Ram S. Puranam

Cloning and expression of a novel Na+-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters

This work was supported by Community of Madrid (Grupo Estrategico 2000-2003), NIH, grant R01CA77575, and SAF 2001-2245.The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.Supported by Grant GM-08041 from the National Institutes of Health, United States Public Health Service.The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.Aided by grants from the National Institutes of Health U.S. Public Health Service, and E. I. Du Pont de Neumours and Company, Inc.This work was supported in part by NRSA, National Institutes of Health Grants NS09463 and NS32501 and from National Science Foundation Grant 9310965.We have recently developed a new method to detect and characterize single base substitutions in transcribed genes which is based on the ability of RNAse A to recognize and cleave single base mismatches in RNA:RNA heteroduplexes. The RNAse A misrnatch cleavage assay was applied to screen human colon carcinoma cell lines and primary tumors for the presence of mutant e-X-ras oncogenes. We have determined that the mutant e-X-ras allele is overexpressed and amplified relative to the normal in the SX-CO-l human colon carcinoma cell lineo The oncogene mutation has been characterized by this method as a glycine to valine substitution at codon 12 of the e-X-ras gene. This result was confirmed by cloning and sequencing. We have previously reported that about 40% of primary human colon tumors contain e-X-ras genes mutant at codon 12 (Forrester et al, Nature 327: 298, 1987). We report here the characterization by molecular cloning and sequencing of the mutation in the e-X-ras oneogene from two of these tumors (tumors 3 and 28). We also describe the histopathologieal eharaeterization of these two tumors and demonstrate, by Southern blot hybridization of NIH3T3 transformants, the simultaneous presenee of mutant e-X-ras and N-ras oncogenes in villous adenoma 28. Our results provide evidence for the frequent assoeiation of ras somatie mutational aetivation in the early stages of tumor development in this common type of human eaneer.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 from the National Institutes o f Health, United States Public Health Service, and E. I. Du Pont de Nemours and Company, Inc. A preliminary report o f this work was presented at the Second Meeting o f the Federation o f European Biochemical Societies (symposium on “Ribonucleic Acid-Structure and Function”), Vienna, April 21 to 24, 1965.1 pagina.-- Trabajo presentado al: 4th International Meeting on Apicomplexa in Farm Animals. (Madrid, Spain. 11-14 October ,2017).Supported by Grant GM-08041 from the National Institutes of Health, United States Public Health Service.Resumen del trabajo presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.Poster presentado al Annual Biomedical Research Conference for Minority Students celebrado en California (US) del 7 al 10 de noviembre de 2012.The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.Resumen del poster presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production and retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these actions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H(2)O(2) production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups. However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR-induced decreases in ROS production.To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozymez substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min 21 ) were typically 15-fold greater than cleavage rates (0.2 min 21 ), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on 5 4 3 10 8 M 21 min 21 ), and the ribozymez substrate complex is very stable (K D < 25 pM ;k off < 0.01 min 21 ). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.Trabajo presentado al Annual Biomedical Research Conference for Minority Students celebrada en Nashville (US) del 13 al 16 de noviembre de 2013.A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATTs apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

Fibroblast Growth Factor Homologous Factor 13 Regulates Na+ Channels and Conduction Velocity in Murine Hearts

Rationale: Fibroblast growth factor homologous factors (FHFs), a subfamily of fibroblast growth factors (FGFs) that are incapable of functioning as growth factors, are intracellular modulators of Na+ channels and have been linked to neurodegenerative diseases. Although certain FHFs have been found in embryonic heart, they have not been reported in adult heart, and they have not been shown to regulate endogenous cardiac Na+ channels or to participate in cardiac pathophysiology. Objective: We tested whether FHFs regulate Na+ channels in murine heart. Methods and Results: We demonstrated that isoforms of FGF13 are the predominant FHFs in adult mouse ventricular myocytes. FGF13 binds directly to, and colocalizes with, the NaV1.5 Na+ channel in the sarcolemma of adult mouse ventricular myocytes. Knockdown of FGF13 in adult mouse ventricular myocytes revealed a loss of function of NaV1.5-reduced Na+ current density, decreased Na+ channel availability, and slowed NaV1.5-reduced Na+ current recovery from inactivation. Cell surface biotinylation experiments showed ≈45% reduction in NaV1.5 protein at the sarcolemma after FGF13 knockdown, whereas no changes in whole-cell NaV1.5 protein or in mRNA level were observed. Optical imaging in neonatal rat ventricular myocyte monolayers demonstrated slowed conduction velocity and a reduced maximum capture rate after FGF13 knockdown. Conclusion: These findings show that FHFs are potent regulators of Na+ channels in adult ventricular myocytes and suggest that loss-of-function mutations in FHFs may underlie a similar set of cardiac arrhythmias and cardiomyopathies that result from NaV1.5 loss-of-function mutations.

Autoimmunity to munc-18 in Rasmussen's encephalitis.

Rasmussens encephalitis (RE) is a rare disease of the central nervous system characterized by severe epileptic seizures, progressive degeneration of a single cerebral hemisphere, and autoimmunity directed against glutamate receptor subunit, GluR3. We report here the identification of high-titer autoantibodies directed against munc-18 in the serum of a single patient with RE previously shown to have anti-GluR3 antibodies. Munc-18 is an intracellular protein residing in presynaptic terminals, which is required for secretion of neurotransmitters. These findings are consistent with the possibility of intermolecular epitope spreading between GluR3, a postsynaptic cell surface protein, and munc-18, a presynaptic intracellular protein. Immune attack on these two proteins, which participate at distinct steps of synaptic transmission, could act in an additive or synergistic manner to impair synaptic function and lead to seizures and neuronal death.

Seizure disorders in mutant mice: Relevance to human epilepsies

The rate at which mutant genes producing an epileptic phenotype in mice have been identified over the past few years has been astounding. Manipulating the genome of mice has led to identification of a diversity of genes whose absence or modification either causes epileptic seizures or, conversely, limits epileptogenesis. In addition, positional cloning of genes in which spontaneously arising mutations cause epilepsy in mice has led to the identification of genes encoding voltage- and ligand-gated ion channels. Finally, engineering a mutation that mimics a rare form of human epilepsy has led to a mouse line with a phenotype similar to that of the human disease. Taken together, these discoveries promise to shed light on the mechanisms underlying genetic control of neuronal excitability, suggest candidate genes underlying genetic forms of human epilepsy, and provide a valuable model with which to elucidate how the genotype produces the phenotype of a rare form of human epilepsy.

Disruption of Fgf13 Causes Synaptic Excitatory–Inhibitory Imbalance and Genetic Epilepsy and Febrile Seizures Plus

We identified a family in which a translocation between chromosomes X and 14 was associated with cognitive impairment and a complex genetic disorder termed “Genetic Epilepsy and Febrile Seizures Plus” (GEFS+). We demonstrate that the breakpoint on the X chromosome disrupted a gene that encodes an auxiliary protein of voltage-gated Na+ channels, fibroblast growth factor 13 (Fgf13). Female mice in which one Fgf13 allele was deleted exhibited hyperthermia-induced seizures and epilepsy. Anatomic studies revealed expression of Fgf13 mRNA in both excitatory and inhibitory neurons of hippocampus. Electrophysiological recordings revealed decreased inhibitory and increased excitatory synaptic inputs in hippocampal neurons of Fgf13 mutants. We speculate that reduced expression of Fgf13 impairs excitability of inhibitory interneurons, resulting in enhanced excitability within local circuits of hippocampus and the clinical phenotype of epilepsy. These findings reveal a novel cause of this syndrome and underscore the powerful role of FGF13 in control of neuronal excitability.

A locus for generalized tonic-clonic seizure susceptibility maps to chromosome 10q25-q26

Inheritance patterns in twins and multiplex families led us to hypothesize that two loci were segregating in subjects with juvenile myoclonic epilepsy (JME), one predisposing to generalized tonic‐clonic seizures (GTCS) and a second to myoclonic seizures. We tested this hypothesis by performing genome‐wide scan of a large family (Family 01) and used the results to guide analyses of additional families. A locus was identified in Family 01 that was linked to GTCS (10q25‐q26). Model‐based multipoint analysis of the 10q25‐q26 locus showed a logarithm of odds (LOD) score of 2.85; similar results were obtained with model‐free analyses (maximum nonparametric linkage [NPL] of 2.71; p = 0.0019). Analyses of the 10q25‐q26 locus in 10 additional families assuming heterogeneity revealed evidence for linkage in four families; model‐based and model‐free analyses showed a heterogeneity LOD (HLOD) of 2.01 (α = 0.41) and maximum NPL of 2.56 (p = 0.0027), respectively, when all subjects with GTCS were designated to be affected. Combined analyses of all 11 families showed an HLOD of 4.04 (α = 0.51) and maximum NPL score of 4.20 (p = 0.000065). Fine mapping of the locus defined an interval of 4.45Mb. These findings identify a novel locus for GTCS on 10q25‐q26 and support the idea that distinct loci underlie distinct seizure types within an epilepsy syndrome such as JME. Ann Neurol 2005;58:449–458

Chromosomal localization of gene for human glutamate receptor subunit-7

We isolated a human glutamate receptor subunit 7 (GluR-7) cosmid after high stringency screening of a human genomic placental library using a rat GluR-7 cDNA as a probe. A 614-bp fragment of the GluR-7 cosmid was sequenced, and an exon that encodes 53 amino acids was found between two introns. The exon exhibited 89% and 96% identity at the nucleotide and amino acid levels, respectively, with the corresponding region of rat GluR-7. The human GluR-7 was classified as a kainate subtype glutamate receptor based on its homology to rat GluR-7. Using somatic cell hybrid analysis, human GluR-7 was localized to chromosome 1. Fine mapping analysis using FISH localized the gene to 1p33–34. Since glutamate receptors of the kainate subtype have been implicated in neurodegenerative disorders, establishing the precise map position of human GluR-7 subunit is an important step towards evaluating this locus as a candidate for mutations in neurodegenerative disorders.

Epilepsy genetics: an abundance of riches for biologists.

Twenty-five genes have been identified in which mutations cause epileptic seizures in mice. The gene for a Na+/H+ exchanger has recently been found to underlie the spontaneous mutant slow wave epilepsy. Studies of such mutants should help elucidate the mechanisms that control neuronal excitability.

Evaluation of the α2A-adrenergic receptor gene in a heritable form of temporal lobe epilepsy

An autosomal dominant form of human temporal lobe epilepsy (TLE) has been mapped to a region of chromosome 10q that contains the intronless α2A-adrenergic receptor (α2AAR) gene. Because mutation of the α2AAR gene in the mouse fosters epileptogenesis, we developed methods for analysis of the α2AAR coding region applicable to any pathophysiologic state in which the α2AAR could be implicated in the disease mechanism. This study rules out mutations in the α2AAR coding region as causal for this form of autosomal dominant TLE.

Stargazing nets new calcium channel subunit

Epilepsy, a brain disorder manifested by recurrent seizures, afflicts approximately 1% of humans. A seizure is a fleeting change in behaviour caused by disordered, rhythmic, and synchronous firing of populations of neurons in the central nervous system (CNS). The behavioural features of different types of seizures are determined by the distinct populations of CNS neurons that fire abnormally. Together, the epilepsies are an extraordinarily diverse collection of disorders, as is testified by the more than 40 distinct forms of human epileptic syndromes1. Genetic susceptibility contributes to at least one third of human epilepsies, but the identity of the susceptibility genes is largely unknown. On page 340 of this issue2, Verity Lett and colleagues identify the gene mutation that underlies a spontaneously arising form of genetic epilepsy in a mouse mutant called ‘stargazer’, a name inspired by its distinctive head movement, presumably due to a defect in vestibular function3. The gene encodes a γ subunit of a voltagesensitive calcium channel (VSCC) and is the first γ subunit known to be expressed in the nervous system. The stargazer mouse is one of several spontaneously arising mutants that exhibit spike-wave epilepsy; the spike-wave electroencephalogram of stargazer closely resembles that observed in humans with absence (or petit-mal) epilepsy. VSCCs constitute a family of diverse proteins that mediate Ca2+ entry through the plasma membrane4, thereby controlling release of neurotransmitters, intracellular signaling pathways and gene expression5. They are composed of an α1 subunit that serves as the membrane pore and voltage sensor. Auxiliary subunits α2, δ and β modulate the kinetics of activation, inactivation, current density and drug binding4. In addition, the presence of the γ subunit in skeletal muscle regulates both the peak currents and rates of activation at more negative potentials6.