RESUMO
BACKGROUND: In past decades, liver transplant (LT) patients were not routinely screened for hepatitis E virus (HEV) infection, and thus it might have been misdiagnosed as an acute rejection episode. Our aim was to analyze a real-world cohort of LT patients who presented with at least 1 episode of biopsy-proven acute rejection (BPAR) and suffered from persistent elevated transaminases, to evaluate the frequency of HEV infection misdiagnosed as a rejection episode. METHODS: Data from 306 patients transplanted between 1997 and 2017, including 565 liver biopsies, were analyzed. Biopsies from patients suffering from hepatitis C (n = 79; 25.8%) and from patients who presented with a Rejection Activity Index <5 (n = 134; 43.8%) were excluded. A subgroup of 74 patients (with 134 BPAR) with persistently elevated liver enzymes was chosen for further HEV testing. RESULTS: Positive HEV IgG was detectable in 18 of 73 patients (24.7%). Positive HEV RNA was diagnosed in 3 of 73 patients with BPAR (4.1%). Patients with HEV infection showed no difference in etiology of the liver disease, type of immunosuppression, or median Rejection Activity Index. CONCLUSION: Few HEV infections were misdiagnosed as acute rejection episodes in this real-world cohort. Thus, HEV infection is an infrequent diagnosis in cases with persistent elevated liver enzymes and BPAR after LT.
Assuntos
Rejeição de Enxerto/diagnóstico , Hepatite E/complicações , Hepatite E/diagnóstico , Transplante de Fígado , Adulto , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Anticorpos Anti-Hepatite/sangue , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The present work reports on the development of a new generation of Lab-on-a-chip (LOC) to perform in-situ and real-time potentiometric measurements in flowing water. The device consisted of two differentiated parts: a poly (dimethylsiloxane) (PDMS) microfluidic structure obtained by soft lithography and a fully integrated chemical sensing platform including four working microelectrodes, two reference microelectrodes and one counter microelectrode for detecting ammonium in a continuous mode. The performance of the device was evaluated following its potentiometric response when analyzing ammonium containing samples. As a key parameter, its time of response was compared to that of a commercially available electrical conductivity sensor used as reference sensor during tests in laboratory using flowing tap water and technical scale using flowing wastewater. As a result, the LOC showed a slope of 55â¯mV/decade, a limit of detection of 4·10-5â¯M and a time of full response between 10 and 12â¯s. It was demonstrated that the device can provide fast and reliable data at real time when immersed in a laminar flow of water. Moreover, the test of robustness showed that it was still functional after immersion in sewage for at least 15â¯min. Besides, the LOC reported here can be helpful for a wide variety of flowing-water applications such as aqua culture outlets control, in-situ and continuous analysis of rivers effluents and sea waters monitoring among others.
RESUMO
Essentials Antiphospholipid antibodies (aPL) are heterogeneous and induce different cellular responses. We analyzed signaling events induced by different monoclonal and patient aPL in monocytes. Two major signaling pathways involving either NADPH-oxidase or LRP8 were identified. Our data suggest that these two pathways mediate the majority of aPL effects on monocytes. SUMMARY: Background Antiphospholipid antibodies (aPLs) contribute to the pathogenesis of the antiphospholipid syndrome (APS) by induction of an inflammatory and procoagulant state in different cell types, and several signaling pathways have been described. Objectives To investigate whether signaling depends on the epitope specificity of aPLs. Methods Cellular effects of three human monoclonal aPLs with distinctly different epitope specificities were analyzed in vitro. Expression of tumor necrosis factor-α mRNA by mouse and human monocytes was the major readout. Analysis included cells from genetically modified mice, and the use of specific inhibitors in human monocytes. Data were validated with IgG isolated from 20 APS patients. Results Cofactor-independent anticardiolipin aPLs activated monocytes by induction of endosomal NADPH oxidase. Activation could be blocked by hydroxychloroquine (HCQ). Anti-ß2 -glycoprotein I aPL activated monocytes by interacting with LDL receptor-related protein 8 (LRP8). This could be blocked by rapamycin. Analysis of 20 APS patients' IgG showed that all IgG fractions activated the same two pathways as the monoclonal aPL, depending on their epitope patterns as determined by ELISA. Monocyte activation by APS IgG could be blocked completely by HCQ and/or rapamycin, suggesting that in most, if not all, APS patients there is no other relevant signaling pathway. Conclusions aPLs activate two major proinflammatory signal transduction pathways, depending on their epitope specificity. HCQ and rapamycin, either alone or in combination, completely suppress signaling by APS IgG. These observations may provide a rationale for specific treatment of APS patients according to their aPL profile.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Adulto , Animais , Anticorpos Anticardiolipina/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Síndrome Antifosfolipídica/etiologia , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Monócitos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Genome-wide association studies have identified and repeatedly confirmed the association of rs3197999 in MST1 with inflammatory bowel disease (IBD). However, the underlying pathophysiology remains unclear. rs3197999 is a non-synonymous single-nucleotide polymorphism which modifies the function of macrophage stimulating protein-1 (MST1). We show by haplotyping that rs3197999 is in linkage disequilibrium with rs1050450 in GPX1, with almost complete cosegregation of the minor alleles. As shown by immunoassay, rs3197999 influences the MST-1 level in serum. But also rs1050450 causes an amino acid exchange in glutathione peroxidase 1 (GPx-1) and reduced activity of this antioxidant enzyme. The association of GPx deficiency and IBD in mice was already shown. We propose that GPx-1 is a better candidate than MST1 for the pathophysiologic link between IBD locus 12 and IBD.
Assuntos
Glutationa Peroxidase/genética , Doenças Inflamatórias Intestinais/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Animais , Feminino , Glutationa Peroxidase/metabolismo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/fisiopatologia , Desequilíbrio de Ligação , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Glutationa Peroxidase GPX1RESUMO
Crohn's disease and ulcerative colitis, the two main types of inflammatory bowel disease (IBD), were reported to be associated with a variety of genetic polymorphisms. A subset of these polymorphisms was identified in both diseases and only three of them were found in primary sclerosing cholangitis (PSC). rs3197999 (Arg689Cys) located in the MST1 gene is one of the most convincingly replicated IBD/PSC-associated polymorphisms but its functional consequences have not been investigated, yet. We expressed both MST1 gene variants (Arg(689) (MSP(wt)) and Cys(689) (MSP(mut)) in a eukaryotic cell system and compared their stimulatory effects on macrophage-like THP-1 cells. Except for the rate of apoptosis that remained unchanged, MSP(mut) significantly increased the stimulatory effect of MSP (macrophage-stimulating protein) on chemotaxis and proliferation by THP-1 cells, indicating a gain of function associated with the Arg689Cys exchange. A broad set of evidence reported previously suggests that pro-inflammatory changes in macrophage function have a major role in the initiation of the inflammatory process in IBD and PSC. Therefore, the gain of function observed with rs3197999 in MST1 might provide a cellular mechanism for the consistent association of this polymorphism with an increased risk for IBD and PSC.
Assuntos
Colangite Esclerosante/genética , Fator de Crescimento de Hepatócito/metabolismo , Doenças Inflamatórias Intestinais/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose , Células CHO , Movimento Celular , Proliferação de Células , Quimiotaxia , Colangite Esclerosante/imunologia , Colangite Esclerosante/metabolismo , Cricetinae , DNA Complementar/genética , DNA Complementar/metabolismo , Células Hep G2 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Interferon gama/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The design and use of a pilot microarray for transcriptome analysis of the symbiotic, nitrogen-fixing Bradyrhizobium japonicum is reported here. The custom-synthesized chip (Affymetrix GeneChip) features 738 genes, more than half of which belong to a 400-kb chromosomal segment strongly associated with symbiosis-related functions. RNA was isolated following an optimized protocol from wild-type cells grown aerobically and microaerobically, and from cells of aerobically grown regR mutant and microaerobically grown nifA mutant. Comparative microarray analyses thus revealed genes that are transcribed in either a RegR- or a NifA-dependent manner plus genes whose expression depends on the cellular oxygen status. Several genes were newly identified as members of the RegR and NifA regulons, beyond genes, which had been known from previous work. A comprehensive transcription analysis was performed with one of the new RegR-controlled genes (id880). Expression levels determined by microarray analysis of selected NifA- and RegR-controlled genes corresponded well with quantitative real-time PCR data, demonstrating the high complementarity of microarray analysis to classical methods of gene expression analysis in B. japonicum. Nevertheless, several previously established members of the NifA regulon were not detected as transcribed genes by microarray analysis, confirming the potential pitfalls of this approach also observed by other authors. By and large, this pilot study has paved the way towards the genome-wide transcriptome analysis of the 9.1-Mb B. japonicum genome.
Assuntos
Bradyrhizobium/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simbiose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Hypocretin-1 (HCRT-1) and hypocretin 2 (HCRT-2), also known as orexin-A and orexin-B, are two neuropeptides derived from the same precursor. Hypocretinergic neurons have been found exclusively in the hypothalamic dorsolateral area. These neurons are implicated in sleep and feeding through activation of specific G-protein-coupled orexin-1 and orexin-2 receptor (OR-R1 and OR-R2). The purpose of this study was to determine the existence of the HCRT peptides in the central input of the rat pineal gland. Further, OR-R1 and OR-R2 expression was determined in the pineal gland and the effect of HCRT-2 on melatonin synthesis and secretion was analysed in dissociated rat pinealocytes. A large contingent of HCRT-positive nerve fibres and terminals were observed in the epithalamus, many of which entered into the pineal parenchyma. A significant number of nerve fibres endowed with positive boutons were identified in the pineal stalk, though the number of positive fibres decreased along the extension of the stalk. So far, no positive fibres have been found in the superficial pineal gland. RT-PCR analysis revealed the expression of OR-R2 mRNA, whereas OR-R1-receptor mRNA was not detected. When tested alone, HCRT-2 had no effect on secretion of melatonin from cultured rat pinealocytes. However, HCRT-2 partially inhibited (by a maximum of 30%) the beta-adrenergic-induced melatonin secretion. The same effect was seen on activation of N-acetyltransferase activity. The distribution and the large number of HCRT-positive fibres together with the effect on noradrenaline-mediated melatonin release through specific receptors suggests that these peptides may be significant central transmitters in pineal function, probably mediating homeostatic signals to the pineal gland.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Melatonina/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/fisiologia , Norepinefrina/fisiologia , Glândula Pineal/fisiologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/biossíntese , Separação Celular , Imuno-Histoquímica , Masculino , Melatonina/biossíntese , Neuropeptídeos/biossíntese , Receptores de Orexina , Orexinas , Glândula Pineal/citologia , Glândula Pineal/metabolismo , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fixação de TecidosRESUMO
A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K(m) (15.2 +/- 2.4 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x s(-1)) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate. LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.
Assuntos
Aminopeptidases/genética , Solanum lycopersicum/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/metabolismo , Sequência de Bases , Catálise , Clonagem Molecular , Primers do DNA , DNA Complementar , Genes de Plantas , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Cnidarians are primitive animals that use neuropeptides as their transmitters. All the numerous cnidarian neuropeptides isolated, so far, have a carboxy-terminal amide group that is essential for their actions. This strongly suggests that alpha-amidating enzymes are essential for the functioning of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in mammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and the 3D structure of PHM are conserved between CP1-A, -B and the PHM part of rat PAM. Furthermore, eight introns in CP1 occur in the same positions and have the same intron phasing as eight introns in the rat PAM gene, showing that the sea anemone PHM is not only structurally, but also evolutionarily related to the PHM part of rat PAM.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Íntrons/genética , Oxigenases de Função Mista/genética , Complexos Multienzimáticos , Anêmonas-do-Mar/enzimologia , Anêmonas-do-Mar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Isoenzimas/química , Isoenzimas/genética , Oxigenases de Função Mista/química , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) from Drosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino acid residues long and shows a striking structural homology with members of the glycoprotein hormone [thyroid-stimulating hormone (TSH); follicle-stimulating hormone (FSH); luteinizing hormone/choriogonadotropin (LH/CG)] receptor family from mammals and with two additional, recently identified mammalian orphan LGRs (LGR-4 and LGR-5). This homology includes the seven transmembrane region (e.g., 49% amino acid identity with the human TSH receptor) and the very large extracellular amino terminus. This amino terminus contains 18 Leu-rich repeats-in contrast with the 3 mammalian glycoprotein hormone receptors and DLGR-1 that contain 9 Leu-rich repeats, but resembling the mammalian LGR-4 and LGR-5 that each have 17 Leu-rich repeats in their amino termini. The DLGR-2 gene is >18.6 kb pairs long and contains 15 exons and 14 introns. Four intron positions coincide with the intron positions of the three mammalian glycoprotein hormone receptors and have the same intron phasing, showing that DLGR-2 is evolutionarily related to these mammalian receptors. The DLGR-2 gene is located at position 34E-F on the left arm of the second chromosome and is expressed in embryos and pupae but not in larvae and adult flies. Homozygous knock-out mutants, where the DLGR-2 gene is interrupted by a P element insertion, die around the time of hatching. This finding, together with the expression data, strongly suggests that DLGR-2 is exclusively involved in development.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes de Insetos/genética , Proteínas de Insetos/genética , Proteínas Musculares/genética , Proteínas/genética , Receptores de Superfície Celular/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Drosophila melanogaster/embriologia , Humanos , Proteínas de Repetições Ricas em Leucina , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , Anêmonas-do-Mar , Alinhamento de SequênciaRESUMO
[formula: see text] Condensation of 3-phenylsulfonyl-1,3-isobenzofuranone (3) with benzocyclohept-6,7-en-5-ones such as 4 and 15 provides a straightforward, general method for synthesis of functionalized benzo[4,5]cyclohepta[1,2-b]naphthalenes (e.g., 5 and 16). This finding was used to achieve a brief and efficient preparation of 6,7-benzo-3,4-(1,4-dimethoxy-2,3-naphtho)-1,5-dioxosuberane (2), an established intermediate to the naturally occurring red pigment radermachol (1).
Assuntos
Cicloeptanos/síntese química , Naftalenos/síntese químicaRESUMO
A chiral synthesis of the aminohydroxy expoxybenzoxocin 6 is described. Enantioselective Friedel-Crafts coupling using a chiral titanium catalyst was employed to produce the optically active atrolactic ester 16a from the phenol 11 and l-menthyl pyruvate (12). The phenolic group in 16a was protected as the benzyl ether and the t-alcohol functionality as the MEM ether to give 20, which after sequential reduction/oxidation provided the aldehyde 22. Addition of the acetylide anion of propargyl aldehyde diethyl acetal (23) to aldehyde 22, followed by oxidation of the resultant diastereoisomeric carbinols, gave the acetylenic ketone 24. Lindlar reduction of 24 afforded the trans-enone 26. Reaction of 26 with thiophenylate anion furnished 27, which was then cyclized to the alpha-methyl pyranoside 29. Oxidation of 29 to the sulfoxide and subsequent thermolysis afforded the hexenulose 30. Sequential epoxidation of 30, reduction of the keto epoxide 31, and reaction of the resultant epoxycarbinol 32 with dimethylamine produced the aminohydroxy pyranose 33a. Debenzylation of 33a to the phenol 33b, followed by intramolecular cyclization, completed the fabrication of the optically active aminohydroxy epoxybenzoxocin 6. The 17-step sequence from the phenol 11 to 6 was achieved in 22% overall yield.
Assuntos
Antibióticos Antineoplásicos/síntese química , Éteres Cíclicos/química , Nogalamicina/análogos & derivados , Éteres Cíclicos/síntese química , Espectroscopia de Ressonância Magnética , EstereoisomerismoRESUMO
Rett's syndrome is a neurodevelopmental disorder which is caused by a mutation on the x-chromosome; thus, it only affects the female sex. After seemingly normal postnatal development affected girls lose already acquired mental, motoric and social skills. The last stage of the syndrome is characterized by microcephaly, severe mental retardation, spastic paraparesis, epilepsia, respiratory dysrhythmia, neurogenic scoliosis, abnormal joint alignment and muscle contractures. Rett's syndrome is probably the leading cause for progressive mental retardation in girls, but still it is relatively unknown. This paper describes Rett syndrome and its pathophysiology. The following case report discusses special anesthesiological implications due to the immature cardiorespiratory system and describes a coagulation disorder following treatment with valproic acid.
Assuntos
Anestesia/métodos , Síndrome de Rett/fisiopatologia , Adulto , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/uso terapêutico , Transtornos da Coagulação Sanguínea/induzido quimicamente , Epilepsia/complicações , Epilepsia/prevenção & controle , Feminino , Humanos , Cálculos Renais/cirurgia , Síndrome de Rett/complicações , Procedimentos Cirúrgicos Urológicos , Ácido Valproico/efeitos adversos , Ácido Valproico/uso terapêuticoRESUMO
Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Cabeça/crescimento & desenvolvimento , Hydra/genética , Hormônios de Invertebrado/genética , Neuropeptídeos/genética , Animais , Cabeça/embriologia , Hydra/crescimento & desenvolvimento , Hibridização In Situ , Neurônios/metabolismo , Fenótipo , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Regeneração/genéticaRESUMO
[formula: see text] A regiospecific total synthesis of (+/-)-biphyscion (1) is described. A novel aspect of the plan was construction of the bianthraquinone ring system from a biphenyl intermediate through the use of a one-pot, double isobenzofuranone condensation.
Assuntos
Antraquinonas/síntese química , Espectroscopia de Ressonância MagnéticaRESUMO
NPY is considered to play an important role in pineal function, because it is co-stored with the dominant pineal transmitter noradrenaline. However, little evidence from the literature suggests that NPY alone is a strong regulator of melatonin synthesis or secretion and it is therefore more likely that NPY modulates noradrenergic neurotransmission in the rat pineal gland. The purpose of the present studies was to determine the nature and origin of NPYergic inputs to, and the type of specific NPY receptor subtypes in, the rat pineal gland. Gel filtration and immunocytochemistry using region-specific antisera revealed that all proNPY present in intrapineal nerve fibres is cleaved to amidated NPY and a C-terminal flanking peptide of NPY (CPON). The vast majority of NPY content in the pineal gland was found to be of sympathetic origin. Receptor autoradiography showed that only a few NPY specific binding sites were present in the superficial pineal gland. A reverse transcriptase polymerase chain reaction detected sequences of only NPY receptor subtype Y1 and not other NPY receptor subtypes in pineal extracts. These results together with the available literature imply that NPY under certain conditions is co-released with noradrenaline and exerts its actions either presynaptically or on the pinealocyte through a Y1 receptor. The available data indicate that NPY has no effect alone, but acts in concert with noradrenaline. A presynaptic action regulating noradrenaline neurotransmission is also possible. NPY has been reported only to act on melatonin secretion in vitro, and it remains to be established what function NPY plays in the pineal gland in vivo. This paper discuss possible modulatory actions of NPY being a predominant sympathetic transmitter.
Assuntos
Neuropeptídeo Y/análise , Glândula Pineal/química , Glândula Pineal/citologia , Receptores de Neuropeptídeo Y/análise , Animais , Autorradiografia , Cromatografia em Gel , Imuno-Histoquímica , Masculino , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Glândula Pineal/metabolismo , Ratos , Ratos Wistar , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Gânglio Cervical Superior/fisiologiaRESUMO
[formula: see text] A regio- and stereospecific total synthesis of (+/-)-methyl rishirilide B (2b), and (alpha)2-macroglobulin inhibitor, is described. A key feature of the synthetic plan was regiospecific construction of a hydroanthracenone intermediate through condensation of a phenylsulfonyl isobenzofuranone with a functionalized 2-cyclohexen-1-one. Introduction of the vicinal trans-hydroxyl groups in the densely functionalized A-ring was accomplished via a novel one-pot procedure that involved oxidation of enolate anions with the Davis reagent.
Assuntos
Antracenos/química , Antracenos/síntese química , Fibrinolíticos/síntese química , alfa-Macroglobulinas/antagonistas & inibidores , Hidrólise , Indicadores e Reagentes , Estereoisomerismo , Streptomyces/químicaRESUMO
Cnidarians (e.g., sea anemones and corals) are the lowest animal group having a nervous system. Previously, we cloned a receptor from sea anemones that showed a strong structural similarity to the glycoprotein hormone (TSH, FSH, LH/CG) receptors from mammals. Here, we determine the genomic organization of this sea anemone receptor. The receptor gene contains eight introns that are all localized within a region coding for the large extracellular N terminus. These introns occur at the same positions and have the same intron phasing as eight introns in the genes coding for the mammalian glycoprotein hormone receptors, indicating that the cnidarian and mammalian receptor genes are evolutionarily related. As with the mammalian receptor genes, the sea anemone receptor gene does not contain introns in the region coding for the transmembrane and intracellular domains. Southern blot analyses show that the cnidarian receptor is coded for by a single gene.
Assuntos
Evolução Molecular , Mamíferos/genética , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas , Receptores de Superfície Celular/genética , Anêmonas-do-Mar/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Éxons , Genoma , Íntrons , Dados de Sequência MolecularRESUMO
After screening the data base of the Berkeley Drosophila Genome Project with a sequence coding for the transmembrane region of a G protein-coupled receptor, we found that Drosophila might contain a gene coding for a receptor that is structurally related to the Gonadotropin-Releasing Hormone (GnRH) receptors from vertebrates. Using the polymerase chain reaction, with Drosophila cDNA as a template, and oligonucleotide probes coding for the presumed exons of this gene, we were able to clone the cDNA coding for this receptor. The transmembrane region of the receptor shows 36% amino acid residue identity with the transmembrane region of the catfish and 31% amino acid residue identity with that of the rat GnRH receptor. The Drosophila receptor gene contains six introns, whereas the rat gene contains two: one intron in the Drosophila gene occurs at exactly the same position and has the same intron phasing as one intron in the rat gene, suggesting that the Drosophila and mammalian GnRH receptor genes are evolutionarily related. Northern blot analyses show that the Drosophila receptor gene is progressively expressed during larval development with a prominent maximum at the 3rd instar larval stage. Pupae contain low amounts of receptor mRNA, while adult flies contain higher levels, with males having about five times more receptor mRNA than females flies. Southern blot analyses show that Drosophila contains only one copy of the receptor gene, which is located at position 27A2-B1 of chromosome 2. This paper is the first report on the molecular cloning of a member of the GnRH receptor family from invertebrates.