RESUMO
The severe cutaneous adverse reaction epidermal necrolysis (EN) which includes toxic epidermal necrolysis and the milder Stevens-Johnson syndrome is characterized by epidermal loss due to massive keratinocyte apoptosis and/or necroptosis. EN is often caused by a drug mediating a specific TCR-HLA interaction via the (pro)hapten, pharmacological interaction or altered peptide loading mechanism involving a self-peptide presented by keratinocytes. (Memory) CD8 + T cells are activated and exhibit cytotoxicity against keratinocytes via the perforin/granzyme B and granulysin pathway and Fas/FasL interaction. Alternatively drug-induced annexin release by CD14 + monocytes can induce formyl peptide receptor 1 death of keratinocytes by necroptosis. Subsequent keratinocyte death stimulates local inflammation, activating other immune cells producing pro-inflammatory molecules and downregulating regulatory T cells. Widespread epidermal necrolysis and inflammation can induce life-threatening systemic effects, leading to high mortality rates. Research into genetic susceptibility aims to identify risk factors for eventual prevention of EN. Specific HLA class I alleles show the strongest association with EN, but risk variants have also been identified in genes involved in drug metabolism, cellular drug uptake, peptide presentation and function of CD8 + T cells and other immune cells involved in cytotoxic responses. After the acute phase of EN, long-term symptoms can remain or arise mainly affecting the skin and eyes. Mucosal sequelae are characterized by occlusions and strictures due to adherence of denuded surfaces and fibrosis following mucosal inflammation. In addition, systemic pathology can cause acute and chronic hepatic and renal symptoms. EN has a large psychological impact and strongly affects health-related quality of life among EN survivors.
Assuntos
Síndrome de Stevens-Johnson , Epiderme , Humanos , Queratinócitos , Qualidade de Vida , Pele , Síndrome de Stevens-Johnson/genéticaRESUMO
Because of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously that rapamycin inhibits the outgrowth of primary skin carcinomas induced by UV radiation. However, the tumors that did grow out showed an altered p53 mutation spectrum. Here, we investigated whether this shift in p53 mutations already occurred in the smallest tumors, which were not affected in onset. We found that rapamycin did not alter the mutational spectrum in small tumors and in preceding microscopic clusters of cells expressing mutant-p53. However, rapamycin did reduce the number of these cell clusters. As this reduction did not affect tumor onset, we subsequently investigated whether rapamycin merely suppressed expression of mutated p53. This was not the case, as we could demonstrate that switching from a diet with rapamycin to one without, or vice versa, did not affect the number of existing mutant-p53 expressing cell clusters. Hence, rapamycin actually reduced the formation of mutant-p53 cell clusters. In wild-type and p53-mutant mice, we could not measure a significant enhancement of UV-induced apoptosis, but we did observe clear enhancement in human skin equivalents. This was associated with a clear suppression of HIF1α accumulation. Thus, we conclude that rapamycin reduces the formation of mutant-p53-expressing cell clusters without affecting tumor onset, suggesting that tumors grow out of a minor subset of cell clusters, the formation of which is not affected by rapamycin.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Genes p53 , Mutação , Neoplasias Induzidas por Radiação/prevenção & controle , Sirolimo/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/análise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Camundongos Pelados , Neoplasias Induzidas por Radiação/genética , Raios UltravioletaRESUMO
AIM: Recent studies suggested a role for IL31 in the pathogenesis of pruritus and disease severity in patients with cutaneous T cell lymphomas (CTCL). However, discrepant results were reported for IL31 serum levels, transcriptional expression levels or immunohistochemistry studies and its relation to pruritus intensity and/or disease severity in CTCL. Most studies did not distinguish between different CTCL variants. We investigated IL31 serum levels in different subtypes of CTCL, including Mycosis Fungoides (MF) (typically not pruritic), Folliculotropic Mycosis Fungoides (FMF) and Sézary syndrome (SS) (both often pruritic). METHODS: From 54 CTCL patients (17 SS, 21 FMF and 16 classic MF) serum samples were analyzed with a high sensitivity V-PLEX immunoassay for IL31. The study group included 35/54 (65%) patients with complaints of pruritus. Thirty-five patients had advanced stage disease (≥stage IIB). A visual analog scale score (VAS score) for pruritus was available in 29 CTCL patients (7 SS, 9 FMF and 13 classic MF) and in other cases complaints of pruritus were retrieved from medical records. qPCR analyses for IL31 expression were performed in lesional skin biopsies from 8 CTCL patients. Serum samples from 4 healthy individuals without pruritus and from 5 atopic dermatitis (AD) patients with severe pruritus were included as controls. RESULTS: In 11/54 (20%) of CTCL patients low serum levels of IL31 were detected (mean 0.48 pg/mL, range 0.20-1.39 pg/mL) including 6/17 (35%) SS patients (mean 0.57 pg/mL) and 5/21 (24%) FMF patients (mean 0.33 pg/mL). All 11 patients with detectable levels of IL31 reported complaints of moderate to severe pruritus and 9/11 patients presented with advanced stage disease (≥IIB). qPCR analyses resulted in lowly expressed IL31 expression levels in 4 of 8 patients; these patients all suffered from pruritus and advanced stage disease. CONCLUSIONS: Translational and transcriptional expression levels of IL31 were very low or undetectable in CTCL patients. Detectable low IL31 serum levels were exclusively observed in SS and FMF patients and not in patients with classic MF. However, these marginal IL31 levels in a small proportion of CTCL patients do not support an essential role for IL31 in CTCL patients.
RESUMO
Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (>or=4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.
Assuntos
Imunossupressores/administração & dosagem , Ácido Micofenólico/análogos & derivados , Neoplasias Induzidas por Radiação/tratamento farmacológico , Sirolimo/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proteínas Angiogênicas/genética , Animais , Western Blotting , Dieta , Feminino , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Pelados , Mutação/genética , Ácido Micofenólico/administração & dosagem , Neoplasias Induzidas por Radiação/irrigação sanguínea , Neoplasias Induzidas por Radiação/genética , Neoplasias Induzidas por Radiação/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Irradiação Corporal TotalAssuntos
Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Deleção de Genes , Transtornos Linfoproliferativos/diagnóstico , Micose Fungoide/diagnóstico , Neoplasias Cutâneas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica , Feminino , Genes p16 , Humanos , Antígeno Ki-1 , Transtornos Linfoproliferativos/genética , Masculino , Pessoa de Meia-Idade , Micose Fungoide/genética , Prognóstico , Neoplasias Cutâneas/genéticaRESUMO
Polymorphic light eruption (PLE) is a putative delayed-type allergic reaction to (solar) ultraviolet (UV) exposure. Inadequate immune suppression after UVB-induced sunburn appears to be associated with reduced trafficking of Langerhans cells (LCs) out of and neutrophils into the epidermis of patients sensitive to UVB provocation of PLE. Therefore, we investigated whether pro-inflammatory and chemotactic cytokines are differentially expressed in UVB-irradiated skin of UVB-provocable PLE patients (n = 6) and age- and gender-matched healthy controls (n = 6). Interstitial interleukin-1alpha (IL-1alpha), IL-1beta, IL-1Ra, IL-4, IL-8, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), MIP-1beta and monocyte chemotactic protein-1 (MCP-1) were measured in suction blister fluid raised 16 h after exposure to 0, three and six minimal erythemal UVB doses. In unirradiated skin, the IL-1Ra levels were significantly lower in the PLE patients than in controls (P < 0.05). IL-8 and TNF-alpha levels increased strongly upon UVB irradiation in both groups. No differential shifts in cytokine profiles were found that could explain a reduced trafficking of Langerhans cells and neutrophils in PLE patients. Dose-trend analyses showed that UVB irradiation caused significant increases in IL-1alpha in both groups, and that the levels of IL-1alpha and IL-1beta were on average twofold higher in the PLE group (P = 0.03 and P = 0.004, respectively.). Accordingly, the ratios of IL-1Ra over IL-1alpha and over IL-1beta were overall lower in the skin of PLE patients (P = 0.015 and P < 0.001, respectively.). This shift in cytokines in UVB-irradiated skin of PLE patients reveals an amplified early pro-inflammatory cytokine response, which may contribute to the allergic reaction to UVB radiation.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1/metabolismo , Transtornos de Fotossensibilidade/metabolismo , Pele/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta , Adulto , Estudos de Casos e Controles , Movimento Celular/efeitos da radiação , Feminino , Humanos , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Células de Langerhans/patologia , Células de Langerhans/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Neutrófilos/efeitos da radiação , Transtornos de Fotossensibilidade/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Lymnaea/fisiologia , Proteínas de Membrana/fisiologia , Ocitocina/análogos & derivados , Ocitocina/fisiologia , Receptores de Vasopressinas/fisiologia , Vasopressinas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/farmacologia , Canais de Cloreto/fisiologia , Clonagem Molecular , Condutividade Elétrica , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Oócitos/fisiologia , Especificidade de Órgãos , Ocitocina/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Ocitocina/química , Receptores de Vasopressinas/química , Receptores de Vasopressinas/genética , Alinhamento de Sequência , XenopusRESUMO
CXCL 11, encoded by the cDNA sequences designated beta-R1, H-174, or I-TAC, is a CXC chemokine ligand for CXCR3 and assumed to be involved in inflammatory diseases characterized by the presence of activated T-cells. We here describe the genomic organization (four exons interrupted by three introns of 585, 98 and 230 bp) and sequence including 960 bp from the immediate 5'-upstream region of the human CXCL 11 gene. Within the promoter region, consensus sequences for regulatory elements (ISRE, GAS, NF-kappaB) important for cytokine-induced gene transcription were identified. The effect of (pro)inflammatory cytokines on CXCL 11 mRNA expression in monocytic cell lines (THP-1, U937) and primary cultures of dermal fibroblasts and endothelial cells were examined using Northern blot analysis. For these cell types, IFN-gamma was a potent inducer of CXCL 11 transcription, which was synergistically enhanced by TNF-alpha.
Assuntos
Quimiocinas CXC/genética , Sequência de Bases , Linhagem Celular , Quimiocina CXCL11 , Quimiocinas CXC/química , Éxons , Biblioteca Genômica , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
CD163 is a member of the group B scavenger receptor cysteine-rich (SRCR) superfamily. This study describes aspects of the tissue distribution, the regulation of expression, and signal transduction after cross-linking of this receptor at the cell surface of macrophages. CD163 showed an exclusive expression on resident macrophages (e.g., red pulp macrophages, alveolar macrophages). The expression was inducible on monocyte-derived macrophages by glucocorticoids but not by interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma. The combination of IL-4 or GM-CSF with glucocorticoids resulted in a further increase. Subcellular analysis of alveolar macrophages by immunoelectron microscopy showed a plasma membrane localization of the antigen. Cross-linking of CD163 with monoclonal antibody induced a protein tyrosine kinase-dependent signal that resulted in (1) slow-type calcium mobilization, (2) inositol triphosphate production, and (3) secretion of IL-6 and GM-CSF. The data suggest a function for the SRCR-superfamily receptor CD163 in the regulation of inflammatory processes by macrophages.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Receptores de Superfície Celular , Transdução de Sinais , Animais , Antígenos de Diferenciação Mielomonocítica/genética , Reagentes de Ligações Cruzadas , Citocinas/biossíntese , Humanos , Immunoblotting , Líquido Intracelular , Macrófagos/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Depuradores , Frações Subcelulares/metabolismo , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Células U937RESUMO
Differentiation between allergic and irritant contact dermatitis reactions is difficult, as both inflammatory diseases are clinically, histologically, and immunohistologically very similar. Previous studies in mice revealed that the chemokine IP-10 is exclusively expressed in allergic contact dermatitis reactions. In the present study, we investigated whether the mRNA expression of IP-10 and the related CXCR3 activating chemokines, Mig and IP-9 are also differentially expressed in human allergic contact dermatitis and irritant contact dermatitis reactions. Skin biopsies from allergic (13 cases) and sodium lauryl sulfate-induced irritant patch test reactions (13 cases), obtained 1-72 h after patch testing, were studied by means of an in situ hybridization technique. Results of chemokine mRNA expression were correlated with clinical scoring, histology, and immunohistochemical data including the proportion of inflammatory cells expressing CXCR3, the receptor for IP-10, Mig, and IP-9, and ICAM-1 and HLA-DR expression on keratinocytes. IP-10, Mig, and IP-9 mRNA were detected in seven of nine allergic contact dermatitis reactions after 24-72 h, but not in sodium lauryl sulfate-induced irritant contact dermatitis reactions. ICAM-1 expression by keratinocytes was only found in allergic contact dermatitis reactions and correlated with chemokine expression. Moreover, up to 50% of the infiltrating cells in allergic contact dermatitis expressed CXCR3, in contrast to only 20% in irritant contact dermatitis reactions. In conclusion, we have demonstrated differences in chemokine expression between allergic contact dermatitis and irritant contact dermatitis reactions, which might reflect different regulatory mechanisms operating in these diseases and may be an important clue for differentiation between allergic contact dermatitis and irritant contact dermatitis reactions.
Assuntos
Quimiocinas CXC/genética , Dermatite Alérgica de Contato/imunologia , Dermatite Irritante/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Testes do Emplastro , RNA Mensageiro/análise , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Antígenos HLA-DR/análise , Humanos , Molécula 1 de Adesão Intercelular/análise , Interferon gama/farmacologia , Queratinócitos/química , Receptores CXCR3 , Receptores de Quimiocinas/análiseRESUMO
Epidermal infiltration by neoplastic CD4+ T cells is a characteristic histologic feature of early stage mycosis fungoides, the most common type of cutaneous T cell lymphoma (CTCL). The mechanisms involved in epidermotropism are unknown. It has been suggested that the CXC chemokines IL-8 and interferon-gamma inducible protein 10 (IP-10) may play a role, but evidence that these chemokines are produced within the epidermis in epidermotropic CTCL is lacking. In this study skin biopsies from 17 CTCL patients, including 12 mycosis fungoides, four pleomorphic CTCL, and one CD8+ CTCL, were investigated for epidermal IL-8 and IP-10 mRNA expression by RNA in situ hybridization. In addition, the expression of monokine induced by gamma-interferon (Mig) mRNA, a CXC chemokine closely related to IP-10, was studied as well. The expression of IL-8 receptors A and B (CXCR1 and CXCR2, respectively) was investigated by immunohistochemistry. The results were correlated with the number and phenotype of epidermotropic T cells. Epidermal expression of IP-10 and Mig mRNA was detected in 10 of 11 and seven of 11 epidermotropic CTCL, respectively, but not in five nonepidermotropic CTCL biopsies or normal human skin. Epidermal IP-10 and Mig mRNA expression correlated with epidermal infiltration of CD4+ T cells, but not of CD8+ T cells. IL-8 mRNA was demonstrated in the epidermis of only two of 15 CTCL biopsies, and was associated, in both cases, with accumulation of neutrophils. Consistently, immunostaining of the (intraepidermal) T cells with antibodies against CXCR1 and CXCR2 was not observed. In conclusion, the results of this study indicate that IP-10, and to a lesser extent Mig, but not IL-8 is involved in the preferential infiltration of neoplastic CD4+ T cells in CTCL.
Assuntos
Quimiocinas CXC/genética , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/farmacologia , Interleucina-8/genética , Linfoma Cutâneo de Células T/metabolismo , RNA Mensageiro/análise , Neoplasias Cutâneas/metabolismo , Antígenos CD/fisiologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Receptores de Quimiocinas/fisiologia , Receptores de Interleucina/fisiologia , Receptores de Interleucina-8A , Receptores de Interleucina-8BRESUMO
Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.
Assuntos
Quimiocinas CXC/metabolismo , Queratinócitos/metabolismo , Receptores de Citocinas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiologia , Quimiotaxia , Clonagem Molecular , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Hibridização In Situ , Inflamação/metabolismo , Ligantes , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Receptores de Citocinas/genética , Linfócitos T/citologiaRESUMO
In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.
Assuntos
Copulação/fisiologia , Lymnaea/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Neuropeptídeos/análise , Neurotransmissores/análise , Animais , Imuno-Histoquímica , Lymnaea/citologia , Masculino , Modelos NeurológicosRESUMO
The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Transporte/genética , Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genética , Sistemas Neurossecretores/citologia , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Feminino , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Nephropidae , Sistemas Neurossecretores/fisiologia , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Sondas RNA , RNA Mensageiro/análiseRESUMO
With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.
Assuntos
Nephropidae/metabolismo , Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Hiperglicemia/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neuropeptídeos/química , Mapeamento de Peptídeos , Homologia de Sequência do Ácido Nucleico , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
As in Lymnaea stagnalis NPY plays a key role in regulating energy flows but has no effect on food intake, two important questions arise: 1) How is the amount of food consumed related to energy storage? 2) Can we give a molecular explanation for this alteration in function of NPY during evolution? Recent data have shown that also in Lymnaea a leptin-like factor is produced by glycogen storing cells which inhibits food intake, a Lymnaea storage feedback factor (LySFF). So, food consumption seems in balance with the amount of energy stored in this animal. We suppose that NPY neurons in Lymnaea have receptors for LySFF so that their activity in regulating energy homeostasis reflects the amount of stored energy. By comparing the molecular structure of NPYs in invertebrates it became clear that only molluscan and arthropod NPY are synthesized from a prohormone similar to vertebrate NPYs and should be considered as real invertebrate homologs of NPY. Based on pharmacological data we suppose that the identified Lymnaea NPY receptor is a Y1 subtype. This might explain that LyNPY has no effect on food intake in Lymnaea as this function of NPY in mammals is regulated through the Y5 subtype receptor.
Assuntos
Evolução Molecular , Neuropeptídeo Y/química , Neuropeptídeo Y/genética , Sequência de Aminoácidos , Animais , Artrópodes , Bases de Dados Factuais , Drosophila , Leptina/química , Lymnaea , Modelos Biológicos , Dados de Sequência Molecular , Moluscos , Neuropeptídeo Y/fisiologia , Filogenia , Homologia de Sequência de AminoácidosRESUMO
The primary structure of the major form of CHH from sinus glands of the crayfish, Orconectes limosus, was determined by manual Edman microsequencing. It is a 72-residue peptide with a calculated Mr of 8400 Da. In the number of residues, it is identical to the CHH of Carcinus maenas and very similar to MIH (moult inhibiting hormone) of Homarus americanus. All three peptides have pGlu as N-terminus in common, and Val-NH2 is the C-terminal residue in Orconectes and Carcinus CHH. Six Cys residues occupy identical position in the three peptides. There is a 61% sequence identity with Carcinus CHH, and an 81% identity with Homarus MIH.
Assuntos
Astacoidea , Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Braquiúros , Cromatografia Líquida de Alta Pressão , Endopeptidases , Hormônios de Invertebrado/química , Dados de Sequência Molecular , Nephropidae , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , TripsinaRESUMO
The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.
Assuntos
Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Braquiúros , Cromatografia Líquida de Alta Pressão , Hormônios de Invertebrado/isolamento & purificação , Hormônios de Invertebrado/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Mapeamento de Peptídeos , Conformação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Homologia de Sequência do Ácido Nucleico , TripsinaRESUMO
BACKGROUND: Previous studies demonstrating that the neoplastic cells in Sézary syndrome and tumor stage mycosis fungoides express interleukin 4 (IL-4), IL-5, and IL-10 have resulted in the concept that cutaneous T-cell lymphomas are derived from CD4(+) T cells with a T(H)2 type cytokine profile. OBJECTIVE: To determine the cytokine profile in CD30(-) primary cutaneous large T-cell lymphomas, which represent a subgroup of cutaneous T-cell lymphoma with an aggressive clinical behavior (5-year survival rate of 15%). DESIGN AND METHODS: Seven biopsy specimens were taken from 4 patients with CD30(-) primary cutaneous large T-cell lymphomas and studied for the expression of T(H)1 (IL-2 and interferon gamma) and T(H)2 (IL-4, IL-5, IL-10) cytokines using a reverse transcription-polymerase chain reaction technique. Skin biopsy specimens from patients with Sézary syndrome, mycosis fungoides, atopic dermatitis, or psoriasis were included as controls. RESULTS: In the 7 CD30(-) primary cutaneous large T-cell lymphomas showing an almost pure population of large tumor cells (>90%), no expression of IL-4 was found, and IL-5 was only found in 1 of 7 cases. In control biopsy specimens, expression of IL-4 and/or IL-5 was demonstrated in atopic dermatitis (3/3), tumor stage mycosis fungoides (2/2), and Sézary syndrome (3/3), but not in plaque stage mycosis fungoides. CONCLUSION: Our results demonstrate that CD30(-) primary cutaneous large T-cell lymphomas do not produce T(H)2 cytokines, illustrating that not all cutaneous T-cell lymphomas have a T(H)2 cytokine profile.