RESUMO
beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.
Assuntos
Antivirais/farmacologia , Carcinoma Hepatocelular/metabolismo , Desoxicitidina/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Timidina/farmacologia , Cromatografia Líquida de Alta Pressão , Desoxicitidina/metabolismo , Meia-Vida , Hepatite B/virologia , Humanos , Fosforilação , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.
Assuntos
Antivirais/uso terapêutico , Hepatite B/tratamento farmacológico , Nucleosídeos/uso terapêutico , Animais , Antivirais/farmacocinética , Modelos Animais de Doenças , Humanos , Testes de Sensibilidade Microbiana , Nucleosídeos/farmacocinéticaRESUMO
A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.
Assuntos
Antivirais/farmacologia , Desoxirribonucleosídeos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Animais , Antivirais/química , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Desoxicitidina/química , Desoxicitidina/farmacologia , Desoxirribonucleosídeos/química , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Vírus da Hepatite B da Marmota/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/tratamento farmacológico , Humanos , Relação Estrutura-Atividade , Especificidade por Substrato , Timidina/química , Timidina/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.
Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Didesoxiadenosina/farmacologia , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Cromatografia Líquida de Alta Pressão , DNA Polimerase Dirigida por DNA/metabolismo , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/metabolismo , Didesoxinucleotídeos , HIV/enzimologia , HIV/fisiologia , Meia-Vida , Células-Tronco Hematopoéticas/efeitos dos fármacos , Vírus da Hepatite B/enzimologia , Vírus da Hepatite B/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Lamivudina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Marmota/sangue , Marmota/virologia , Inibidores da Síntese de Ácido Nucleico , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Células Tumorais CultivadasRESUMO
beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.
Assuntos
Desoxiadenosinas/toxicidade , Mutagênicos/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Antivirais/toxicidade , Ensaio Cometa/métodos , Didesoxiadenosina/toxicidade , Hepatite B/tratamento farmacológico , Humanos , Linfócitos/efeitos dos fármacos , Testes de Mutagenicidade , Salmonella typhimurium/genéticaRESUMO
A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Nucleosídeos/farmacologia , Animais , Fármacos Anti-HIV/farmacologia , Antivirais/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/farmacologia , Desoxiadenosinas/uso terapêutico , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , HIV-1/efeitos dos fármacos , Hepatite B/virologia , Humanos , Masculino , Marmota , Nucleosídeos/uso terapêutico , Células-Tronco/efeitos dos fármacos , Timidina/farmacologia , Timidina/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Bovine mitochondrial ribosomes are presented as a model system for mammalian mitochondrial ribosomes. An alternative system for identifying individual bovine mitochondrial ribosomal proteins (MRPs) by RP-HPLC is described. To identify and to characterize individual MRPs proteins were purified from bovine liver, separated by RP-HPLC, and identified by 2D PAGE techniques and immunoblotting. Molecular masses of individual MRPs were determined. Selected proteins were subjected to N-terminal amino acid sequencing. The peptide sequences obtained were used to screen different databases to identify several corresponding MRP sequences from human, mouse, rat, and yeast. Signal sequences for mitochondrial import were postulated by comparison of the bovine mature N-termini determined by amino acid sequencing with the deduced mammalian MRP sequences. Significant sequence similarities of these new MRPs to known r-proteins from other sources, e.g., E. coli, were detected only for two of the four MRP families presented. This finding suggests that mammalian mitochondrial ribosomes contain several novel proteins. Amino acid sequence information for all of the bovine MRPs will prove invaluable for assigning functions to their genes, which would otherwise remain unknown.
Assuntos
Mitocôndrias/química , Proteínas Ribossômicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Proteína Ribossômica L3 , Proteínas Ribossômicas/química , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Partículas Submitocôndricas/químicaRESUMO
A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.
Assuntos
Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza B/crescimento & desenvolvimento , Vacinas contra Influenza , Influenza Humana/prevenção & controle , Vacinas Atenuadas , Adaptação Fisiológica , Temperatura Baixa , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vírus da Influenza B/genética , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Vacinas Atenuadas/imunologiaRESUMO
The distribution and nasal clearance of 99Tcm-labelled albumin (18.5 MBq), used as a mucosal vaccine surrogate for FluMist, was determined in three volunteers. The subjects were randomized in a cross-over clinical study design to receive either large-particle aerosal (nasal spray) followed by nose drops, or nose drops followed by the nasal spray, 1 week apart. Gamma scintigraphy was used to measure the distribution and clearance. The 'vaccine' delivered as drops was cleared from the nose into the oesophagus and upper stomach at very variable rates. In contrast, the nasal spray was uniformly distributed and cleared from the nasopharynx with a 50% mean clearance time of 50 min (range 40-60 min) and was not detected in the lungs.
Assuntos
Vacinas contra Influenza/farmacocinética , Nasofaringe/diagnóstico por imagem , Agregado de Albumina Marcado com Tecnécio Tc 99m/farmacocinética , Vacinação/métodos , Administração Intranasal , Adolescente , Adulto , Aerossóis , Esôfago/diagnóstico por imagem , Esôfago/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Humanos , Vacinas contra Influenza/administração & dosagem , Masculino , Mucosa Nasal , Nasofaringe/metabolismo , Cintilografia , Estômago/diagnóstico por imagem , Agregado de Albumina Marcado com Tecnécio Tc 99m/administração & dosagemRESUMO
The frequency of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants in virus populations not previously exposed to drug was determined in vitro by using HIV-1RF and the protease inhibitor SC-55389A. Two variants with single mutations responsible for drug resistance (V82A and N88S) were quantifiably isolated after only one round of replication, yielding a crude frequency estimate of at least 1 SC-55389A-resistant variant per 3.5 x 10(5) wild-type infectious units.
Assuntos
Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Ureia/análogos & derivados , Resistência Microbiana a Medicamentos/genética , HIV-1/genética , Humanos , Mutação Puntual , Ureia/farmacologiaRESUMO
The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação/fisiologia , Ureia/análogos & derivados , Sequência de Aminoácidos , Clonagem Molecular , Resistência Microbiana a Medicamentos , Protease de HIV/metabolismo , HIV-1/enzimologia , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ureia/farmacologiaRESUMO
MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.
Assuntos
Aciltransferases/antagonistas & inibidores , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Antifúngicos/química , Candida albicans/enzimologia , Humanos , Relação Estrutura-AtividadeRESUMO
The purpose of this study was to design and test a model that could identify and define which cellular adhesion molecules (CAMs) present on peripheral blood leukocytes were depressed in spinal cord injury (SCI) patients. CAMs on peripheral blood cells of SCI patients with pressure ulcers were measured by flow cytometry and compared with those of age-matched healthy controls and SCI patients on physical rehabilitation therapy (PRT) protocols without pressure ulcers. The latter patients had normal levels (97%) of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) and a low incidence of infection. By contrast, prior to undergoing rehabilitation therapy, SCI patients with pressure ulcers had significantly diminished LFA-1 levels (62%). Very late antigen 4 (VLA-4; alpha 4 beta 1; 34%) levels (i.e., alpha 4 = 34% and beta 1 = 44%) were approximately half those in controls (72%). Expression of alpha 2 and alpha 3 was also diminished in patients. Patients receiving PRT after debridement developed increased levels of LFA-1 and VLA-4 by the 6th week but alpha 2 and alpha 3 remained relatively low. These results combined with data from previous studies suggest that patients not receiving PRT developed severe pressure ulcers which required debridement surgery and healed more slowly due, in part, to reduced levels of CAMs.
Assuntos
Moléculas de Adesão Celular/sangue , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/reabilitação , Cicatrização/fisiologia , Adulto , Feminino , Citometria de Fluxo , Humanos , Integrina alfa4beta1 , Integrinas/sangue , Antígeno-1 Associado à Função Linfocitária/sangue , Masculino , Úlcera por Pressão/sangue , Receptores de Retorno de Linfócitos/sangue , Receptores de Antígeno muito Tardio/sangueRESUMO
Multiple communicative pathways among the nervous, endocrine and immune systems facilitate physiological immunoregulation. Spinal cord injury (SCI) patients have decreased natural (NK cell) and adaptive (T cell) immune function and reduced blood levels of cellular adhesion molecules (CAMs) that participate in immune function and wound healing. We found decreased LFA-1 and VLA-4 on peripheral blood leukocytes in SCI patients and lower levels of CAMs in SCI patients with pressure ulcers than in those without them. SCI might affect immune cells and immune responsiveness by: (1) disrupting the outflow of signals from the sympathetic nervous system to lymphoid tissues and their blood vessels as well as the returning afferent signals from these tissues to the brain; (2) immunosuppression caused by the stressors affecting SCI patients; (3) interrupting returning signals to the CNS from the periphery thereby reducing facilitation of immunoregulatory CNS neurons and decreasing their activity; or a combination of all three. SCI patients may develop dysregulation of the sympathetic nervous system that is intimately involved in immune function. Chronic stress mediates immunosuppression by corticosteroids, catecholamines, endorphins and met-enkephalin. The hypothalamus coordinates the response to stress through the release of soluble products from the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. Whereas the nervous and endocrine systems are not concerned with immunological specificity, they do influence the intensity, kinetics and localization of immune responses. Products of an activated immune system may generate feedback circuits capable of inhibiting, enhancing or regulating neuronal input. Immune system cells can produce neurologically active peptides including ACTH, CRF, growth hormone, thyrotropin, prolactin, human chorionic gonadotropin, endorphin, enkephalins, substance P, somatostatin and VIP. Cytokines are likely important mediators of the HPA response to immune stimuli.
Assuntos
Neuroimunomodulação/imunologia , Sistemas Neurossecretores/imunologia , Traumatismos da Medula Espinal/imunologia , Humanos , Sistemas Neurossecretores/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologiaAssuntos
Aciltransferases/metabolismo , Ácidos Mirísticos/metabolismo , Fatores de Ribosilação do ADP , Acil Coenzima A/metabolismo , Aciltransferases/biossíntese , Aciltransferases/química , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Humanos , Indicadores e Reagentes , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Mirístico , Plasmídeos , Mutação Puntual , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção/métodos , TrítioRESUMO
The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.
Assuntos
Citomegalovirus/enzimologia , Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Endopeptidases/genética , Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Histidina/genética , Humanos , Isoflurofato/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Análise de Sequência , Proteínas Virais/genéticaRESUMO
The murine cytomegalovirus UL80 open reading frame was cloned and the predicted amino acid sequence compared with those from other herpesviruses. The open reading frame encodes a fused protease-capsid assembly protein precursor and maintains conserved features including the active site serine, conserved regions CD1 through CD5, the release and maturation sites, and a potential internal cleavage site within the protease. However, the murine cytomegalovirus protease differs in comparison with the other proteases because it contains a unique 15-16 amino acid insertion between CD3 and CD1. The assembly protein sequences are relatively divergent, but they can be arranged into groups defined by herpesvirus subfamily, with each group possessing a conserved motif at its carboxyl terminus.
Assuntos
Capsídeo/genética , Citomegalovirus/genética , Endopeptidases/genética , Genes Virais , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/biossíntese , Clonagem Molecular , Sequência Conservada , Endopeptidases/biossíntese , Herpesviridae/genética , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Viral/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.