Mystique is a new insulin-like growth factor-I-regulated PDZ-LIM domain protein that promotes cell attachment and migration and suppresses Anchorage-independent growth

By comparing differential gene expression in the insulin-like growth factor (IGF)-IR null cell fibroblast cell line (R- cells) with cells overexpressing the IGF-IR (R+ cells), we identified the Mystique gene expressed as alternatively spliced variants. The human homologue of Mystique is located on chromosome 8p21.2 and encodes a PDZ LIM domain protein (PDLIM2). GFP-Mystique was colocalized at cytoskeleton focal contacts with alpha-actinin and beta1-integrin. Only one isoform of endogenous human Mystique protein, Mystique 2, was detected in cell lines. Mystique 2 was more abundant in nontransformed MCF10A breast epithelial cells than in MCF-7 breast carcinoma cells and was induced by IGF-I and cell adhesion. Overexpression of Mystique 2 in MCF-7 cells suppressed colony formation in soft agarose and enhanced cell adhesion to collagen and fibronectin. Point mutation of either the PDZ or LIM domain was sufficient to reverse suppression of colony formation, but mutation of the PDZ domain alone was sufficient to abolish enhanced adhesion. Knockdown of Mystique 2 with small interfering RNA abrogated both adhesion and migration in MCF10A and MCF-7 cells. The data indicate that Mystique is an IGF-IR-regulated adapter protein located at the actin cytoskeleton that is necessary for the migratory capacity of epithelial cells.     

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.     

Population genetic approaches to neurological disease: Parkinson's disease as an example

Parkinson's disease (PD) is a common, progressive, incurable disabling condition. The cause is unknown but over the past few years tremendous progress in our understanding of the genetic bases of this condition has been made. To date, this has almost exclusively come from the study of relatively rare Mendelian forms of the disease and there are no currently, widely accepted common variants known to increase susceptibility.The role that the "Mendelian" genes play in common sporadic forms of PD is unknown. Moreover, most studies in PD can really be described as candidate polymorphism studies rather than true and complete assessments of the genes themselves. We provide a model of how one might tackle some of these issues using Parkinson's disease as an illustration. One of the emerging hypotheses of gene environment interaction in Parkinson's disease is based on drug metabolizing (or xenobiotic) enzymes and their interaction with putative environmental toxins. This motivated us to describe a tagging approach for an extensive but not exhaustive list of 55 drug metabolizing enzyme genes. We use these data to illustrate the power, and some of the limitations of a haplotype tagging approach. We show that haplotype tagging is extremely efficient and works well with only a modest increase in effort through different populations. The tagging approach works much less well if the minor allele frequency is below 5%. However, it will now be possible using these tags to evaluate these genes comprehensively in PD and other neurodegenerative conditions.     

Neurogenetic interactions and aberrant behavioral co-morbidity of attention deficit hyperactivity disorder (ADHD): dispelling myths

Background

Attention Deficit Hyperactivity Disorder, commonly referred to as ADHD, is a common, complex, predominately genetic but highly treatable disorder, which in its more severe form has such a profound effect on brain function that every aspect of the life of an affected individual may be permanently compromised. Despite the broad base of scientific investigation over the past 50 years supporting this statement, there are still many misconceptions about ADHD. These include believing the disorder does not exist, that all children have symptoms of ADHD, that if it does exist it is grossly over-diagnosed and over-treated, and that the treatment is dangerous and leads to a propensity to drug addiction. Since most misconceptions contain elements of truth, where does the reality lie?

Results

We have reviewed the literature to evaluate some of the claims and counter-claims. The evidence suggests that ADHD is primarily a polygenic disorder involving at least 50 genes, including those encoding enzymes of neurotransmitter metabolism, neurotransmitter transporters and receptors. Because of its polygenic nature, ADHD is often accompanied by other behavioral abnormalities. It is present in adults as well as children, but in itself it does not necessarily impair function in adult life; associated disorders, however, may do so. A range of treatment options is reviewed and the mechanisms responsible for the efficacy of standard drug treatments are considered.

Conclusion

The genes so far implicated in ADHD account for only part of the total picture. Identification of the remaining genes and characterization of their interactions is likely to establish ADHD firmly as a biological disorder and to lead to better methods of diagnosis and treatment.

     

Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.     

Molecular docking of potential inhibitors with the mTOR protein

The mTOR (mammalian or mechanistic Target of Rapamycin) is linked with oral cancer. Therefore, it is of interest to study the molecular docking-based binding of paclitaxel (a FDA approved drug for oral cancer) and its analogues with mTOR. Hence, we report the binding features of 10-Deacetyltaxol, 7-Epi-10-deacetyltaxol, 7-Epi-Taxol and 6alpha-Hydroxypaclitaxel with mTOR for further consideration.     

Virtual histology of transgenic mouse embryos for high-throughput phenotyping

A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X-ray computed tomography to image whole embryos, isometric resolutions of 27 μm or 8 μm were achieved with scan times of 2 h or 12 h, respectively, using mid-gestation E9.5–E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity.     

Proteomic analysis of recurrent joint inflammation in juvenile idiopathic arthritis

The synovial fluid proteome in juvenile idiopathic arthritis was investigated to isolate joint-specific biomarkers that are expressed in patients displaying recurrent joint inflammation. To identify the synovial specific proteome, matched synovial fluid and plasma samples were subjected to protein separation by 2-dimension electrophoresis (2DE). Forty-three protein spots, overexpressed in the joint, were identified. Synovial fluids from children with single-event knee joint inflammation were then compared with a group with recurrent knee disease. Nine synovial specific proteins were significantly differentially expressed in the recurrent group. Proteolytic fragments of collagen X, fibrin beta-chain, and T-cell receptor alpha-region have been identified among this protein cluster. Putative biomarkers, overexpressed in the joint and differentially expressed in children with recurrent joint inflammation, have been identified. These proteins may play a significant role determining the pathological state within the chronically inflamed joint and influence disease progression in JIA. This is the first study of the synovial proteome in children.     

Basal chromodorid sperm ultrastructure (Nudibranchia, Gastropoda, Mollusca)

The relationship between three genera considered basal in the Chromodorididae (Cadlina, Tyrinna, Cadlinella) has not yet been resolved by traditional morphological means. Here we examined the sperm ultrastructure of Tyrinna nobilis, Tyrinna evelinae, Cadlina flavomaculata and Cadlina cf. nigrobranchiata, with the expectation of finding phylogenetically informative characters. No Tyrinna or Cadlina species showed sperm similarities to Cadlinella. Both Cadlina species and Tyrinna nobilis (but not T. evelinae) exhibited coarse striations in the acrosomal pedestal. The putative fibers that occurred between the coarse striations of the pedestal are condensed into a layer in Cadlina and Tyrinna, but not in other species that also have coarse striations (Gymnodoris), and may constitute evidence for a close relationship. Tyrinna evelinae possessed fine acrosomal striations, which was shared with other Chromodorididae, Actinocyclidae and the cryptobranchs Rostanga and Aphelodoris. We also examined the sperm ultrastructure of ‘Chromodoris’ ambiguus, an animal which has shown molecular affinities to species of Cadlina, and not Chromodoris. The sperm of ‘C.’ ambiguus did not exhibit the typical Cadlina characteristics, but also showed important differences to other investigated Chromodoris species.