RESUMO
The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Olho/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Imuno-Histoquímica , Zíper de Leucina , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.
Assuntos
Proteínas de Homeodomínio/genética , Mutação Puntual , Retinose Pigmentar/genética , Deleção de Sequência , Transativadores/genética , Adulto , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Códon , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Feminino , Genes Dominantes , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/metabolismo , Humanos , Íntrons , Escore Lod , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Retina/patologia , Retinose Pigmentar/patologia , Retinose Pigmentar/fisiopatologia , Transativadores/biossíntese , Transativadores/metabolismo , Acuidade VisualAssuntos
Proteínas de Ligação a DNA/genética , Proteínas do Olho/genética , Mutação , Retinose Pigmentar/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica , Genes Dominantes , Ligação Genética , Marcadores Genéticos , Análise Heteroduplex , Humanos , Zíper de Leucina/genética , Masculino , Linhagem , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Blood pressure and nutritional profiles in the 'Oraon' tribal community of India living in rural and urban areas were studied between 1981-85 after a house to house survey of 4523 rural tribal people (RT) and 935 of their urban tribal counterparts (UT). Prevalence of hypertension was found to be 4.8/1000 males and 4.3/1000 females in rural tribal group giving an overall rate of 4.6/1000. In contrast the same were 27.1/1000 males and 21.4/1000 females in UT group, overall rate being 25.6/1000. Average calorie consumption were 1750 and 2280 and mean 24 hour-urinary sodium excretions 58 and 118 milliequivalents in RT and UT groups respectively. Of the total of 21 subjects in RT and 24 in UT detected to be hypertensive, 7 had common family inheritance. Increased mean arterial pressure correlated with increased sodium consumption and body weight. Hypertensives from both the groups showed higher urinary sodium excretion (P < 0.05). This epidemiologic study proves the role of a genetic factor/defect complicated by higher salt consumption in causation of increased blood pressure.
Assuntos
Pressão Sanguínea , Hipertensão/epidemiologia , População Rural , Sódio/urina , População Urbana , Adulto , Idoso , Peso Corporal , Dieta , Ingestão de Energia , Feminino , Humanos , Hipertensão/etnologia , Hipertensão/etiologia , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
Lethal toxin (LT) secreted by Bacillus anthracis consists of two proteins, protective antigen (PA) and lethal factor (LF). LT causes lysis of macrophages and derived cell lines at low concentrations. PA binds to the cell surface receptors and mediates translocation of LF into cytosol of mammalian cells. Internalization of LF into cytosol by osmotic lysis of pinocytic vesicles requires high concentration of LF for cell lysis. To examine the possible cell lysis by LF at low concentration, we introduced LF directly into cytosol of J774A.1 cells through reconstituted Sendai virus envelopes. The introduction of LF lysed J774A.1 cells in a concentration dependent manner. Internalization of PA alone through virosome had no toxic effect on J774A.1 cells. In the process of cytotoxicity LF was not cleaved by cellular proteases. Unlike many protein toxins, golgi was not involved in the expression of lethal toxin activity. These results indicate that LF is the toxic component of anthrax lethal toxin and prior proteolytic processing or trafficking through golgi is not required for its activity.
Assuntos
Antígenos de Bactérias , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/toxicidade , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/metabolismo , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microinjeções , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Respirovirus , VirulênciaAssuntos
Hipertensão/epidemiologia , Adulto , Idoso , Etnicidade , Comportamento Alimentar/etnologia , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , População Rural , População UrbanaRESUMO
Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds to the receptor on mammalian cells and facilitates translocation of EF or LF into the cytosol. PA is the primary component of several anthrax vaccines. In this study we expressed and purified PA from Escherichia coli. The purification of PA from E. coli was possible after transporting the protein into the periplasmic space using the outer membrane protein A signal sequence. The purification involved sequential chromatography through hydroxyapatite, DEAE Sepharose CL-4B, followed by Sephadex G-100. The typical yield of purified PA from this procedure was 500 microg/liter. PA expressed and purified from E. coli was similar to the PA purified from Bacillus anthracis in its ability to lyse a macrophage cell line (J774A.1). The present results suggest that a signal sequence is required for the efficient translocation of PA into E. coli periplasmic space.
Assuntos
Antígenos de Bactérias , Bacillus anthracis , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Sequência de Aminoácidos , Toxinas Bacterianas/metabolismo , Sequência de Bases , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Plasmídeos/genética , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/isolamento & purificação , Deleção de Sequência/genéticaRESUMO
The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.
Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Endocitose/imunologia , Animais , Transporte Biológico , Linhagem Celular , Mamíferos , Oligopeptídeos/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , SoluçõesRESUMO
Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Rodopsina/genética , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Zíper de Leucina , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Rodopsina/metabolismo , Saccharomyces cerevisiae , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
NRL, a bZIP transcription factor of the Maf subfamily, interacts with the homeodomain protein CRX and synergistically regulates rhodopsin expression. Here we report that six isoforms of NRL (29-35 kDa) are generated by phosphorylation and expressed specifically in the mammalian retina. The anti-NRL antibody also cross-reacts with a cytosolic 45-kDa protein, which is detected in neuronal tissues but is not encoded by the NRL gene. In both human retinal cell cultures and sections of fetal and adult human retina, NRL is present in the nuclei of developing and mature rods but not cones. We propose that NRL regulates rod photoreceptor-specific gene expression and is involved in rod differentiation.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Fosfatase Alcalina/farmacologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Células COS , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas , Proteínas Recombinantes/metabolismo , Retina/embriologia , Retina/metabolismo , Rodopsina/biossíntese , Fatores de Tempo , TransfecçãoRESUMO
The retinitis pigmentosa GTPase regulator (RPGR) gene encodes a protein homologous to the RCC1 guanine nucleotide exchange factor and is mutated in 20% of patients with X-linked retinitis pigmentosa. We have characterized the full-length and variant cDNAs corresponding to the mouse homolog of the RPGR gene (mRpgr). Comparison with the human cDNA revealed sequence identity primarily in the region of RCC1 homology repeats. As in humans, the mRpgr gene maps within 50 kilobases from the 5'-end of the Otc gene. The mRpgr transcripts are detected as early as E7 during embryonic development and are expressed widely in the adult mice. Variant mRpgr isoforms are generated by alternative splicing and by utilizing two in-frame initiation codons. The products of mRpgr cDNAs migrate aberrantly in SDS-polyacrylamide gels because of a charged domain. In transfected COS cells, the mRpgr protein is isoprenylated and is localized in the Golgi complex. This subcellular distribution is not observed after treatments with brefeldin A or mevastatin and when the conserved isoprenylation sequence (CTIL) at the carboxyl terminus is deleted or mutagenized. These studies suggest a role for the mRpgr protein in Golgi transport and form the basis for investigating the mechanism of photoreceptor degeneration in X-linked retinitis pigmentosa.