Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background  

Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.     

Mapping Plant Interactomes Using Literature Curated and Predicted Protein–Protein Interaction Data Sets

Most cellular processes are enabled by cohorts of interacting proteins that form dynamic networks within the plant proteome. The study of these networks can provide insight into protein function and provide new avenues for research. This article informs the plant science community of the currently available sources of protein interaction data and discusses how they can be useful to researchers. Using our recently curated IntAct Arabidopsis thaliana protein–protein interaction data set as an example, we discuss potentials and limitations of the plant interactomes generated to date. In addition, we present our efforts to add value to the interaction data by using them to seed a proteome-wide map of predicted protein subcellular locations.For well over two decades, plant scientists have studied protein interactions within plants using many different and evolving approaches. Their findings are represented by a large and growing corpus of peer-reviewed literature reflecting the increasing activity in this area of plant proteomic research. More recently, a number of predicted interactomes have been reported in plants and, while these predictions remain largely untested, they could act as a useful guide for future research. These studies have allowed researchers to better understand the function of protein complexes and to refine our understanding of protein function within the cell (Uhrig, 2006; Morsy et al., 2008). The extraction of protein interaction data from the literature and its standardized deposition and representation within publicly available databases remains a challenging task. Aggregating the data in databases allows researchers to leverage visualization, data mining, and integrative approaches to produce new insights that would be unachievable when the data are dispersed within largely inaccessible formats (Rodriguez et al., 2009).Currently, there are three databases that act as repositories of plant protein interaction data. These are IntAct (http://www.ebi.ac.uk/intact/; Aranda et al., 2010), The Arabidopsis Information Resource (TAIR; http://www.Arabidopsis.org/; Poole, 2007), and BioGRID (http://www.thebiogrid.org/; Breitkreutz et al., 2008). These databases curate experimentally established interactions available from the peer-reviewed literature (as opposed to predicted interactions, which will be discussed below). Each repository takes its own approach to the capture, storage, and representation of protein interaction data. TAIR focuses on Arabidopsis thaliana protein–protein interaction data exclusively; BioGRID currently focuses on the plant species Arabidopsis and rice (Oryza sativa), while IntAct attempts to capture protein interaction data from any plant species. Unlike the other repositories, IntAct follows a deep curation strategy that captures detailed experimental and biophysical details, such as binding regions and subcellular locations of interactions using controlled vocabularies (Aranda et al., 2010). While the majority of plant interaction data held by IntAct concern protein–protein interaction data in Arabidopsis, there is a small but growing content of interaction data relating to protein–DNA, protein–RNA, and protein–small molecule interactions, as well as interaction data from other plant species.Using the IntAct Arabidopsis data set as an example, we outline how the accumulating knowledge captured in these repositories can be used to further our understanding of the plant proteome. We compare the characteristics of predicted interactomes with the IntAct protein–protein interaction data set, which consists entirely of experimentally measured protein interactions, to gauge the predictive accuracy of these studies. Finally, we show how the IntAct data set can be used together with a recently developed Divide and Conquer k-Nearest Neighbors Method (DC-kNN; K. Lee et al., 2008) to predict the subcellular locations for most Arabidopsis proteins. This data set predicts high confidence subcellular locations for many unannotated Arabidopsis proteins and should act as a useful resource for future studies of protein function. Although this article focuses on the IntAct Arabidopsis protein–protein interaction data set, readers are also encouraged to explore the resources offered by our colleagues at TAIR and BioGRID.Each database employs its own system to report molecular interactions, as represented in the referenced source publications, and each avoids making judgments on interaction reliability or whether two participants in a complex have a direct interaction. Thus, the user should carefully filter these data sets for their specific purpose based on the full annotation of the data sets. In particular, the user should consider the experimental methods and independent observation of the same interaction in different publications when assessing the reliability and type of interaction of the proteins (e.g., direct or indirect). Confidence scoring schemes for interaction data are discussed widely in the literature (Yu and Finley, 2009).     

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T^mCG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.     

Human dendritic cells transduced with herpes simplex virus amplicons encoding human immunodeficiency virus type 1 (HIV-1) gp120 elicit adaptive immune responses from human cells engrafted into NOD/SCID mice and confer partial protection against HIV-1 challenge

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.     

On averaging power for genetic association and linkage studies

A power calculation is crucial in planning genetic studies. In genetic association studies, the power is often calculated using the expected number of individuals with each genotype calculated from an assumed allele frequency under Hardy-Weinberg equilibrium. Since the allele frequency is often unknown, the number of individuals with each genotype is random and so a power calculation assuming a known allele frequency may be incorrect. Ambrosius et al. recently showed that the power ignoring this randomness may lead to studies with insufficient power and proposed averaging the power due to the randomness. We extend the method of averaging power in two directions. First, for testing association in case-control studies, we use the Cochran-Armitage trend test and find that the time needed for calculating the averaged power is much reduced compared to the chi-square test with two degrees of freedom studied by Ambrosius et al. A real study is used for illustration of the method. Second, we extend the method to linkage analysis, where the number of identical-by-descent alleles shared by siblings is random. The distribution of identical-by-descent numbers depends on the underlying genetic model rather than the allele frequency. The robust test for linkage analysis is also examined using the averaged powers. We also recommend a sensitivity analysis when the true allele frequency or the number of identical-by-descent alleles is unknown.     

Neuroregulatory events follow adaptive immune-mediated elimination of HIV-1-infected macrophages: studies in a murine model of viral encephalitis

HIV-1-specific cellular immunity serves to eliminate infected cells and disease. However, how this process specifically affects the CNS is poorly understood. To mirror the regulatory events that occur in human brain after HIV-1 infection, a murine model of viral encephalitis was used to study relationships, over time, among lymphocyte-mediated infected cell elimination, innate immune responses, and neuropathology. Nonobese diabetic SCID mice were reconstituted with human PBL and a focal encephalitis induced by intracranial injection of autologous HIV-1-infected, monocyte-derived macrophages (MDM). On days 7, 14, and 21 after MDM injection into the basal ganglia, the numbers of human lymphocytes and mouse monocytes, virus-infected MDM, glial (astrocyte and microglial) responses, cytokines, inducible NO (iNOS), neurotrophic factors, and neuronal Ags were determined in brain by immunohistochemistry, real-time PCR, and Western blot assays. Microglia activation, astrocytosis, proinflammatory cytokines, and iNOS expression accompanied the loss of neuronal Ags. This followed entry of human lymphocytes and mouse monocytes into the brain on days 7 and 14. Elimination of virus-infected human MDM, expression of IL-10, neurotropins, and a down-regulation of iNOS coincided with brain tissue restoration. Our results demonstrate that the degree of tissue damage and repair parallels the presence of infected macrophages and effectors of innate and adaptive immunity. This murine model of HIV-1 encephalitis can be useful in elucidating the role played by innate and adaptive immunity in disease progression and resolution.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.