RESUMO
PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.
Assuntos
Corpo Estriado/citologia , Neurônios/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Frações Subcelulares/enzimologia , Animais , Western Blotting/métodos , Calbindina 2 , Colina O-Acetiltransferase/metabolismo , Corpo Estriado/enzimologia , Masculino , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Óxido Nítrico Sintase Tipo I/metabolismo , Parvalbuminas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Frações Subcelulares/ultraestrutura , Sinaptossomos/enzimologia , Sinaptossomos/ultraestruturaRESUMO
CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiperda (Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1'-hydroxylaseand dextromethorphan O-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1'-hydroxylase, apparent Km values were 4.9, 3.7, and 2.5 microM and the Vmax values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphan O-demethylase, apparent Km values were 0.6, 0.6, and 2.0 microM and the Vmax values were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1'-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/química , Animais , Linhagem Celular , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Cães , Ativação Enzimática , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Cinética , Masculino , Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/biossíntese , NADPH-Ferri-Hemoproteína Redutase/genética , Especificidade de Órgãos , Coelhos , SpodopteraRESUMO
Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.
Assuntos
Quimiocinas CXC/metabolismo , Ativação Linfocitária , Receptores de Quimiocinas/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Astrócitos , Sequência de Bases , Cálcio/metabolismo , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiotaxia de Leucócito , Cromossomos Humanos Par 4 , Clonagem Molecular , DNA Complementar/genética , Dessensibilização Imunológica , Humanos , Interferon gama/farmacologia , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/biossíntese , Receptores CXCR3 , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacosRESUMO
N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.
Assuntos
Antibacterianos/metabolismo , Escherichia coli/metabolismo , Glicilglicina/metabolismo , Ribossomos/metabolismo , Resistência a Tetraciclina/fisiologia , Antibacterianos/farmacologia , Bioensaio , Sistema Livre de Células , Doxiciclina/farmacologia , Escherichia coli/genética , Escherichia coli/ultraestrutura , Biossíntese Peptídica , Resistência a Tetraciclina/genéticaRESUMO
The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.
Assuntos
Genes Fúngicos/genética , Piruvato Quinase/genética , Leveduras/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sondas de DNA/genética , Genoma Fúngico , Íntrons/genética , Dados de Sequência Molecular , Piruvato Quinase/biossíntese , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Transformação Genética , Leveduras/enzimologiaRESUMO
Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.
Assuntos
Escherichia coli/metabolismo , Interleucina-6/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Linfócitos B/imunologia , Sequência de Bases , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/biossíntese , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/isolamento & purificação , Interleucina-6/farmacologia , Cinética , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.
Assuntos
Interleucina-6/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Camundongos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII). These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation. Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type. Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes. The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII. The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria. Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55. Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system. Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence. The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Núcleo Celular/metabolismo , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação da Expressão Gênica , Genes Fúngicos , Genes , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Substâncias Macromoleculares , Mitocôndrias/enzimologia , Mutação , RNA Mensageiro/genética , Saccharomyces cerevisiae/enzimologia , Transcrição GênicaRESUMO
The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Genes , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Saccharomyces cerevisiae/metabolismoRESUMO
The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494.