Unexpected Identification Research Articles

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Heparanase: Structure, Biological Functions, and Inhibition by Heparin-Derived Mimetics of Heparan Sulfate

Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothelial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (L-iduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis, angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single functional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.

A Genetic Screen for Maternal-Effect Suppressors of decapentaplegic Identifies the eukaryotic translation initiation factor 4A in Drosophila

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, we conducted a genetic screen for maternal-effect suppressors of dpp haplo-insufficiency. We screened approximately 7000 EMS-mutagenized genomes and isolated and mapped seven independent dominant suppressors of dpp, Su(dpp), which were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp(H46), a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. Here we report the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). We show that Su(dpp)(YE9) maps to eIF4A and that this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling.

Unexpected identification of two interstitial deletions in a patient with a pericentric inversion of a chromosome 4 and an abnormal phenotype

Interstitial deletions and pericentric inversions of chromosome 4 appear to be unusual phenomena. Here, we report the case of a 14-year-old boy with severe psychomotor retardation with a de novo 46,XY,der(4)del(p15.2p15.31)inv(4)(p15.2q13.3)del(4)(q13.2q13.2) karyotype. We used FISH analysis with YAC and BAC clones to characterise the inversion's breakpoints. A complex event with six breakpoints was found, characterised by a pericentric inversion and two deletions, the first on the short arm of chromosome 4 (4p) and the second on the long arm of chromosome 4 (4q). The deletion events had removed two segments, one of approximately 5 Mb, from 4p, outside the inversion, and the other 2 Mb from 4q, inside the inversion. These rearrangements were not found in the parents. Microsatellite marker analysis showed that the inversion carrying chromosome 4 was derived from the father. Bioinformatic analysis of the human genome sequence allowed us to identify several hemizygotic genes in the patient, which might be involved in the pathogenesis of this clinical phenotype.

Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism.

The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin-AMP), a mechanistically important intermediate. Molybdopterin-AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.

Death takes a holiday

At times, the DIAP-1 anti-apoptotic protein is not what it seems. Erika Geisbrecht and Denise Montell (Johns Hopkins School of Medicine, Baltimore, MD) show that, in the fly ovary, this caspase inhibitor is needed for cell migration but not survival. Figure Border cells (arrow) migrate to the wrong spot in DIAP-1 mutants. Montell's group studies border cells in the fly egg chamber, which migrate to help establish the sperm entry site. Rac activity is needed for this; so to identify more players, the group screened for proteins that restore migration when Rac activity is reduced. They found DIAP-1. Although DIAP-1 is essential for cell survival in the embryo, its loss in the ovary caused migration defects but not cell death. Extra actin or profilin, presumably leading to increased actin polymerization, also compensated for the loss of Rac. DIAP-1 may also promote actin polymerization. To block apoptosis, DIAP-1 inhibits the fly caspase-9 homologue, called Dronc. Dronc is probably the DIAP-1 target in border cell migration as well, as reducing its activity suppressed the loss of Rac. Possible Dronc substrates include Rac and actin, which are cleaved by caspases during apoptosis, or profilin. The unexpected identification of DIAP-1 as a suppressor emphasizes the importance of random screens. “The whole point is to find something that you can't even imagine would be involved,” says Montell. Tumor cells that manage to increase their levels of DIAP-1–like proteins could gain both a survival advantage and the ability to migrate. ▪ Reference: Geisbrecht, E., and D. Montell. 2004. Cell. 118:111–125. [PubMed]

Urinary tract involvement by colorectal cancer.

Involvement of the urinary tract by colorectal cancer is sufficiently rare to be encountered by an individual surgeon on an infrequent basis. The aim of this review is to highlight technical and oncologic issues that should be considered when dealing with complex colorectal cancer that involves the urinary tract. The relevant literature from 1975 to 2001 was identified using the MEDLINE database of the U.S. National Library of Medicine and reviewed. Because of the diversity of forms of presentation of urologic involvement, few randomized, controlled trials are available, with most evidence derived from retrospective studies. Three distinct clinical situations in which the urinary tract may be affected by colorectal cancer were identified: involvement by primary colorectal cancer, involvement by recurrent cancer, and unexpected intraoperative findings of urinary tract involvement. Management strategies to identify and treat locally advanced primary or recurrent colorectal cancer involving the urinary tract improve survival with acceptable morbidity and mortality. Careful preoperative assessment of all patients with colorectal cancer will reduce unexpected identification of urinary tract invasion at the time of surgery. In patients in whom cure is not possible, endourologic techniques combined with judicious surgical resection can provide high-quality palliation. Optimal care of many of these conditions is facilitated by specialist urologic advice. The wide spectrum of possible urinary tract involvement by colorectal cancer requires individual patient-specific and disease-specific consideration. The literature offers important guidelines that aid decision making and improve management of these challenging problems.

Alpha-synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma.

Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of alpha-synuclein protein (alpha-syn) inside brain cells. It is likely that the formation of alpha-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding alpha-syn have been found in inherited forms of PD. alpha-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of alpha-syn in conditioned culture media from untransfected and alpha-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-alpha-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, alpha-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to alpha-syn. Our findings suggest that cells normally secrete alpha-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular alpha-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.

GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity.

The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca(2+) concentration ([Ca(2+)](i)). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane-anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca(2+)](i) in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca(2+)](i) elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-delta(1) isoform, and a concomitant increase in inositol-1,4,5-trisphosphate (IP(3)) formation. Collectively, these findings indicate that hypo-osmotic activation of GAP43 induces Ca(2+) release from IP(3)-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho inositide metabolism, spontaneous triggering of cytosolic Ca(2+) transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.

P1232 Unexpected identification of two new KCNQ1 mutations in the general population

P1232 Unexpected identification of two new KCNQ1 mutations in the general population

859 Unexpected identification of the B lymphocyte transcription factor PAX5 in mouse immortalized biliary epithelial cells (IBECS) stimulated with lipopolysaccharide or proinflammatory cytokines

Biliary epithelial cells (BECs) of chronic non-suppurative destructive cholangitis (CNSDC) in primary biliary cirrhosis (PBC) reportedly express p21WAF1/Cip1 and p16INK4a, which may induce cell cycle arrest and are related to progressive loss of BECs of PBC. Given that the ATM pathway plays a role in the induction of p21WAF1/Cip1, we examined its possible involvement in bile duct damage of PBC. The expression of phosphorylated-ATM (p-ATM) reflecting the activation of ATM, p21WAF1/Cip1 and 8-hydroxy-deoxyguanosine (8-OHdG), an oxidative stress marker, was examined immunohistochemically in the liver tissues of 20 cases of stage 1/2 PBC, 9 extrahepatic biliary obstruction (EBO), 35 chronic viral hepatitis (CVH), 17 nonalcoholic steatohepatitis (NASH), and 18 histologically normal liver. p21WAF1/Cip1, p-ATM and 8-OHdG were frequently and extensively co-expressed in the nuclei of CNSDC in PBC, and their expressions were correlated. In contrast, the expression of these three molecules was absent or faint in small bile ducts in normal livers, CVH, and EBO, and these molecules were clearly expressed in the nuclei of hepatocytes of NASH, in which oxidative stress is involved in hepatocellular damage. In conclusion, oxidative stress-induced p21WAF1/Cip1 expression in BECs in PBC is closely associated with activation of the ATM pathway and the resultant reduced regeneration or cell cycle arrest of BECs may be related to the progressive loss of small bile ducts of PBC.

Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development

Cleavage of heparan sulphate proteoglycans (HSPGs) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Heparanase, degrading heparan sulphate (HS) at specific intrachain sites, is synthesized as a latent ∼65 kDa protein that is processed at the N-terminus into a highly active ∼50 kDa form. The heparanase enzyme is preferentially expressed in human tumours and its overexpression in low-metastatic tumour cells confers a highly invasive phenotype in experimental animals. Heparanase also releases angiogenic factors and accessory fragments of HS from the tumour microenvironment and induces an angiogenic response in vivo. Heparanase may thus facilitate tumour cell invasion, vascularization and survival in a given microenvironment, all critical events in cancer progression. These observations, the anticancerous effect of heparanase-inhibiting molecules, and the unexpected identification of a single predominant functional heparanase suggest that the enzyme is a promising target for drug development.

Two Tunes: Jean Rhys' Voyage in the Dark

Anna Morgan, the first person narrator of Jean Rhys' Voyage in the Dark (I934)1 has often been described either a weak, irresponsible and parasitic woman who consequently slides down the social scale one of the dispossessed urban spinsters2, as a member of the group of single women, without family ties ... [who], albeit reluctantly, find their own jobs or, finally, a character that is mainly based on Jean Rhys' own life. Any of these definitions taken exclusively would fall far short of her complex personality. In order to fully understand her motivation and behaviour, one has to take into account that her upbringing a coloniser in a colonial environment, her unexpected identification with the colonised, her gender and the way she is treated in consequence of the above all contribute to an image that is much the result of an idiosyncratic inclination of the way she is treated by her social environment. Having been brought up in the Caribbean the daughter of a white former slave-owner and a Creole mother positions Anna between the two cultures from the very start. Her taking sides through sympathy for and identification with the Black population does not make her childhood and youth an easy one with clearly defined role-models, because her family's living conditions together with her white skin colour set her off and betray her slave-owner's history.

From the ER to the Golgi: Insights from the Study of Combined Factors V and VIII Deficiency

The unexpected identification of ERGIC-53 as the gene responsible for combined FV/FVIII deficiency clearly establishes ERGIC-53 as a component of the ER-Golgi transport machinery that is required for the efficient export of coagulation factors V and VIII. This is the first demonstration of a cargo-specific pathway for protein export from the ER in mammalian cells. However, the apparently normal levels observed in these patients for most other plasma proteins demonstrate that ERGIC-53 is not essential for the integrity of the intermediate compartment or the more general process of protein export. The identification of the genetic defect in the subset of patients with FV/VIII deficiency but with intact ERGIC-53 may unmask additional critical components of this unique transport pathway.

Human H-kininogen Is a Ferritin-binding Protein

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.

AA recovery and spirituality: An addiction medicine perspective

KHANTZIAN AND MACK have done their usual fine job with their review on how Alcoholics Anonymous (AA) works. This paper makes an important contribution to the literature in helping clinicians understand why the 12-step recovery process has succeeded with the desire of addiction where many traditional medical approaches have failed. There are important omissions in their paper. However, all these omissions may have been motivated by their desire to make the AA response palpable to the clinicians, as I know both of these fine scholars are familiar with the AA literature. Their section on spirituality misses both the essence and the foundation of spirituality in AA as articulated by Carl Jung in his historic interaction with the cofounder of AA, Bill Wilson, in which Jung said: “The compulsion to use alcohol is so great that it is likely that only a spiritual experience could overwhelm that compulsion” (Alcoholics Anonymous, 1985). The “Big Book” of Alcoholics Anonymous (1976) uses the terms spiritual experience and spiritual awakening, manifesting itself in many ways using the 12 steps as a guide to describe what happens to bring about a personality change sufficient to induce recovery. Although some of these experiences may be immediate and overwhelmingly associated with the experience of hitting bottom and taking the first step, admitting they were powerless over alcohol and other drugs, most are of the educational variety of revelation developing slowly over time. The core of this process is the tapping of an unexpected inner resource and identification of this resource as “their own concep

Amyloid β-peptide is produced by cultured cells during normal metabolism

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

The structure of the murine Fc receptor for IgG. Assignment of intrachain disulfide bonds, identification of N-linked glycosylation sites, and evidence for a fourth form of Fc receptor.

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.