they widen the spectrum of <it>REEP1 </it>mutations.</p

Progress in Three-Dimensional Coherent X-Ray Diffraction Imaging

The Fourier inversion of phased coherent diffraction patterns offers images without the resolution and depth-of-focus limitations of lens-based tomographic systems. We report on our recent experimental images inverted using recent developments in phase retrieval algorithms, and summarize efforts that led to these accomplishments. These include ab-initio reconstruction of a two-dimensional test pattern, infinite depth of focus image of a thick object, and its high-resolution (~10 nm resolution) three-dimensional image. Developments on the structural imaging of low density aerogel samples are discussed.Comment: 5 pages, X-Ray Microscopy 2005, Himeji, Japa

MED12 regulates a transcriptional network of calcium-handling genes in the heart

The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart

An assessment of the resolution limitation due to radiation-damage in x-ray diffraction microscopy

X-ray diffraction microscopy (XDM) is a new form of x-ray imaging that is being practiced at several third-generation synchrotron-radiation x-ray facilities. Although only five years have elapsed since the technique was first introduced, it has made rapid progress in demonstrating high-resolution threedimensional imaging and promises few-nm resolution with much larger samples than can be imaged in the transmission electron microscope. Both life- and materials-science applications of XDM are intended, and it is expected that the principal limitation to resolution will be radiation damage for life science and the coherent power of available x-ray sources for material science. In this paper we address the question of the role of radiation damage. We use a statistical analysis based on the so-called "dose fractionation theorem" of Hegerl and Hoppe to calculate the dose needed to make an image of a lifescience sample by XDM with a given resolution. We conclude that the needed dose scales with the inverse fourth power of the resolution and present experimental evidence to support this finding. To determine the maximum tolerable dose we have assembled a number of data taken from the literature plus some measurements of our own which cover ranges of resolution that are not well covered by reports in the literature. The tentative conclusion of this study is that XDM should be able to image frozen-hydrated protein samples at a resolution of about 10 nm with "Rose-criterion" image quality.Comment: 9 pages, 4 figure

GATA-3 in Human T Cell Helper Type 2 Development

The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell–mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/− individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell–mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo

T-cell subpopulations αβ and γδ in cord blood of very preterm infants : The influence of intrauterine infection

Open Access: This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPreterm infants are very susceptible to infections. Immune response mechanisms in this group of patients and factors that influence cord blood mononuclear cell populations remain poorly understood and are considered insufficient. However, competent immune functions of the cord blood mononuclear cells are also described. The aim of this work was to evaluate the T-cell population (CD3+) with its subpopulations bearing T-cell receptor (TCR) αβ or TCR γδ in the cord blood of preterm infants born before 32 weeks of gestation by mothers with or without an intrauterine infection. Being a pilot study, it also aimed at feasibility check and assessment of an expected effect size. The cord blood samples of 46 infants age were subjected to direct immunofluorescent staining with monoclonal antibodies and then analyzed by flow cytometry. The percentage of CD3+ cells in neonates born by mothers with diagnosis of intrauterine infection was significantly lower than in neonates born by mothers without infection (p = 0.005; Mann-Whitney U test). The number of cells did not differ between groups. Infection present in the mother did not have an influence on the TCR αβ or TCR γδ subpopulations. Our study contributes to a better understanding of preterm infants' immune mechanisms, and sets the stage for further investigations.Peer reviewedFinal Published versio

Retargeted adenoviruses for radiation-guided gene delivery

The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment

A Salmonid EST Genomic Study: Genes, Duplications, Phylogeny and Microarrays

Background: Salmonids are of interest because of their relatively recent genome duplication, and their extensive usein wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different speciesprovide valuable genomic information for one of the most widely studied groups of fish.Results: 298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in thisstudy and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmonclones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, genepredictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbowtrout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4,92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchusand Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae.Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of theavailable EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-specieshybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species.Conclusion: An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon,Atlantic salmon and charr are 94–96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent withan ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, providevaluable new tools to study salmonids