RESUMO
A strategy of mutagenesis followed by yeast two-hybrid assay was used to determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the major site for interaction with PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure, based on the structural similarity of RACK1 to other beta-propeller WD-repeat proteins, indicated that the majority of the amino acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Mutação Puntual , Receptores de Quinase C Ativada , Sequências Repetitivas de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Criptorquidismo/patologia , Espermatozoides/patologia , Testículo/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/análise , Animais , Apoptose , Western Blotting , Criptorquidismo/etiologia , Criptorquidismo/cirurgia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Epitélio/patologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/patologiaRESUMO
Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.
Assuntos
Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/efeitos da radiação , Fenômenos Biofísicos , Biofísica , Transferência de Energia , Radicais Livres/química , Herpesvirus Humano 1/enzimologia , Holoenzimas/química , Holoenzimas/efeitos da radiação , Peso Molecular , Subunidades Proteicas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação , Ribonucleotídeo Redutases/químicaRESUMO
The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Isoenzimas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Catálise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Dados de Sequência Molecular , Fosforilação , Ligação ProteicaRESUMO
The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique "signature" regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR1 and UCR2 are located between the extreme amino-terminal region and the catalytic region of the PDE4 enzymes. The UCR1 of the PDE4D3 isoform was used as a "bait" in a two-hybrid screen, which identified a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and "pull down" analysis of constructs incorporating various regions of the PDE4D3 cDNA demonstrated that the carboxyl-terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2. The interaction was blocked by mutations of two positively charged amino acids (Arg-98 and Arg-101 to alanine) located within an otherwise largely hydrophobic region of UCR1. Mutation of three negatively charged amino acids in UCR2 (Glu-146, Glu-147, and Asp-149, all to alanine) also blocked the interaction. The phosphorylation of UCR1 by cAMP-dependent protein kinase (PKA) in vitro attenuated the ability of UCR1 to interact with UCR2. Mutation of the PKA substrate site in UCR1 (Ser-54) to aspartic acid, which mimics the activation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 and UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic interactions and which may be disrupted by PKA phosphorylation. We suggest that the UCR1 and UCR2 domains may form a module that interacts with and regulates the PDE4 catalytic region.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Sequência Conservada , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA Complementar , Células HeLa , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
The Pde4a gene is a mammalian homolog of the dunce learning and memory gene of Drosophila melanogaster and encodes cAMP-specific phosphodiesterases, targets for drugs with antidepressant and anti-inflammatory actions in humans. We have analyzed the intron/exon and promoter structure of the murine Pde4a gene. Pde4a encodes at least two different transcripts, each generated by alternative mRNA splicing and the use of alternative promoters. The majority of Pde4a exons are tightly clustered at the 3' end of the gene. The 5' region of the gene contains at least one widely separated exon, which encodes the 5' end of a distinct mRNA transcript and contains a separate promoter and transcriptional start site. Analysis of YAC clones determined that the Pde4a gene maps to the 4-cM region of Chromosome (Chr) 9, close to Ldlr and Epor, in a region syntenic to human PDE4A.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , Mapeamento Físico do Cromossomo , Regiões Promotoras Genéticas/genética , 3',5'-AMP Cíclico Fosfodiesterases/química , Animais , Sequência de Bases , Southern Blotting , Cromossomos Artificiais de Levedura/química , Cromossomos Artificiais de Levedura/genética , Primers do DNA/química , Sondas de DNA/química , Enzimas de Restrição do DNA/química , Éxons/genética , Biblioteca Gênica , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Biogen, in collaboration with Merck & Co, is developing late activator VLA-4 (alpha4beta1) integrin antagonists for the potential treatment of inflammatory conditions [271194]. Merck has begun phase II trials with the lead compound, BIO-1211, for asthma, Biogen is still conducting preclinical research for its designated indications [317648,319225]. Under the collaborative agreement, each company has worldwide rights to certain indications; Merck has rights for asthma and Biogen retains the rights to a number of smaller indications, including multiple sclerosis, inflammatory bowel disease, renal indications and most diseases in which the US patient population is less than 200,000 [271194]. VLA-4 inhibitors show anti-inflammatory action by inhibition of binding between adhesion factors and leukocytes, but with no loss of basophil function, and they have the advantage of specificity not seen with existing drugs [273417]. In February 1999, Lehman Brothers predicted 40% probabilities that the compound would reach the US and ex-US markets for the asthma indication (Merck), and launch onto these markets by 2003. Peak annual sales of US dollar 500 million (US) and US dollar 500 million (outside US) are predicted, both in 2010 [319225].
RESUMO
The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.
Assuntos
Renina/antagonistas & inibidores , Administração Oral , Angiotensina I/biossíntese , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Bloqueadores Ganglionares/farmacologia , Humanos , Técnicas In Vitro , Losartan/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Renina/farmacologia , Fatores de TempoRESUMO
Of the five PDE4D isoenzymes, only the PDE4D4 cAMP specific phosphodiesterase was able to bind to SH3 domains. Only PDE4D4 and PDE4A5, but not any other PDE4A, B, C and D isoforms expressed in rat brain, bound to src, lyn and fyn kinase SH3 domains. Purified PDE4D4 could bind to purified lyn SH3. PDE4D4 and PDE4A5 both exhibited selectivity for binding the SH3 domains of certain proteins. PDE4D4 did not bind to WW domains. We suggest that an important function of the unique N-terminal region of PDE4D4 may be to allow for association with certain SH3 domain-containing proteins.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/química , AMP Cíclico/farmacologia , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Proteínas de Transporte/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Isoenzimas/metabolismo , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Quinases da Família src/metabolismoRESUMO
The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/análise , Animais , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Isoenzimas , Peptídeos/análise , Testes de Precipitina , Receptores de Quinase C Ativada , Receptores de Superfície Celular/análise , Especificidade por Substrato , Leveduras/enzimologiaRESUMO
A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.
Assuntos
Amidas/química , Renina/antagonistas & inibidores , Administração Oral , Amidas/farmacocinética , Amidas/farmacologia , Animais , Disponibilidade Biológica , Humanos , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Renina/sangue , Espectrofotometria Infravermelho , Relação Estrutura-AtividadeRESUMO
Acute intraperitoneal infection of weanling BALB/c mice with murine cytomegalovirus (MCMV) resulted in an inoculum titer-dependent weight loss, mortality and elevation of plasma transaminases (ALT: alanine transaminase and AST: aspartate transaminase). Three days post infection (p.i.) with 10(4.85) plaque forming units (pfu) there was 90% mortality with a mean death day p.i. of 4.1 +/- 0.2. Plasma levels of ALT and AST were elevated 24- and 15-fold, respectively. Organ titers of virus (log10 pfu/g tissue) were 6.16 in the liver, 6.05 in the spleen, 4.0-4.7 in the lung, heart, kidney and intestine and undetectable in the muscle and brain. Organ concentrations (units/g wet-weight) of ALT were highest in the liver, whilst for AST the highest levels were found in the heart. The concentrations of ALT but not AST were reduced (35-55%) in the infected liver; the concentrations of ALT and AST were not changed in other infected organs. There were excellent correlations (r > 0.95) between viral titers in the liver, increases of plasma ALT and depletion of liver ALT. HPMPC and ganciclovir administered either p.o. or s.c. reduced mortality, increases in plasma transaminases and viral burdens in the liver and prevented depletion of liver ALT. HPMPC was approximately 10-fold more potent than ganciclovir. These results strongly suggest that intraperitoneal infection of the BALB/c mouse with MCMV represents an animal model of CMV hepatitis that can be monitored by measuring plasma ALT.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Citosina/análogos & derivados , Modelos Animais de Doenças , Ganciclovir/uso terapêutico , Hepatite Viral Animal/tratamento farmacológico , Muromegalovirus/fisiologia , Organofosfonatos , Compostos Organofosforados/uso terapêutico , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Peso Corporal , Cidofovir , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Citosina/uso terapêutico , Feminino , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adipócitos/metabolismo , Hormônio do Crescimento/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adipócitos/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Dados de Sequência MolecularAssuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Antidepressivos/uso terapêutico , Transtorno Depressivo/enzimologia , Inibidores Enzimáticos/uso terapêutico , Inflamação/enzimologia , Inibidores de Fosfodiesterase/uso terapêutico , 3',5'-AMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Transtorno Depressivo/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Using the SKH-1 hairless mouse (HM) we have addressed the issue as to whether topically applied acyclovir (ACV) may mediate some of its antiviral actions by a systemic effect. When topically applied in a formulation consisting of polyvinyl alcohol (25% w/v):DMSO:cremophor EL:linoleic acid (63:16:16:5, v/v/v/v), ACV penetrated hairless mouse skin in a concentration-dependent manner and dose-dependently reduced cutaneous herpes simplex virus 1 (HSV-1) KOS infection. Topically applied ACV also effectively reduced the mortality associated with disseminated HSV-2 HG-52 infection. At 1 h following topical application of 1.7% w/v ACV the plasma and skin concentrations of ACV were 5.5 nmoles/ml and 120 nmoles/g. At 1 h following an oral dose of ACV with antiviral efficacy comparable to topically applied ACV (1.7% w/v) the plasma and skin concentrations of ACV were 21.3 nmoles/ml and 51 nmoles/g. These findings imply that when applied topically to the HM, ACV can mediate a portion of its antiviral activity through a systemic mode of action.
Assuntos
Aciclovir/farmacocinética , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/efeitos dos fármacos , Dermatopatias Infecciosas/tratamento farmacológico , Aciclovir/sangue , Aciclovir/metabolismo , Administração Cutânea , Administração Oral , Animais , Herpes Simples/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Pelados , Absorção Cutânea/efeitos dos fármacos , Dermatopatias Infecciosas/metabolismoAssuntos
3',5'-AMP Cíclico Fosfodiesterases , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Pirrolidinonas/farmacologia , Domínios de Homologia de src , Processamento Alternativo , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 5 , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Cinética , Dados de Sequência Molecular , Família Multigênica , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Ratos , RolipramRESUMO
We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IVPDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 microM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 microM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.