Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia.

The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC)n.(GCC)n, are correlated with the ability of the individual G- and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the G- and C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C.C paired. The presence of a C.C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.

Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.

Efeito da estimulação purinérgica sobre a produção de melatonina em macrófagos da linhagem RAW 264.7

A melatonina é um hormônio produzido de forma rítmica e no período de escuro pela glândula pineal bem como de forma não rítmica por diversos tecidos e células imunocompetentes. É sintetizada pela acetilação e metilação da serotonina pela ação das enzimas arilalquilamina N-acetiltransferase (AA-NAT) e acetilserotonina -O-metiltransferase (ASMT) que levam à formação de N-acetilserotonina (NAS) e melatonina (MEL), respectivamente. Nos últimos anos temos demonstrado que síntese de melatonina pela pineal pode ser negativamente modulada por mediadores inflamatórios e pelo ATP que atua como co-transmissor juntamente com a noradrenalina liberada no terminal nervoso simpático que a inerva. Perifericamente, contudo, estes mediadores inflamatórios apresentam um efeito contrário induzindo a produção de melatonina em células imunocompetentes. Estas observações levaram à criação da hipótese de um eixo imune-pineal. Esse trabalho teve como objetivo verificar o efeito do ATP sobre produção de melatonina em macrófagos da linhagem RAW 264.7 Os dados desse trabalho mostram que o ATP é capaz de induzir de maneira dose dependente a produção de melatonina em macrófagos através da modulação das enzimas AA-NAT e ASMT. Foi demostrado também que esse efeito é mediado pelo receptor P2X7 e que a melatonina produzida age autocrina e paracrinamente aumentando a fagocitose de particulas de zimosan. Com isso, podemos concluir que o ATP é um ativador endógeno do eixo imune-pineal

Estudo de associação entre disfunção neurocognitiva, estresse oxidativa e polimorfismos em pacientes jovens com Transtornos Bipolar tipo I

O Transtorno Bipolar (TB) tipo I é uma doença caracterizada por episódios de mania e depressão recorrentes com importante prejuízo do funcionamento global e comprometimento das funções cognitivas. Além disso, sabe-se que o número de episódios de humor patológico ao longo da vida pode também influenciar o funcionamento cognitivo destes sujeitos. Neste cenário, ocorreu a necessidade de se investigar marcadores genéticos para disfunção cognitiva no TB com o objetivo de estudar este fenômeno. Dentre os potenciais genes responsáveis por influenciar a cognição destacam-se os polimorfismos funcionais do fator neurotrófico derivado do cérebro (BDNF), da catecol-O-metiltransferase (COMT), da apolipoproteína-E (APOE) e do canal de cálcio de baixa voltagem subunidade 1-C (CACNA1C). Sabe-se, também, que no TB os marcadores de estresse oxidativo estão aumentados durante todas as fases da doença, entretanto, não é claro qual impacto destes na disfunção cognitiva de indivíduos com TB. O objetivo dessa tese foi avaliar o desempenho cognitivo de pacientes jovens com bipolaridade tipo I e sua associação com o genótipo de BDNF, COMT, APOE e CACNA1C e também com os níveis plasmáticos de oxidação da guanosina (8-OHdG) e citosina (5-Mec) durante os episódios de humor, eutimia e em controles. Para investigar essa associação foram incluídos 116 pacientes (79 em episódio de humor patológico e 37 eutímicos) com diagnóstico de TB tipo I (DSMIV-TR); 97 controles saudáveis foram submetidos à avaliação neuropsicológica e coleta de sangue para extração de DNA visando genotipagem para BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 e rs7412), CACNA1C (rs1006737), 8-OhdG e 5-Mec. A análise dos dados obtidos revelou que pacientes portadores do genótipo Met/Met rs4680/rs165599 do COMT apresentam comprometimento cognitivo mais grave (função executiva, fluência verbal, memória e inteligência) comparado ao genótipo Val/Met ou Val/Val durante episódios maníacos ou mistos. Na mesma direção destes resultados, verificou-se que pacientes portadores do alelo Met rs4680 do COMT apresentam comprometimento do reconhecimento de emoções faciais em episódios de mania e depressão. Nenhum efeito do COMT foi observado em controles. O alelo de risco Met do CACNA1C se associou a um pior comprometimento executivo independente dos sintomas maníacos ou depressivos no TB, porém nenhum efeito se observou nos controles. O alelo Met do BDNF rs6265 ou a presença do alelo 4 da APOE não representa um fator que identifique um grupo com desempenho cognitivo diferenciado durante as fases do TB ou em controles. Sujeitos com TB apresentaram níveis mais elevados de 8-OHdG e tais níveis eram diretamente proporcionais ao número de episódios maníacos ao longo da vida, sugerindo um papel dos episódios hiperdopaminérgicos na oxidação das bases de DNA. Concluiu-se que a genotipagem para COMT e CACNA1C em pacientes com TB pode identificar um grupo de pacientes associados a pior disfunção cognitiva durante as fases maníacas e mistas do TB. Tal dado pode ser um indicador do envolvimento do sistema dopaminérgico e dos canais de cálcio de baixa voltagem na fisiopatologia da disfunção cognitiva no TB e deve ser explorado em outros estudos

Biologic therapies and non-melanoma skin cancer rates in three chronic immune-mediated diseases

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The policy and ethical implications of genetic research on attention deficit hyperactivity disorder

Objective: To review the policy and ethical implications of recent research on the molecular genetics of attention deficit hyperactivity disorder (ADHD). Method: MEDLINE and psycINFO database searches were used to identify studies on the genetics of ADHD. The implications of replicated candidate genes are discussed. Results: The findings for most genes have been inconsistent but several studies have implicated the genes in the dopaminergic pathway in the aetiology of ADHD. Conclusions: The current evidence on the genetics of ADHD is insufficient to justify genetic screening tests but it will provide important clues as to the aetiology of ADHD. Genetic information on susceptibility to ADHD has the potential to be abused and to stigmatize individuals. Researchers and clinicians need to be mindful of these issues in interpreting and disseminating the results of genetic studies of ADHD.

Multicenter linkage study of schizophrenia loci on chromosome 22q

The hypothesis of the existence of one or more schizophrenia susceptibility loci on chromosome 22q is supported by reports of genetic linkage and association, meta-analyses of linkage, and the observation of elevated risk for psychosis in people with velocardiofacial syndrome, caused by 22q11 microdeletions. We tested this hypothesis by evaluating 10 microsatellite markers spanning 22q in a multicenter sample of 779 pedigrees. We also incorporated age at onset and sex into the analysis as covariates. No significant evidence for linkage to schizophrenia or for linkage associated with earlier age at onset, gender, or heterogeneity across sites was observed. We interpret these findings to mean that the population-wide effects of putative 22q schizophrenia susceptibility loci are too weak to detect with linkage analysis even in large samples.

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN > 44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide- linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design. Crown Copyright (c) 2005 Published by Elsevier B.V. All rights reserved.

The phasevarion: A genetic system controlling coordinated, random switching of expression of multiple genes

Several host-adapted bacterial pathogens contain methyltransferases associated with type III restriction-modification (R-M) systems that are subject to reversible, high-frequency on/off switching of expression (phase variation). To investigate the role of phase-variable expression of R-M systems, we made a mutant strain lacking the methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae and analyzed its phenotype. By microarray analysis, we identified a number of genes that were either up- or down-regulated in the mod mutant strain. This system reports the coordinated random switching of a set of genes in a bacterial pathogen and may represent a widely used mechanism.

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L(-/-) germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L(-/-) meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.