RESUMEN

Chronic exposure to low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmoset monkeys was used to model the prodromal stage of Parkinson's disease (PD), and to investigate mechanisms underlying disease progression and recovery. Marmosets were subcutaneously injected with MPTP for a period of 12weeks, 0.5mg/kg once per week, and clinical signs of Parkinsonism, motor- and non-motor behaviors were recorded before, during and after exposure. In addition, postmortem immunohistochemistry and proteomics analysis were performed. MPTP-induced parkinsonian clinical symptoms increased in severity during exposure, and recovered after MPTP administration was ended. Postmortem analyses, after the recovery period, revealed no alteration of the number and sizes of tyrosine hydroxylase (TH)-positive dopamine (DA) neurons in the substantia nigra. Also levels of TH in putamen and caudate nucleus were unaltered, no differences were observed in DA, serotonin or nor-adrenalin levels in the caudate nucleus, and proteomics analysis revealed no global changes in protein expression in these brain areas between treatment groups. Our findings indicate that parkinsonian symptoms can occur without detectable damage at the cellular or molecular level. Moreover, we show that parkinsonian symptoms may be reversible when diagnosed and treated early.

Asunto(s)

1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Conducta Animal/efectos de los fármacos , Progresión de la Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neostriado/metabolismo , Neurotoxinas/farmacología , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/metabolismo , Recuperación de la Función , Sustancia Negra/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , Animales , Callithrix , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Femenino , Intoxicación por MPTP/inducido químicamente , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Masculino , Neostriado/patología , Neurotoxinas/administración & dosificación , Enfermedad de Parkinson Secundaria/patología , Proteómica , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología

RESUMEN

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Asunto(s)

Copulación/fisiología , Trastornos del Desarrollo Sexual/fisiopatología , Lateralidad Funcional/fisiología , Lymnaea/fisiología , Péptidos/genética , Potenciales de Acción/fisiología , Animales , Axones/patología , Sistema Nervioso Central/citología , Trastornos del Desarrollo Sexual/patología , Femenino , Ganglios de Invertebrados/citología , Lymnaea/citología , Lymnaea/genética , Masculino , Neuronas/fisiología , Níquel/metabolismo , Pene/inervación , Pene/patología , Pene/fisiopatología , Péptidos/metabolismo , Análisis de la Célula Individual , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

Asunto(s)

Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Hipocampo/fisiopatología , Mutación Missense , Neuronas/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Animales , Células Cultivadas , Condicionamiento Psicológico/fisiología , Trastorno Depresivo Mayor/genética , Trastorno Depresivo Mayor/fisiopatología , Conducta Exploratoria/fisiología , Miedo/fisiología , Conducta Alimentaria/fisiología , Técnicas de Sustitución del Gen , Hipocampo/citología , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Neuronas/citología , Técnicas de Placa-Clamp , Inhibición Prepulso/fisiología , Reflejo de Sobresalto/fisiología , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología

RESUMEN

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Asunto(s)

Biosíntesis de Proteínas , Proteoma/metabolismo , Sinaptosomas/metabolismo , Secuencia de Aminoácidos , Animales , Autorradiografía , Sistema Nervioso Central/química , Sistema Nervioso Central/metabolismo , Decapodiformes , Electroforesis en Gel Bidimensional , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Hibridación in Situ , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Células Fotorreceptoras/metabolismo , Terminales Presinápticos/metabolismo , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Análisis de Secuencia de Proteína , Sinaptosomas/química

RESUMEN

BACKGROUND: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. OBJECTIVES: Proteomics were used to search for new markers of which the expression changes after mild irritation. METHODS: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. RESULTS: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. CONCLUSIONS: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.

Asunto(s)

Alternativas a las Pruebas en Animales/métodos , Dermatitis por Contacto/diagnóstico , Proteínas de Choque Térmico , Proteínas de Neoplasias/metabolismo , Proteoma , Biomarcadores/análisis , Núcleo Celular/metabolismo , Técnicas de Cultivo , Dermatitis por Contacto/metabolismo , Dermatitis por Contacto/patología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico HSP27 , Humanos , Chaperonas Moleculares , Dodecil Sulfato de Sodio/administración & dosificación

RESUMEN

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 , Transfección

RESUMEN

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.

Asunto(s)

Cadmio/metabolismo , Metalotioneína/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Expresión Génica , Insectos/metabolismo , Metalotioneína/genética , Metalotioneína/aislamiento & purificación , Metales/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/metabolismo

RESUMEN

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Asunto(s)

Acetilcolina/metabolismo , Proteínas Portadoras/metabolismo , Lymnaea , Neuroglía/metabolismo , Neuronas/metabolismo , Transmisión Sináptica , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Técnicas de Cocultivo , Concentración 50 Inhibidora , Ligandos , Lymnaea/química , Lymnaea/genética , Lymnaea/fisiología , Modelos Neurológicos , Datos de Secuencia Molecular , Neuroglía/química , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Unión Proteica , Señales de Clasificación de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Alineación de Secuencia , Serotonina/metabolismo , Serotonina/farmacología , Transmisión Sináptica/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Quimiocinas CXC/metabolismo , Queratinocitos/metabolismo , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Quimiotaxis , Clonación Molecular , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Hibridación in Situ , Inflamación/metabolismo , Ligandos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Receptores de Citocinas/genética , Linfocitos T/citología

RESUMEN

The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd-binding, cysteine-rich peptides were isolated from whole-body homogenates, using gel filtration and reversed-phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989-Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139-Da protein was unsuccessful, probably due to N-terminal blockage. Using different PCR techniques (3' and 5' RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C-terminal part of this protein. The 4139-Da protein is probably encoded by the N-terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post-translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.

Asunto(s)

Cadmio/farmacología , Sistema Digestivo/metabolismo , Insectos/efectos de los fármacos , Metalotioneína/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Resistencia a Medicamentos , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Suelo/parasitología

RESUMEN

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y-receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y-receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y-receptor-encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size-fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.

Asunto(s)

Lymnaea/metabolismo , Receptores de Neuropéptido Y/genética , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células CHO , Clonación Molecular , Colforsina/farmacología , Cricetinae , AMP Cíclico/biosíntesis , ADN Complementario/genética , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Neuropéptido Y/metabolismo , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Asunto(s)

Proteínas de Unión al GTP/genética , Lymnaea/genética , Neuropéptidos/genética , Receptores de Neuropéptido/genética , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Clonación Molecular , Sondas de ADN , ADN Complementario , Electrofisiología , Proteínas de Unión al GTP/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Corazón/inervación , Datos de Secuencia Molecular , Fibras Nerviosas/química , Fibras Nerviosas/metabolismo , Sistema Nervioso/química , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Neuropéptidos/metabolismo , Oocitos/fisiología , ARN Mensajero/análisis , Receptores de Neuropéptido/análisis , Receptores de Neuropéptido/metabolismo , Xenopus

RESUMEN

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.

Asunto(s)

Lymnaea/química , Neuronas/química , Neuropéptidos/análisis , Animales , Cromatografía Liquida , Peso Molecular , Neuropéptidos/aislamiento & purificación , Fragmentos de Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Asunto(s)

Proteínas del Tejido Nervioso/metabolismo , Hipófisis/metabolismo , Cloruro de Sodio Dietético/administración & dosificación , Secuencia de Aminoácidos , Animales , Espectrometría de Masas , Datos de Secuencia Molecular , Ratas

RESUMEN

The alpha6A and alpha6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the alpha6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of alpha6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the alpha6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant alpha6A cDNAs were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1 at levels comparable to that of wild-type alpha6Abeta1. A single alpha6 polypeptide chain (150 kDa) was precipitated from transfectants expressing alpha6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded alpha6A subunits that were cleaved once into a heavy and a light chain, whereas alpha6A subunits with mutations in one of the two RK sequences were, like wild-type alpha6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of alpha6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two alpha6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because alpha6(RKKG), alpha6(GKKR) and alpha6(RGGR) subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. alpha6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas alpha6A mutants with the RKKR sequence shifted to other positions in the alpha6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, alpha6A mutants with an alpha5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-beta1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved alpha6Abeta1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant alpha6Abeta1, did not activate uncleaved alpha6Abeta1. Thus uncleaved alpha6Abeta1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of alpha6 is required to generate a proper conformation that enables the affinity modulation of the alpha6Abeta1 receptor by PMA.

Asunto(s)

Antígenos CD/química , Antígenos CD/metabolismo , Integrinas/química , Integrinas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/aislamiento & purificación , Secuencia de Bases , Sitios de Unión , Pollos , Cartilla de ADN , Humanos , Integrina alfa6 , Integrina alfa6beta1 , Integrinas/aislamiento & purificación , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales Cultivadas , Xenopus

RESUMEN

Neuropeptides are known to be important signaling molecules in several neural systems of the pond snail Lymnaea stagnalis. Although the functions of these peptides have been studied in many neurons, the nature of the postsynaptic signal transduction is mainly unknown. The cloning and characterization of neuropeptide receptors in Lymnaea thus would be very valuable in further elucidating peptidergic pathways. Indirect evidence suggests that these neuropeptides operate via G-protein-coupled mechanisms indicating the presence of G-protein-coupled receptors as the initial postsynaptic targets. Here we describe the cloning of a neuropeptide receptor from Lymnaea and the isolation of an endogenous ligand. This peptide, PSFHSWSamide, belongs to the leucokinin family of peptides, and, thus, this Lymnaea receptor is the first example of a leucokinin-like neuropeptide receptor, representing a new subfamily of G-protein-coupled neuropeptide receptors.

Asunto(s)

Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Lymnaea/metabolismo , Oligopéptidos/metabolismo , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Ratas , Receptores de Neuropéptido/genética

RESUMEN

We have identified two novel peptide toxins from molluscivorous Conus species that discriminate subtypes of high voltage-activated (HVA) calcium currents in molluscan neurons. The toxins were purified using assays on HVA calcium currents in the caudodorsal cells (CDCs) of the snail Lymnaea stagnalis. The CDC HVA current consists of a rapidly inactivating, transient current that is relatively insensitive to dihydropyridines (DHPs) and a slowly inactivating, DHP-sensitive L-current. The novel toxins, designated omega-conotoxins PnVIA and PnVIB, completely and selectively block the transient HVA current in CDCs with little (PnVIA) or no (PnVIB) effect on the sustained L-type current. The block is rapid and completely reversible. It is noteworthy that both PnVIA and PnVIB reveal very steep dose dependences of the block, which may imply cooperativity in toxin action. The amino acid sequences of PnVIA (GCLEVDYFCGIPFANNGLCCSGNCVFVCTPQ) and of PnVIB (DDDCEPPGNFCGMIKIGPPCCSGWCFFACA) show very little homology to previously described omega-conotoxins, although both toxins share the typical omega-conotoxin cysteine framework but have an unusual high content of hydrophobic residues and net negative charge. These novel omega-conotoxins will facilitate selective analysis of the functions of HVA calcium channels and may enable the rational design of drugs that are selective for relevant subtypes.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Dihidropiridinas/farmacología , Venenos de Moluscos/farmacología , Neuronas/fisiología , omega-Conotoxinas , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/química , Canales de Calcio/efectos de los fármacos , Técnicas In Vitro , Lymnaea , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Venenos de Moluscos/química , Neuronas/efectos de los fármacos , Nimodipina/farmacología , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad

RESUMEN

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Asunto(s)

Lymnaea/química , Factores de Crecimiento Nervioso/fisiología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Calcio/metabolismo , Hemolinfa/química , Humanos , Datos de Secuencia Molecular , Neuronas Motoras/ultraestructura , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/aislamiento & purificación , Factores de Crecimiento Nervioso/metabolismo , Neuritas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Factor de Crecimiento Nervioso , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas

RESUMEN

A novel calcium channel blocking peptide designated omega-conotoxin-Tx VII has been characterized from the venom of the molluscivorous snail Conus textile. The amino acid sequence (CKQADEPCDVFSLDCCTGICLGVCMW) reveals the characteristic cysteine framework of omega-conotoxins, but it is extremely hydrophobic for this pharmacological class of peptides and further unusual in its net negative charge (-3). It is further striking that the sequence of TxVII, a calcium current blocker, is 58% identical to that of delta-conotoxin-TxVIA, which targets sodium channels. TxVII effects were examined in the caudodorsal cell (CDC) neurons from the mollusc Lymnaea stagnalis. The toxin has no significant effect on sodium or potassium currents in these cells, but it clearly blocks the calcium currents. TxVII most prominently blocks the slowly inactivating, dihydropyridine- (DHP-) sensitive current in CDCs, while blockade of the rapidly inactivating current is less efficient. This novel omega-conotoxin is apparently targeted to DHP-sensitive calcium channels and thereby provides a lead for future design of selective conopeptide probes for L-type channels.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Dihidropiridinas/farmacología , Péptidos/farmacología , omega-Conotoxinas , Secuencia de Aminoácidos , Animales , Canales de Calcio/fisiología , Lymnaea , Potenciales de la Membrana , Sondas Moleculares , Datos de Secuencia Molecular

RESUMEN

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

Asunto(s)

Neuropéptidos/farmacología , Oxitocina/análogos & derivados , Conducto Deferente/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Hibridación in Situ , Lymnaea , Masculino , Datos de Secuencia Molecular , Oxitocina/farmacología , Conducta Sexual Animal/efectos de los fármacos