Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.

Genomic organization of the human e1af gene, a member of Ets transcription factors

The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19 kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7 kb. The 5'-untranslated region is about 0.3 kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF. (C) 1999 Elsevier Science B.V. All rights reserved.

A scalable strategy for the parallelization of multiphysics unstructured mesh-iterative codes on distributed-memory systems

Realizing scalable performance on high performance computing systems is not straightforward for single-phenomenon codes (such as computational fluid dynamics [CFD]). This task is magnified considerably when the target software involves the interactions of a range of phenomena that have distinctive solution procedures involving different discretization methods. The problems of addressing the key issues of retaining data integrity and the ordering of the calculation procedures are significant. A strategy for parallelizing this multiphysics family of codes is described for software exploiting finite-volume discretization methods on unstructured meshes using iterative solution procedures. A mesh partitioning-based SPMD approach is used. However, since different variables use distinct discretization schemes, this means that distinct partitions are required; techniques for addressing this issue are described using the mesh-partitioning tool, JOSTLE. In this contribution, the strategy is tested for a variety of test cases under a wide range of conditions (e.g., problem size, number of processors, asynchronous / synchronous communications, etc.) using a variety of strategies for mapping the mesh partition onto the processor topology.

Computational modeling of mold filling and related free-surface flows in shape casting: an overview of the challenges involved

Accurate representation of the coupled effects between turbulent fluid flow with a free surface, heat transfer, solidification, and mold deformation has been shown to be necessary for the realistic prediction of several defects in castings and also for determining the final crystalline structure. A core component of the computational modeling of casting processes involves mold filling, which is the most computationally intensive aspect of casting simulation at the continuum level. Considering the complex geometries involved in shape casting, the evolution of the free surface, gas entrapment, and the entrainment of oxide layers into the casting make this a very challenging task in every respect. Despite well over 30 years of effort in developing algorithms, this is by no means a closed subject. In this article, we will review the full range of computational methods used, from unstructured finite-element (FE) and finite-volume (FV) methods through fully structured and block-structured approaches utilizing the cut-cell family of techniques to capture the geometric complexity inherent in shape casting. This discussion will include the challenges of generating rapid solutions on high-performance parallel cluster technology and how mold filling links in with the full spectrum of physics involved in shape casting. Finally, some indications as to novel techniques emerging now that can address genuinely arbitrarily complex geometries are briefly outlined and their advantages and disadvantages are discussed.

HPLC-ESI-MS method development for the elucidation of nicardipine degradation products

Purpose: Nicardipine is a member of a family of calcium channel blockers named dihydropiridines that are known to be photolabile and may cause phototoxicity. It is therefore vital to develop analytical method which can study the photodegradation of nicardipine. Method: Forced acid degradation of nicardipine was conducted by heating 12 ml of 1 mg/ml nicardipine with 3 ml of 2.5 M HCl for two hours. A gradient HPLC medthod was developed using Agilent Technologies 1200 series quaternary system. Separation was achieved with a Hichrome (250 x 4.6 mm) 5 μm C18 reversed phase column and mobile phase composition of 70% A(100%v/v water) and 30% B(99%v/v acetonitrile + 1%v/v formic acid) at time zero, composition of A and B was then charged to 60%v/v A;40%v/v B at 10minutes, 50%v/v A; 50%v/v B at 30minutes and 70%v/v A; 30%v/v B at 35minutes. 20μl of 0.8mg/ml of nicardipine degradation was injected at room temperature (25oC). The gradient method was transferred onto a HPLC-ESI-MS system (HP 1050 series - AQUAMAX mass detector) and analysis conducted with an acid degradation concentration of 0.25mg/ml and 20μl injection volume. ESI spectra were acquired in positive ionisation mode with MRM 0-600 m/z. Results: Eleven nicardipine degradation products were detected in the HPLC analysis and the resolution (RS) between the respective degradants where 1.0, 1.2, 6.0, 0.4, 1.7, 3.7, 1.8, 1.0, and 1.7 respectively. Nine degradation products were identified in the ESI spectra with the respective m/z ratio; 171.0, 166.1, 441.2, 423.2, 455.2, 455.2, 331.1, 273.1, and 290.1. The possible molecular formulae for each degradants were ambiguously determined. Conclusion: A sensitive and specific method was developed for the analysis of nicardipine degradants. Method enables detection and quantification of nicardipine degradation products that can be used for the study of the kinetics of nicardipine degradation processes.

Spectroscopic studies of oligomers containing 2,5-trans furanoid sugar amino acids

Sugar amino acids and their oligomers, known as carbopeptoids, are commonly studied as foldamers. However, study of their conformational preference is often challenging when the adopted conformations are extended and/or disordered. This study is the first to explore the disordered nature of such carbopeptoids by utilizing a family of 2,5-trans carbopeptoids. An array of spectroscopic techniques has been used to investigate the conformational preference of these carbopeptoids. However, using this data alone it has not been possible to assign conformational preference as an ordered extended conformation or as a disordered family of closely related conformations. Computational methods need to be employed to achieve reliable interpretation of the spectroscopic data. Chirality, 2008. © 2008 Wiley-Liss, Inc.

An alpha 2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha sub(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha sub(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.

The type I antifreeze protein gene family in Pleuronectidae

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis.

Metastasis-inducing DNA Regulates the Expression of the Osteopontin Gene by Binding the Transcription Factor Tcf-4

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

The 2,5 Oligoadenylate Synthetase/RNaseL pathway is a novel effector of BRCA1 - and interferon-gamma-mediated apoptosis

I discovered that 2,5OAS family of proteins was transcriptionally upregulated by BRCA1 and interferon gamma in a synergistic manner. This correlated with synergistically induced apoptosis and both the induction of 2,5OAS and the accompanying apoptosis could be inhibited by 2,5OAS specific siRNA proving 2,5OAS was the apoptotic effector.

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

Properties of SEPT9 Isoforms and the requirement for GTP binding

Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions. Alterations in the levels of Sept9 protein by overexpression of individual isoforms can clearly perturb cellular behaviour and may thus provide a mechanistic explanation for observations of deranged septin expression in neoplasia. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pelophylaxins: novel antimicrobial peptide homologs from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: identification by “shotgun” cDNA cloning and sequence analysis.

Amphibian skin secretions are rich in antimicrobial peptides that act as important components of an innate immune system. Here, we describe a novel “shotgun” skin peptide precursor cloning technique that facilitates rapid access to these genetically encoded molecules and effects their subsequent identification and structural characterization from the secretory peptidome. Adopting this approach on a skin secretion-derived library from a hitherto unstudied Chinese species of frog, we identified a family of novel antimicrobial peptide homologs, named pelophylaxins, that belong to previously identified families (ranatuerins, brevinins and temporins) found predominantly in the skin secretions from frogs of the genus Rana. These data further substantiate the scientifically robust nature of applying parallel transcriptome and peptidome analyses on frog defensive skin secretions that can be obtained in a non-invasive, non-destructive manner. In addition, the present data illustrate that rapid structural characterization of frog skin secretion peptides can be achieved from an unstudied species without prior knowledge of primary structures of endogenous peptides.

The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.