RESUMEN
PURPOSE: Gamete donors and recipients of such donations have been explored by previous studies, which mostly focus on post-donation scenarios. Our study analyses the general willingness to donate oocytes or sperm and focuses on differences between potential female and male donors in attitudes, meanings, and motives in a pre-donation setting. METHODS: An electronic survey (n = 555 students) was used in this anonymous observational study. To enable comparisons between men and women regarding their attitudes, meanings, and motives and their willingness to donate gametes, we designed two separate questionnaires. RESULTS: The sample was divided into three groups based on the willingness to donate: potential donors (n = 133; women: 48.1%, men: 51.9%); doubtful donors (n = 207; women: 75.8%, men: 24.2%); and non-donors (n = 215; women: 68.3%, men: 31.7%). The group of potential male donors (39.2%) was significantly larger than the group of potential female donors (16.9%). Significant differences regarding altruism, the meaning of one's self-worth, and passing on the own genes were found between doubtful and potential donors. Potential donors attached less value to altruism but more value to the enhancement of one's self-worth and passing on one's genes than doubtful donors. The motive of passing on one's genes and altruistic motives were more important to men than to women. CONCLUSION: This study helps to create a better understanding of potential donors in the existing donation framework and supports the evaluation of the given regimes in the context of designing an improved framework.
Asunto(s)
Donación de Oocito/tendencias , Oocitos/crecimiento & desarrollo , Espermatozoides/crecimiento & desarrollo , Obtención de Tejidos y Órganos/tendencias , Adulto , Altruismo , Actitud , Austria/epidemiología , Femenino , Humanos , Masculino , Donación de Oocito/éticaRESUMEN
Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B-substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 µM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.
Asunto(s)
Biopterinas/análogos & derivados , Trasplante de Páncreas/métodos , Daño por Reperfusión/prevención & control , Donantes de Tejidos , Animales , Biopterinas/uso terapéutico , Isquemia Fría , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación , Modelos Animales , Páncreas/irrigación sanguínea , Páncreas/patología , Trasplante de Páncreas/fisiología , Ácido Peroxinitroso/biosíntesis , Trasplante Isogénico , Tirosina/análogos & derivados , Tirosina/biosíntesisRESUMEN
Much attention has been paid in initial biochemical studies on the ability of indoleamine 2,3-dioxygenase to use superoxide as substrate to cleave tryptophan to N-formyl kynurenine. This ability, however, is limited to the ferric form of the enzyme only, whereas the ferrous form requires oxygen rather than superoxide as substrate. As long as the enzyme is held in the ferrous form, high yield formation of product proceeds from the ferrous oxygen tryptophan ternary complex without the participation of superoxide. Enzyme assays in homogenates are carried out in presence of Methylene Blue, ascorbate and catalase. Ascorbate can be replaced by other reductants like e.g. tetrahydrobiopterin. Experiments with alteration of intracellular tetrahydrobiopterin concentrations in intact interferon-gamma treated cells clearly showed that tetrahydrobiopterin is not required for the indoleamine 2,3-dioxygenase reaction. In homogenates of interferon-gamma treated T-24 cells, substrates of xanthine oxidase did not stimulate the indoleamine 2,3-dioxygenase reaction, nor did allopurinol inhibit the reaction, nor did superoxide dismutase alter indoleamine 2,3-dioxygenase activity irrespective of the reductant used. From these experiments we concluded that molecular oxygen rather than superoxide is used in cell homogenates by indoleamine 2,3-dioxygenase to cleave L-tryptophan. A detailed analysis of available reports on oxygen and superoxide utilization by indoleamine 2,3-dioxygenase gives a comprehensive picture that the enzyme uses oxygen bound to the ferrous enzyme for cleavage of tryptophan, that the enzyme needs to be held by reductants in the ferrous state in enzyme incubations, and that superoxide is one of the reductants capable performing this reduction.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/farmacología , Animales , Línea Celular Tumoral , Colorantes/metabolismo , Humanos , Azul de Metileno/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
The immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) is activated by interferon-gamma (IFN-gamma) and via tryptophan depletion, suppresses adaptive T cell-mediated immunity in inflammation, host immune defense, and maternal tolerance. Its role in solid organ transplantation is still unclear. Therefore, we investigated the usefulness of IDO-mediated tryptophan catabolism in the evaluation of kidney allograft rejection. Blood, urine, and tissue samples were collected from 34 renal transplant patients without rejection and from nine patients with biopsy-confirmed episodes of acute rejection (n=12). Concentrations of kynurenine and tryptophan in serum and urine were analyzed by high-pressure liquid chromatography. Kynurenine to tryptophan ratio (kyn/trp) was calculated to estimate IDO activity. Immunostaining for IDO was performed on renal biopsies. Neopterin was assessed using radioimmunoassay. Kyn/trp and neopterin were detectable at low levels in serum of healthy volunteers and were increased in non-rejecting allograft recipients. Serum levels of kyn/trp were higher in recipients with rejection compared to non-rejectors as early as by day 1 post-surgery. Rejection episodes occurring within 13+/-5.9 days after transplantation were accompanied by elevated kyn/trp in serum (114+/-44.5 micromol/mmol, P=0.001) and urine (126+/-65.9 micromol/mmol, P=0.02) compared to levels during stable graft function. Kyn/trp correlated significantly with neopterin suggesting an IFN-gamma-induced increase in IDO activity. Immunostaining showed upregulation of IDO in rejection biopsies, localized in tubular-epithelial cells. Non-rejected grafts displayed no IDO expression. Acute rejection is associated with simultaneously increased serum and urinary kyn/trp in patients after kidney transplantation. Thus, IDO activity might offer a novel non-invasive means of immunomonitoring of renal allografts.
Asunto(s)
Rechazo de Injerto/diagnóstico , Rechazo de Injerto/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/fisiología , Enfermedad Aguda , Adulto , Anciano , Creatinina/sangre , Células Epiteliales/enzimología , Femenino , Rechazo de Injerto/patología , Humanos , Inmunohistoquímica , Riñón/enzimología , Riñón/patología , Trasplante de Riñón/patología , Quinurenina/sangre , Masculino , Persona de Mediana Edad , Neopterin/sangre , Triptófano/sangreRESUMEN
In this study we investigated the effect of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases, on ischemia-reperfusion injury (IRI) following murine pancreas transplantation. Pancreatic grafts were exposed to prolonged cold ischemia times (CIT) and different treatment regimens: normal saline (S), S + 16 h CIT, BH4 50 mg/kg + 16 h CIT. Nontransplanted animals served as controls. Graft microcirculation was analyzed by means of functional capillary density (FCD) and capillary diameters (CD) after 2 h reperfusion using intravital microscopy. Quantification of inflammatory responses (mononuclear infiltration) and endothelial disintegration (edema formation) was done by histology (hematoxylin and eosin), and peroxynitrite formation assessed by nitrotyrosine immunostaining. FCD was significantly reduced after prolonged CIT, paralleled by increased peroxynitrite formation as compared with controls (all p < 0.05). Microcirculatory changes correlated significantly with intragraft peroxynitrite generation (Spearman: r = -0.56; p < 0.01). Pancreatic grafts treated with BH4 displayed markedly higher FCD values (p < 0.01) and abrogated nitrotyrosine staining (p = 0.03). CD were not significantly different in any group. Histology showed increased inflammation, interstitial edema, hemorrhage, acinar vacuolization and focal areas of necrosis after 16 h CIT, which was diminished by BH4 administration (p < 0.01). BH4 treatment significantly reduces post-ischemic deterioration of microcirculation as well as histologic damage and might be a promising novel strategy in attenuating IRI following pancreas transplantation.
Asunto(s)
Biopterinas/análogos & derivados , Microcirculación/efectos de los fármacos , Microcirculación/patología , Trasplante de Páncreas , Daño por Reperfusión/inducido químicamente , Daño por Reperfusión/patología , Animales , Biopterinas/efectos adversos , Biopterinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación/metabolismo , Ácido Peroxinitroso/metabolismo , Daño por Reperfusión/metabolismo , Trasplante Homólogo/patología , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Tetrahydrobiopterin (H4-biopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan, of ether lipid oxidase, and of the three nitric oxide synthase (NOS) isoenzymes. As a consequence, H4-biopterin plays a key role in a vast number of biological processes and pathological states associated with neurotransmitter formation, vasorelaxation, and immune response. In mammals, its biosynthesis is controlled by hormones, cytokines and certain immune stimuli. This review aims to summarize recent developments concerning regulation of H4-biopterin biosynthetic and regulatory enzymes and pharmacological effects of H4-biopterin in various conditions, e.g. endothelial dysfunction or apoptosis of neuronal cells. Also, approaches towards gene therapy of diseases like the different forms of phenylketonuria or of Parkinson's disease are reviewed. Additional emphasis is given to H4-biopterin biosynthesis and function in non-mammalian species such as fruit fly, zebra fish, fungi, slime molds, the bacterium Nocardia as well as to the parasitic protozoan genus of Leishmania that is not capable of pteridine biosynthesis but has evolved a sophisticated salvage network for scavenging various pteridine compounds, notably folate and biopterin.
Asunto(s)
Biopterinas/análogos & derivados , Biopterinas/biosíntesis , Animales , Bacterias/metabolismo , Biopterinas/deficiencia , Biopterinas/farmacología , Biopterinas/fisiología , Diferenciación Celular/fisiología , Células Eucariotas/metabolismo , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Terapia Genética , HumanosRESUMEN
The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered.
Asunto(s)
Quimiocinas CXC/genética , Cromosomas Humanos Par 4/genética , Hibridación Fluorescente in Situ/métodos , Péptidos y Proteínas de Señalización Intercelular , Ratones/genética , Familia de Multigenes , Animales , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Evolución Molecular , Humanos , Especificidad de la EspecieRESUMEN
Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.
Asunto(s)
Quimiocinas CXC/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Quimiocina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas CXC/química , Quimiocinas CXC/aislamiento & purificación , Quimiotaxis , Cricetinae , Humanos , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Receptores CXCR3 , Receptores de Quimiocina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Linfocitos T/citología , Linfocitos T/metabolismo , TransfecciónRESUMEN
Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I-TAC, SCYB11, ss-R1, H174, IP-9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP-10/crg-2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3-expressing human T lymphocytes as well as for 300--19 pre-B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage-like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow-derived dendritic cells) and stimulus (interferons, LPS, IL-1 beta and TNF-alpha), an up to 10,000-fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real-time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up-regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN-gamma which were induced with similar kinetics by several orders of magnitude.
Asunto(s)
Quimiocinas/farmacología , Rechazo de Injerto , Receptores de Quimiocina/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Regulación hacia Arriba , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas/antagonistas & inhibidores , Quimiocinas/genética , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/genética , Quimiocinas CXC/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Cricetinae , Relación Dosis-Respuesta a Droga , Trasplante de Corazón , Humanos , Interferón gamma/genética , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR3 , Receptores de Quimiocina/genética , Linfocitos T/metabolismo , TransfecciónRESUMEN
We tested the 4-amino analogue of tetrahydrobiopterin (H(4)aminobiopterin), a novel pterin-based inhibitor of nitric oxide synthases, for its efficacy in a murine cardiac-transplant model employing an improved cuff technique. We treated groups of 5 animals each for the first 7 post-operative days with various doses of H(4)aminobiopterin, with Cyclosporin A (15 mg/kg/day), or no treatment. H(4)aminobiopterin (3 times 50 mg/kg/day) proved to be as efficient as high-dose Cyclosporin A (15 mg/kg/day) in prolonging allograft survival and in suppressing histologic changes caused by the immunoreaction. Surprisingly, the doses of H(4)aminobiopterin effective in prolonging allograft survival did not change the plasma nitrite plus nitrate, or the expression of inducible nitric oxide synthase, interferon-gamma, tumor necrosis factor-alpha, and B7-1 (CD80), indicating that H(4)aminobiopterin may act through a novel, yet undiscovered mechanism.
Asunto(s)
Biopterinas/farmacología , Inhibidores Enzimáticos/farmacología , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Biopterinas/análogos & derivados , Biopterinas/farmacocinética , Ciclosporina/administración & dosificación , Ciclosporina/farmacocinética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Semivida , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacocinética , Interferón gamma/análisis , Interferón gamma/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Revascularización Miocárdica/métodos , Miocardio/patología , Necrosis , Tolerancia al Trasplante/efectos de los fármacosRESUMEN
The myxomycete Physarum polycephalum expresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares < 39% amino acid identity with known NOSs but contains conserved binding motifs for all NOS cofactors. It lacks the sequence insert responsible for calcium dependence in the calcium-dependent NOS isoenzymes. NOS expression was strongly up-regulated in Physarum macroplasmodia during the 5-day starvation period needed to induce sporulation competence. Induction of both NOS and sporulation competence were inhibited by glucose, a growth signal and known repressor of sporulation, and by L-N6-(1-iminoethyl)-lysine (NIL), an inhibitor of inducible NOS. Sporulation, which is triggered after the starvation period by light exposure, was also prevented by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase. In addition, also expression of lig1, a sporulation-specific gene, was strongly attenuated by NIL or ODQ. 8-Bromo-cGMP, added 2 h before the light exposure, restored the capacity of NIL-treated macroplasmodia to express lig1 and to sporulate. This indicates that the second messenger used for NO signaling in sporulation of Physarum is cGMP and links this signaling pathway to expression of lig1.
Asunto(s)
Proteínas de Ciclo Celular/genética , Óxido Nítrico Sintasa/genética , Physarum polycephalum/enzimología , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Secuencia Conservada , GMP Cíclico/metabolismo , Inducción Enzimática , Glucosa/metabolismo , Luz , Datos de Secuencia Molecular , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/biosíntesis , Filogenia , Physarum polycephalum/genética , Physarum polycephalum/metabolismo , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Transducción de Señal , Esporas/enzimología , Esporas/fisiologíaRESUMEN
GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H(4)-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7 kDa) and 195 (22.1 kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-gamma-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9 kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7 kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3'-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9 kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity.
Asunto(s)
Empalme Alternativo , GTP Ciclohidrolasa/genética , Monocitos/enzimología , Physarum polycephalum/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Exones , GTP Ciclohidrolasa/química , Humanos , Intrones , Datos de Secuencia Molecular , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Células Tumorales CultivadasAsunto(s)
Antioxidantes/uso terapéutico , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón , Animales , Evaluación Preclínica de Medicamentos , Rechazo de Injerto/metabolismo , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Factores de Tiempo , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.
Asunto(s)
Ácido Ascórbico/farmacología , Biopterinas/análogos & derivados , Endotelio Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Pterinas , Biopterinas/metabolismo , Células Cultivadas , Citrulina/metabolismo , GMP Cíclico/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , GTP Ciclohidrolasa/antagonistas & inhibidores , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Humanos , Hipoxantinas/farmacología , Óxido Nítrico Sintasa/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pteridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Soluciones , Cordón UmbilicalRESUMEN
Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.
Asunto(s)
Biopterinas/análogos & derivados , Dihidropteridina Reductasa/efectos de los fármacos , Óxido Nítrico Sintasa/efectos de los fármacos , Fenilalanina Hidroxilasa/efectos de los fármacos , Animales , Biopterinas/química , Biopterinas/farmacología , Dihidropteridina Reductasa/metabolismo , Activación Enzimática , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Fenilalanina Hidroxilasa/metabolismo , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A T-cell attracting CXC chemokine phylogenetically related to MIG and SCYB10 was recently characterized and termed SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11). Here, we cloned the cDNA of the murine homologue of this protein, Scyb11, from interferon-gamma/lipopolysaccharide-stimulated RAW264.7 mouse macrophage-like cells. The nucleotide sequence of Scyb11 shares 63% identity with its human counterpart. It encodes a 100 amino acid immature protein of 11,265 Da which contains a putative signal peptide of 21 amino acids. The molecular mass of the mature protein was calculated to be 9,113 Da. Sequence identity of the murine and human SCYB11 proteins is 68%. Phylogenetic tree analysis of mouse CXC chemokines places SCYB11 together with the murine homologues of MIG and SCYB10 (Crg-2/muIP-10) on an individual branch. A genomic sequence was obtained by genome walking and subcloning DNA fragments from a BAC clone containing Scyb11. Like human SCYB11, Scyb11 contains 4 exons with intron/exon boundaries at positions comparable to the human gene. Whereas introns 2 and 3 are of similar length in the murine and human genes, intron 1 of Scyb11 contains 1,260 bp more than intron 1 of the human gene. Intron 1 of Scyb11 is also characterized by a 201-bp stretch with repetitive sequences of high cryptic simplicity. Using a BAC clone containing Scyb11, this gene could be mapped to chromosome 5 at position 5E3. Since human SCYB11 is localized on 4q21.2, this result confirms the mouse/human homology of the two chromosome regions.
Asunto(s)
Quimiocinas CXC/genética , Mapeo Cromosómico , Exones/genética , Intrones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Quimiocina CXCL11 , Quimiocinas CXC/química , Paseo de Cromosoma , Clonación Molecular , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Histamine, an important inflammatory mediator in allergic diseases and asthma, was reported to have modulatory effects on T cells by down-regulating Th1-type cell cytokines like interleukin 2 (IL-2) and interferon-gamma (IFN-gamma). In this study we examined the effect of histamine and the histamine-receptor antagonists cimetidine and diphenhydramine on the production of neopterin after stimulation with IFN-gamma in the myelomonocytoma cell line THP-1. Increasing concentrations of histamine markedly suppressed IFN-gamma induced neopterin formation. Simultaneous preincubation of THP-1 cells with histamine, IFN-gamma and different concentrations of the H(2)-receptor antagonist cimetidine showed a clear antagonizing effect on neopterin formation. In contrast, the H(1)-receptor antagonist diphenhydramine was not able to abrogate the suppressive effect of histamine on neopterin production. Our results suggest, that histamine may be a potent inhibitor of effects or mechanisms induced by IFN-gamma in monocytes/macrophages. Cimetidine, and possibly other H(2)-receptor antagonists, may reverse down-regulatory actions of endogenously formed histamine on activated monocytic cells.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Histamina/farmacología , Neopterin/antagonistas & inhibidores , Neopterin/biosíntesis , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismo , Cimetidina/farmacología , Difenhidramina/farmacología , Relación Dosis-Respuesta Inmunológica , Antagonistas de los Receptores Histamínicos/farmacología , Humanos , Leucemia Mielomonocítica AgudaRESUMEN
We studied the effects of a novel pterin antagonist of NO synthase, the 4-amino analogue of tetrahydrobiopterin (4-ABH4), in a rat model of endotoxic shock and compared its properties with those of N(G)-monomethyl L-arginine (L-NMMA). Treatment with a bolus dose of 4-ABH4 at 2 h after LPS challenge significantly improved the 6-day survival rate, compared with the controls treated with saline. L-NMMA treatment did not significantly influence the survival rate. This bolus treatment, using either compound, had no effect on the plasma nitrite + nitrate or plasma IL-6 levels. The continuous infusion of 4-ABH4 efficiently suppressed the enhanced calcium-dependent/independent NO synthase activities induced by endotoxin in lung homogenates and completely suppressed the increase in plasma nitrite + nitrate caused by endotoxin at 5 h, with no significant difference compared with the L- NMMA treatment. Treatment of RAW264.7 murine macrophages with 4-ABH4 but not with L-NMMA suppressed endotoxin-induced tumor necrosis factor-alpha release by the cells, whereas nitrite in the supernatant decreased in a dose-dependent fashion in both assay systems. Our data show that 4-ABH4, an inhibitor of inducible NO synthase, significantly improves survival in a rat model of endotoxic shock when administered in a bolus dose that does not reduce plasma total nitrite + nitrate levels. Because we observed no overt signs of toxicity and no influence on organ-specific tetrahydrobiopterin levels, we conclude that the novel compound 4-ABH4 is a promising drug candidate for protection against endotoxin-related mortality.
Asunto(s)
Biopterinas/análogos & derivados , Endotoxemia/prevención & control , Animales , Biopterinas/farmacología , Línea Celular , Endotoxemia/enzimología , Endotoxemia/inmunología , Inhibidores Enzimáticos/farmacología , Interleucina-6/sangre , Lipopolisacáridos/toxicidad , Masculino , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Sprague-Dawley , Choque Séptico/enzimología , Choque Séptico/inmunología , Choque Séptico/prevención & control , Factor de Necrosis Tumoral alfa/metabolismo , omega-N-Metilarginina/farmacologíaRESUMEN
In order to reduce animal testing for quality control of pharmaceutical agents intended for parenteral use, the Limulus amebocyte lysate (LAL) assay is now being accepted in many cases as an alternative to measuring pyrogenic activity of samples in rabbits. However, since the LAL test is specific for cell wall components from Gram-negative bacteria and is sometimes difficult to perform in samples containing large amounts of protein, this alternative still leaves a considerable diagnostic gap. Here, we have optimized a previously established test based on assessing the formation of neopterin or nitrite in interferon-gamma-treated human (THP-1) or murine (J774A.1, RAW264.7) monocytoid cell lines, respectively, in response to bacterial pyrogens. Optimal results were obtained either with THP-1 cells in serum-containing media and using a high concentration of interferon-gamma (IFN-gamma) or with RAW264.7 cells in serum-free media and independent of the IFN-gamma dose. Results were significantly correlated with those obtained by another cell-culture-based assay in which formation of tumor necrosis factor-alpha by THP-1 1G3 cells was assessed. Also in RAW264.7 murine monocytoid cells, formation of nitrite and of tumor necrosis factor-alpha in response to a variety of samples was correlated. Samples shown to be pyrogenic in rabbits in a previous study were unambiguously detected with the test presented here. As expected, the LAL test was negative with cell-free supernatants from Staphylococcus aureus66 kDa). Taken together, these results indicate that the use of monocytoid cell lines and the detection of metabolites which are triggered in the course of immunostimulation could fill the gap left by the LAL test and help to further reduce animal testing for pyrogens.