RESUMO
Here, we investigated the effects of sympathectomy on systemic bacterial loads following infection with Listeria monocytogenes, and on innate and specific immune responses in the peritoneum. Sympathectomy decreased systemic bacterial loads, and increased the number of peritoneal leukocytes and the percentage of peritoneal macrophages three days postinfection. This suggests that sympathectomy-induced decreases systemic bacterial loads are associated with increased recruitment of inflammatory cells into tissues during the innate immune response.
Assuntos
Listeriose/imunologia , Macrófagos Peritoneais/microbiologia , Peritônio/imunologia , Peritonite/imunologia , Animais , Interferon gama/sangue , Macrófagos Peritoneais/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Norepinefrina/metabolismo , Oxidopamina , Peritônio/inervação , Peritonite/microbiologia , Fagócitos/citologia , Fagócitos/microbiologia , Baço/imunologia , Baço/metabolismo , Simpatectomia Química , SimpatolíticosRESUMO
Sympathectomy of BALB/c mice that were injected with either Listeria monocytogenes or saline did not affect the total number of splenic leukocytes measured 1-3 days after injection, but sympathectomy did increase the percentages of neutrophils in the spleens of both infected and uninfected mice. By contrast, sympathectomy was associated with increased numbers of peritoneal exudate cells (PEC) and peritoneal macrophages in both groups of mice. Sympathectomy did not affect tumor necrosis factor-alpha, interleukin-12, or interferon-gamma production in cultured splenocytes or PEC in either infected or uninfected mice.
Assuntos
Leucócitos/fisiologia , Baço/citologia , Simpatectomia Química , Animais , Cromatografia Líquida de Alta Pressão , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Contagem de Leucócitos , Leucócitos/imunologia , Listeriose/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/fisiologia , Norepinefrina/metabolismo , Oxidopamina , Cavidade Peritoneal/patologia , Baço/imunologia , SimpatolíticosRESUMO
OBJECTIVES: We administered antioxidant vitamins to rabbits with pacing-induced cardiomyopathy to assess whether antioxidant therapy retards the progression of congestive heart failure (CHF). BACKGROUND: Although oxidative stress is increased in CHF, whether progression of heart failure could be prevented or reduced by antioxidants is not known. METHODS: Rabbits with chronic cardiac pacing and sham operation were randomized to receive a combination of beta-carotene, ascorbic acid and alpha-tocopherol, alpha-tocopherol alone or placebo over eight weeks. Echocardiography was used to measure cardiac function weekly. Resting hemodynamics and in vivo myocardial beta-adrenergic responsiveness were studied at week 8. Animals were then sacrificed for measuring myocardial beta-receptor density, norepinephrine (NE) uptake-1 site density, sympathetic neuronal marker profiles, tissue-reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and oxidative damage of mitochondrial DNA (mtDNA). RESULTS: Rapid cardiac pacing increased myocardial oxidative stress as evidenced by reduced myocardial GSH/GSSG ratio and increased oxidized mtDNA and produced cardiac dysfunction, beta-adrenergic subsensitivity, beta-receptor downregulation, diminished sympathetic neurotransmitter profiles and reduced NE uptake-1 carrier density. A combination of antioxidant vitamins reduced the myocardial oxidative stress, attenuated cardiac dysfunction and prevented myocardial beta-receptor downregulation and sympathetic nerve terminal dysfunction. Administration of alpha-tocopherol alone produced similar effects, but the effects were less marked than those produced by the three vitamins together. Vitamins produced no effects in sham-operated animals. CONCLUSIONS: Antioxidant vitamins reduced tissue oxidative stress in CHF and attenuated the associated cardiac dysfunction, beta-receptor downregulation and sympathetic nerve terminal abnormalities. The findings suggest that antioxidant therapy may be efficacious in human CHF.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Insuficiência Cardíaca/prevenção & controle , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , alfa-Tocoferol/farmacologia , beta Caroteno/farmacologia , Análise de Variância , Animais , DNA Mitocondrial/isolamento & purificação , Regulação para Baixo , Ecocardiografia , Glutationa/metabolismo , Hemodinâmica , Neurotransmissores/metabolismo , Coelhos , Receptores Adrenérgicos beta/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.
Assuntos
Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Regulação Alostérica , Sítio Alostérico , Substituição de Aminoácidos , Arginina/metabolismo , Citidina Trifosfato/metabolismo , Dimerização , Entropia , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Modelos Moleculares , Nucleotidiltransferases/genética , Ligação Proteica , Conformação Proteica , TitulometriaRESUMO
Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Flavodoxina/metabolismo , NADH NADPH Oxirredutases/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , Proteínas de Bactérias/química , Sítios de Ligação , Escherichia coli/química , Flavodoxina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , NADH NADPH Oxirredutases/química , Oxirredução , Ligação Proteica , Conformação Proteica , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismoRESUMO
Many investigators have shown that ablation of the sympathetic nervous system (SNS) with 6-hydroxydopamine (6-OHDA) can alter cell-mediated and humoral immune responses to antigenic challenge. Fewer studies have examined 6-OHDA-induced changes in natural immunity. In this study, we have examined the effect of chemical sympathectomy on the nonspecific and specific phases of the response to infection with Listeria monocytogenes. Sympathectomy decreased splenic bacterial loads 3 and 5 days post-infection and increased splenic neutrophils 3 days post-infection. Sympathectomy decreased splenocyte numbers and antigen-stimulated cytokine secretion from splenocytes. These results suggest that the SNS influences specific responses by modulating innate responses.
Assuntos
Imunidade Inata/imunologia , Listeriose/imunologia , Baço/imunologia , Baço/microbiologia , Simpatectomia Química , Animais , Interferon gama/metabolismo , Interleucina-2/metabolismo , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fibras Nervosas/metabolismo , Neutrófilos/imunologia , Neutrófilos/microbiologia , Norepinefrina/metabolismo , Oxidopamina , Baço/inervação , Sistema Nervoso Simpático/imunologia , Sistema Nervoso Simpático/microbiologia , Simpatolíticos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
Chronic angiotensin-converting enzyme (ACE) inhibition has been shown to improve cardiac sympathetic nerve terminal function in heart failure. To determine whether similar effects could be produced by angiotensin II AT(1) receptor blockade, we administered the ACE inhibitor quinapril, angiotensin II AT(1) receptor blocker losartan, or both agents together, to rabbits with pacing-induced heart failure. Chronic rapid pacing produced left ventricular dilation and decline of fractional shortening, increased plasma norepinephrine (NE), and caused reductions of myocardial NE uptake activity, NE histofluorescence profile, and tyrosine hydroxylase immunostained profile. Administration of quinapril or losartan retarded the progression of left ventricular dysfunction and attenuated cardiac sympathetic nerve terminal abnormalities in heart failure. Quinapril and losartan together produced greater effects than either agent alone. The effect of renin-angiotensin system inhibition on improvement of left ventricular function and remodeling, however, was not sustained. Our results suggest that the effects of ACE inhibitors are mediated via the reduction of angiotensin II and that angiotensin II plays a pivotal role in modulating cardiac sympathetic nerve terminal function during development of heart failure. The combined effect of ACE inhibition and angiotensin II AT(1) receptor blockade on cardiac sympathetic nerve terminal dysfunction may contribute to the beneficial effects on cardiac function in heart failure.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Norepinefrina/sangue , Terminações Pré-Sinápticas/metabolismo , Sistema Renina-Angiotensina/fisiologia , Tetra-Hidroisoquinolinas , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Imunofluorescência , Coração/inervação , Coração/fisiologia , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Isoquinolinas/farmacologia , Losartan/farmacologia , Neurônios/química , Neurônios/enzimologia , Norepinefrina/análise , Marca-Passo Artificial , Quinapril , Coelhos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Função Ventricular Esquerda/fisiologiaRESUMO
L-Deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, has previously been shown to improve immune responses and restore noradrenergic (NA) nerve fibers in the spleen of old rats. In tumor-bearing rats, L-deprenyl inhibited tumor incidence and enhanced tuberoinfundibular dopaminergic (TIDA) neurotransmission in the hypothalamus. The aim of the present study was to investigate whether alterations in sympathetic NA activity and cellular immune responses in the spleen, and TIDA activity in the hypothalamus, accompany deprenyl-induced regression of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary tumors. Rats with DMBA-induced mammary tumors were treated with 0, 2.5 mg, or 5.0 mg/kg body weight of deprenyl daily for 13 weeks. Saline-treated tumor-bearing rats exhibited reduced splenic IL-2 and IFN-gamma levels, and lowered splenic norepinephrine (NE) concentration and hypothalamic dopaminergic activity, compared to rats without tumors. In contrast, treatment with 2.5 mg/kg and 5.0 mg/kg of deprenyl reduced the number and size of mammary tumors. Deprenyl-induced tumor regression was accompanied by increased immune measures in the spleen, including enhanced IL-2 and IFN-gamma production, and NK cell activity. Neural measures enhanced by deprenyl included NE concentration in the spleen and TIDA neuronal activity in the hypothalamus. These results suggest that (1) mammary tumorigenesis is associated with the inhibition of sympathetic NA activity in the spleen, TIDA activity in the hypothalamus, and cell-mediated immunity, and (2) reversal of the inhibition of catecholaminergic neuronal activities of the central nervous system and peripheral nervous system by deprenyl may enhance anti-tumor immunity.
Assuntos
Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/imunologia , Inibidores da Monoaminoxidase/farmacologia , Neuroimunomodulação/efeitos dos fármacos , Selegilina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinógenos , Divisão Celular/imunologia , Feminino , Citometria de Fluxo , Ácido Hidroxi-Indolacético/metabolismo , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neuroimunomodulação/imunologia , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Baço/citologia , Baço/imunologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Selegiline is a centrally acting sympatholytic agent with neuroprotective properties. It also has been shown to promote sympathetic reinnervation after sympathectomy. These actions of selegiline may be beneficial in heart failure that is characterized by increased sympathetic nervous activity and functional sympathetic denervation. Twenty-seven rabbits with rapid cardiac pacing (360 beats/min, 8 wk) and twenty-three rabbits without pacing were randomly assigned to receive selegiline (1 mg/day, 8 wk) or placebo. Rapid pacing increased plasma norepinephrine (NE) and decreased left ventricular fractional shortening, baroreflex sensitivity, cardiac sympathetic nerve terminal profiles, cardiac NE uptake activity, and myocardial beta-adrenoceptor density. Selegiline administration to animals with rapid ventricular pacing attenuated the increase in plasma NE and decreases in fractional shortening, baroreflex sensitivity, sympathetic nerve profiles, NE uptake activity and beta-adrenoceptor density. Thus selegiline appears to exert a sympatholytic and cardiac neuroprotective effect in pacing-induced cardiomyopathy. The effects are potentially beneficial because selegiline not only improves cardiac function but also increases baroreflex sensitivity in heart failure.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Coração/inervação , Receptores Adrenérgicos beta/efeitos dos fármacos , Selegilina/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Análise de Variância , Animais , Barorreflexo/efeitos dos fármacos , Sítios de Ligação , Estimulação Cardíaca Artificial , Modelos Animais de Doenças , Ecocardiografia , Coração/efeitos dos fármacos , Insuficiência Cardíaca/sangue , Imuno-Histoquímica , Isoproterenol/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Norepinefrina/sangue , Norepinefrina/farmacocinética , Coelhos , Receptores Adrenérgicos beta/metabolismo , Sistema Nervoso Simpático/metabolismo , Simpatolíticos/farmacologiaRESUMO
BACKGROUND: Chronic excessive norepinephrine (NE) causes cardiac sympathetic nerve terminal abnormalities, myocardial beta-adrenergic receptor downregulation, and beta-adrenergic subsensitivity. The present study was carried out to determine whether these changes could be prevented by antioxidants. METHODS AND RESULTS: Ferrets were administered either NE (1.33 mg/d) or vehicle by use of subcutaneous pellets for 4 weeks. Animals were simultaneously assigned to receive either antioxidant vitamins (beta-carotene, ascorbic acid, and alpha-tocopherol) or placebo pellets. NE increased plasma NE 4- to 5-fold but had no effect on heart rate, heart weight, arterial pressure, or left ventricular systolic function. However, myocardial NE uptake activity and NE uptake-1 site density were reduced, as well as cardiac neuronal NE, tyrosine hydroxylase, and neuropeptide Y. In addition, there was a decrease in myocardial beta-adrenergic receptor density with a selective decrease of the beta(1)-receptor subtype, reduction of the high-affinity site for isoproterenol, decreased basal adenylyl cyclase activity, and the adenylyl cyclase responses to isoproterenol, Gpp(NH)p, and forskolin. All of these changes were prevented by antioxidant vitamins. The effects of NE on myocardial beta-adrenergic receptor density, NE uptake-1 carrier site density, and neuronal NE were also prevented by superoxide dismutase or Trolox C. CONCLUSIONS: The toxic effects of NE on the sympathetic nerve terminals are mediated via the formation of NE-derived oxygen free radicals. Preservation of the neuronal NE reuptake mechanism is functionally important, because the antioxidants also prevented myocardial beta-adrenergic receptor downregulation and postreceptor abnormalities. Thus, antioxidant therapy may be beneficial in heart failure, in which cardiac NE release is increased.
Assuntos
Agonistas alfa-Adrenérgicos/farmacocinética , Antioxidantes/farmacologia , Coração/inervação , Norepinefrina/farmacocinética , Receptores Adrenérgicos beta/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Ácido Ascórbico/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cromanos/farmacologia , Furões , Sequestradores de Radicais Livres/farmacologia , Insuficiência Cardíaca/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Masculino , Miocárdio/química , Miocárdio/enzimologia , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Superóxido Dismutase/farmacologia , Vitamina A/farmacologia , Vitamina E/análogos & derivados , Vitamina E/farmacologiaRESUMO
Congestive heart failure is associated with cardiac adrenergic nerve terminal changes and beta-adrenoceptor density downregulation. To study the temporal sequence of these changes, we performed studies in rabbits at 2, 4, and 8 wk of cardiac pacing (360 beats/min) and at 1, 2, and 4 wk after cessation of pacing. Rapid pacing produced left ventricular (LV) dysfunction and an increase in plasma norepinephrine (NE) in 1-2 wk. At week 2, NE uptake activity, NE uptake-1 density, and adenylyl cyclase responses to isoproterenol, 5'-guanylyl imidodiphosphate [Gpp(NH)p], and forskolin reduced. However, immunostained tyrosine hydroxylase profile, beta-adrenoceptor density, and NE histofluorescence did not reduce until 4-8 wk of pacing. After cessation of cardiac pacing, LV function normalized quickly, followed by return of tyrosine hydroxylase and NE profiles in 1 wk and adenylyl cyclase responses to agonists and NE uptake activity in 2 wk. Myocardial beta-adrenoceptor density returned to normal by 4 wk after cessation of pacing. Our results suggest that there is no permanent structural neuronal damage in the myocardium within the first 8 wk of rapid cardiac pacing. Abnormal myocardial NE reuptake mechanism may play an important pathophysiological role in heart failure.
Assuntos
Adenilil Ciclases/metabolismo , Insuficiência Cardíaca/fisiopatologia , Miocárdio/metabolismo , Terminações Nervosas/enzimologia , Norepinefrina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Adenilil Ciclases/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Estimulação Cardíaca Artificial , Colforsina/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Guanilil Imidodifosfato/farmacologia , Hemodinâmica , Miocárdio/citologia , Coelhos , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo , Função Ventricular EsquerdaRESUMO
L-deprenyl, a monoamine oxidase-B inhibitor, partially reversed the age-associated decline in splenic sympathetic noradrenergic (NA) innervation and immune reactivity in old male rats. The purpose of the present study was to examine whether the effects of deprenyl on splenic sympathetic NA nerve fibers and immune functions are mediated through a metabolite of deprenyl, L-desmethyldeprenyl. Old male F344 rats were treated with 0, 0.25, or 1.0 mg L-(-)-deprenyl/kg BW; 0.025, 0.25, or 1.0 mg L-(-)-desmethyldeprenyl/kg BW; and 1.0 mg D-(+)-desmethyldeprenyl/kg BW i.p. daily for 8 weeks. The animals were sacrificed after a 10-day drug wash-out period and the spleens were removed for histofluorescence, immunocytochemistry, neurochemical, and immunological analysis. The volume density of NA nerve fibers was increased in the spleens of deprenyl- and L-desmethyldeprenyl-treated old rats. Con A-induced IFN-gamma production by spleen cells was elevated in 1.0 mg/kg deprenyl- and L-desmethyldeprenyl-treated rats in comparison to saline- and D-desmethyldeprenyl-treated old rats. Deprenyl and desmethyldeprenyl treatment did not alter the percentage of CD5+ T cells, but treatment with 1.0 mg/kg deprenyl and 0.025 mg/kg L-desmethyldeprenyl prevented the decline in the percentage of sIgM(+)B cells in the spleens of old rats. These results suggest that L-desmethyldeprenyl may be as equipotent as deprenyl in preventing age-associated diminution in splenic sympathetic NA innervation and immunocompetence.
Assuntos
Fibras Adrenérgicas/imunologia , Envelhecimento/imunologia , Anfetaminas/farmacologia , Regeneração Nervosa/imunologia , Norepinefrina/fisiologia , Selegilina/farmacologia , Baço/imunologia , Baço/inervação , Fibras Adrenérgicas/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Animais , Masculino , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Baço/efeitos dos fármacosRESUMO
In aged Fischer 344 (F344) rats, sympathetic noradrenergic (NA) innervation of the spleen is markedly diminished compared with young rats. To determine if diminished NA innervation can still provide functional signals to splenic T cells, young (3 months old) and old (17 months old) F344 rats were treated with the NA-selective neurotoxin, 6-hydroxydopamine (6-OHDA) to destroy peripheral NA nerve fibers. In 3-month-old rats, no alterations in spleen cell Con A-induced T cell proliferation, IL-2 or IFN-gamma production were observed up to 15 days after sympathectomy, when splenic NE was maximally depleted. By 21 days post-sympathectomy, when NE levels had partially recovered, Con A-induced proliferation and IFN-gamma production, but not IL-2 production, were reduced in sympathectomized animals. After day 21 post-sympathectomy, no alterations in T cell functions were observed in sympathectomized animals. In 17-month-old rats, spleen cell Con A-induced proliferation and IL-2 production were reduced 5 days after sympathectomy in the absence of changes in CD5+ T cells or IFN-gamma production. Desipramine pretreatment, to block 6-OHDA uptake and prevent sympathectomy, completely blocked the 6-OHDA-induced effects, demonstrating that the destruction of NA nerve fibers is required. After day 5 post-sympathectomy, no sympathectomy-induced alterations in Con A-induced T cell functions were observed in old animals. These differences between young and old rats demonstrate that old animals are more susceptible to loss of sympathetic NA innervation, perhaps because compensatory mechanisms are limited. The sympathectomy-induced reduction in T cell proliferation indicates that splenic NA innervation in old animals, though diminished, can exert a positive regulatory influence on T lymphocyte function. Further study of sympathetic neural-immune interactions in the aged rat may provide a means to improve T cell responsiveness in aging.
Assuntos
Envelhecimento/imunologia , Norepinefrina/metabolismo , Simpatectomia Química , Linfócitos T/imunologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Desipramina/farmacologia , Relação Dose-Resposta a Droga , Ativação Linfocitária/efeitos dos fármacos , Masculino , Oxidopamina/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/inervação , Baço/metabolismo , Fibras Simpáticas Pós-Ganglionares/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Polarização de Fluorescência , Proteínas de Choque Térmico HSP70/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Cardiac sympathetic nerve terminal dysfunction plays an important role in the downregulation of myocardial beta-adrenoceptors in heart failure. To determine whether chronic angiotensin-converting enzyme (ACE) inhibition improved cardiac sympathetic nerve terminal function and hence increased myocardial beta-adrenergic responsiveness, we administered ACE inhibitors to dogs with chronic right-sided heart failure (RHF) produced by tricuspid avulsion and pulmonary artery constriction. The RHF animals exhibited fluid retention, elevated right heart filling pressures, blunted inotropic response to isoproterenol, and reduced beta-adrenoceptor density. These changes were accompanied by decreases in right ventricular norepinephrine (NE) uptake and neuronal NE histofluorescence and tyrosine hydroxylase immunoreactive profiles. ACE inhibitors had no effect on the production of heart failure but greatly reduced the attenuation of cardiac NE uptake, neuronal NE histofluorescence, and tyrosine hydroxylase immunoreactive profiles. ACE inhibition also improved the inotropic response to isoproterenol and restored myocardial beta-adrenoceptor density. The changes probably are caused by reduction of cardiac NE release by ACE inhibition and may contribute to the beneficial effects of ACE inhibitor therapy in patients with chronic heart failure.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Insuficiência Cardíaca/fisiopatologia , Miocárdio/metabolismo , Terminações Nervosas/fisiopatologia , Norepinefrina/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Animais , Cães , Enalapril/farmacologia , Fluorescência , Glioxilatos/farmacologia , Insuficiência Cardíaca/metabolismo , Hemodinâmica , Terminações Nervosas/enzimologia , Peptidil Dipeptidase A/metabolismo , Ramipril/farmacologia , Receptores Adrenérgicos beta/metabolismo , Sistema Nervoso Simpático/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Resistance to several toxic anions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells. In this paper, we report the determination of the overall fold for ArsC, a 16 kDa protein of the ars operon involved in the reduction of arsenate to arsenite, using multidimensional, multinuclear NMR. The protein is found to contain large regions of extensive mobility, particularly in the active site. A model fold, computed on the basis of a preliminary set of NOEs, was found to be structurally homologous to E. coli glutaredoxin, thiol transferases, and glutathione S-transferase. Some kinship to the structure of low molecular weight tyrosine phosphatases, based on rough topological similarity but more so on the basis of a common anion-binding-loop motif H-CX(n)R, was also detected. Although functional, secondary, and tertiary structural homology is observed with these molecules, no significant homology in primary structure was detected. The mobilities of the active site of ArsC and of other enzymes are discussed.
Assuntos
Adenosina Trifosfatases/química , Arsênio/farmacologia , Escherichia coli/química , Bombas de Íon , Complexos Multienzimáticos , Plasmídeos/química , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , ATPases Transportadoras de Arsenito , Sítios de Ligação , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Plasmídeos/genética , Estrutura Secundária de ProteínaRESUMO
This study examined how specific emotions relate to autonomic nervous and immune system parameters and whether cynical hostility moderates this relationship. Forty-one married couples participated in a 15-min discussion about a marital problem. Observers recorded spouses' emotional expressions during the discussion, and cardiovascular, neuroendocrine, and immunologic parameters were assessed throughout the laboratory session. Among men high in cynical hostility, anger displayed during the conflict was associated with greater elevations in systolic and diastolic blood pressure, cortisol, and increases in natural killer cell numbers and cytotoxicity. Among men low in cynical hostility, anger was associated with smaller increases in heart rate and natural killer cell cytotoxicity. These findings suggest that models describing the impact of stress on physiology should be refined to reflect the joint contribution of situational and dispositional variables.
Assuntos
Conflito Psicológico , Células Matadoras Naturais/imunologia , Casamento/psicologia , Adulto , Afeto , Ira , Feminino , Frequência Cardíaca/fisiologia , Hostilidade , Humanos , Linfócitos/imunologia , Masculino , Valor Preditivo dos Testes , PsiconeuroimunologiaRESUMO
The effects of L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, on the concentrations of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-hydroxytryptamine), and 5-hydroxyindoleacetic acid (5-HIAA) in medial basal hypothalamus (MBH), substantia nigra (SN), striatum (Str), and nucleus accumbens (NAc) of young (3 month) and old (21 month) male F344 rats were examined after a 7-day wash-out period following 1, 15, or 30 days of deprenyl treatment in young rats and a 9-day wash-out period after a 10-week deprenyl treatment in old rats. The brain areas were microdissected and the concentrations of neurotransmitters were measured by High Performance liquid chromatography with electrochemical detection (HPLC-EC). Deprenyl administration following the drug wash-out period increased the concentrations of DOPAC in the SN, Str, and in the NAc of young rats but it was decreased in the NAc of old rats. The concentration of HVA was lower in the Str of young deprenyl-treated rats, and in the Str and NAc of old deprenyl-treated rats, but it was higher in the SN of young deprenyl-treated rats. The concentration of 5-HIAA was increased in the MBH, SN, and in the NAc of young deprenyl-treated rats, but it was decreased in the Str and NAc of old deprenyl-treated rats. The concentration of NE was increased in the MBH, SN, Str, and in the NAc of young rats treated with deprenyl and in the MBH of old deprenyl-treated rats. The concentration of 5-HT was increased in the SN of young deprenyl-treated rats. The concentration of DA increased in the Str of both young and old deprenyl-treated rats. We concluded that a drug wash-out period after deprenyl treatment differentially affects the metabolism of catecholamines and indoleamine depending on the region of the brain and that this effect may be due to variation in the kinetics of MAO inhibition.
Assuntos
Química Encefálica/efeitos dos fármacos , Catecolaminas/metabolismo , Indóis/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Selegilina/farmacologia , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Catecolaminas/análise , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , Hipotálamo/química , Hipotálamo/efeitos dos fármacos , Indóis/análise , Masculino , Inibidores da Monoaminoxidase/administração & dosagem , Núcleo Accumbens/química , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Selegilina/administração & dosagem , Substância Negra/química , Substância Negra/efeitos dos fármacosRESUMO
Sympathetic noradrenergic (NA) neuronal activities in the thymus, spleen and mesenteric lymph nodes (MLN) and immune responses in the spleen were examined in young male F344 rats treated daily with 0, 0.25 mg, or 2.5 mg/kg body weight of L-deprenyl, an irreversible monoamine oxidase-B (MAO-B) inhibitor. Rats were treated daily for 1, 15, or 30 days, and sacrificed 7 days after the last deprenyl treatment. Deprenyl treatment increased norepinephrine (NE) content in the spleen without modifying the pattern and density of NA innervation in the splenic white pulp. The concentration of NE was unaltered in the thymus, but it was increased in the MLN of deprenyl-treated rats. One day of treatment with deprenyl decreased splenic NK cell activity while 15 days of deprenyl treatment enhanced splenic NK cell activity. Deprenyl elevated Con A-induced T lymphocyte proliferation following 30 days of treatment, but did not alter spleen cell Con A-induced IL-2 production or the percentage of CD5 + T cells in the spleen. A moderate decrease in the percentage of sIgM + B cells was observed in the spleens of 15- and 30-day deprenyl-treated rats. These results suggest that deprenyl has sympathomimetic action on sympathetic NA nerve fibers in the spleen; the enhancement of NA neuronal activity may contribute to the modulation of immune responses in the spleen.
Assuntos
Fibras Adrenérgicas/química , Tecido Linfoide/inervação , Fármacos Neuroprotetores/farmacologia , Norepinefrina/imunologia , Selegilina/farmacologia , Fibras Adrenérgicas/imunologia , Animais , Linfócitos B/química , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Antígenos CD5/análise , Divisão Celular/imunologia , Concanavalina A/farmacologia , Sistema Nervoso Entérico/química , Sistema Nervoso Entérico/imunologia , Citometria de Fluxo , Interleucina-2/imunologia , Células Matadoras Naturais/química , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/inervação , Tecido Linfoide/imunologia , Masculino , Norepinefrina/análise , Ratos , Ratos Endogâmicos F344 , Baço/imunologia , Baço/inervação , Linfócitos T/química , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Timo/imunologia , Timo/inervaçãoRESUMO
Anti-DNA autoantibodies are a serological hallmark of the autoimmune disorder systemic lupus erythematosus. In a process involving antigen recognition, these antibodies are also believed to mediate the kidney inflammation that results in much of the morbidity and mortality associated with lupus. However, the nature of specific DNA antigens recognized by anti-DNA and the way in which anti-DNA interact with these molecules remains poorly understood. As a first step in defining the molecular basis of anti-DNA interactions, binding site selection experiments were conducted using three clonally related murine monoclonal anti-ssDNA autoantibodies previously isolated from a lupus prone MRL-lpr mouse (Swanson, P. C., Ackroyd, P. C., and Glick, G. D. (1996) Biochemistry 35, 1624-1633). Studying the interaction of these autoantibodies with the selected sequences (and variants) through affinity measurements and footprinting experiments provides evidence for sequence-specific binding of ssDNA. However, despite the similarity in amino acid sequence between the three mAbs, only mAb 11F8 appears to possess sequence specificity. The salient features of the interaction between 11F8 and its selected sequence (e.g., limited dependence of ionic strength upon binding, cross-reactivity, and conformational complementarity) are best described by combining the paradigms invoked to explain protein.nucleic acid and antibody.antigen recognition.