RESUMEN
Heatstroke is a devastating condition that is characterized by severe hyperthermia and central nervous system dysfunction. However, the mechanism of thermoregulatory center dysfunction of the hypothalamus in heatstroke is unclear. In this study, we established a heatstroke mouse model and a heat-stressed neuronal cellular model on the pheochromocytoma-12 (PC12) cell line. These models revealed that HS promoted obvious neuronal injury in the hypothalamus, with high pathological scores. In addition, PC12 cell apoptosis was evident by decreased cell viability, increased caspase-3 activity, and high apoptosis rates. Furthermore, 14 differentially expressed proteins in the hypothalamus were analyzed by fluorescence two-dimensional difference gel electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Expression changes in hippocalcin (HPAC), a downregulated neuron-specific calcium-binding protein, were confirmed in the hypothalamus of the heatstroke mice and heat-stressed PC12 cells by immunochemistry and western blot. Moreover, HPAC overexpression and HPAC-targeted small interfering RNA experiments revealed that HPAC functioned as an antiapoptotic protein in heat-stressed PC12 cells and hypothalamic injury. Lastly, ulinastatin (UTI), a cell-protective drug that is clinically used to treat patients with heatstroke, was used in vitro and in vivo to confirm the role of HPAC; UTI inhibited heat stress (HS)-induced downregulation of HPAC expression, protected hypothalamic neurons and PC12 cells from HS-induced apoptosis and increased heat tolerance in the heatstroke animals. In summary, our study has uncovered and demonstrated the protective role of HPAC in heatstroke-induced hypothalamic injury in mice.
Asunto(s)
Apoptosis , Encefalopatías/metabolismo , Golpe de Calor/metabolismo , Hipocalcina/metabolismo , Hipotálamo/metabolismo , Neuronas/metabolismo , Proteómica , Animales , Apoptosis/efectos de los fármacos , Encefalopatías/etiología , Encefalopatías/patología , Encefalopatías/prevención & control , Modelos Animales de Enfermedad , Glicoproteínas/farmacología , Golpe de Calor/complicaciones , Golpe de Calor/tratamiento farmacológico , Hipocalcina/genética , Hipotálamo/efectos de los fármacos , Hipotálamo/patología , Masculino , Ratones Endogámicos BALB C , Neuronas/efectos de los fármacos , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Células PC12 , Proteómica/métodos , Ratas , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electroforesis Bidimensional Diferencial en GelRESUMEN
Gingko biloba extract 761 (EGb 761) protects neuronal cells from ischemic brain injury via a number of neuroprotective mechanisms. Hippocalcin is a calcium sensor protein that regulates intracellular calcium concentrations and apoptotic cell death. We investigated whether EGb 761 regulates hippocalcin expression in cerebral ischemia. Male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach demonstrated reduction in hippocalcin expression in vehicle-treated animals during MCAO, whereas EGb 761 treatment prevented injury-induced decreases in hippocalcin expression. RT-PCR and Western blot analyses indicated that EGb 761 attenuates injury-induced decrease in hippocalcin. These results suggest that the maintenance of hippocalcin during cerebral ischemia contributes to the neuroprotective role of EGb 761.
Asunto(s)
Animales , Humanos , Masculino , Western Blotting , Encéfalo , Lesiones Encefálicas , Isquemia Encefálica , Calcio , Muerte Celular , Corteza Cerebral , Ginkgo biloba , Hipocalcina , Infarto de la Arteria Cerebral Media , Neuronas , Extractos Vegetales , Ratas Sprague-DawleyRESUMEN
AIM OF THE STUDY: To investigate the effect of ginsenoside Rb(3) on synaptic transmission after oxygen-glucose deprivation in vitro. MATERIALS AND METHODS: The population spike (PS) was recorded in the stratum pyramidale of mouse hippocampal slices using extracellular recordings. RESULTS: Ginsenoside Rb(3) depressed the basal synaptic transmission, which also promoted the recovery amplitude of PS after OGD in a concentration-dependent manner. The GABA(A) receptor agonist muscimol improved the recovery, which was similar to that of ginsenoside Rb(3). Moreover, the effect of ginsenoside Rb(3) in combination with muscimol was not additive. Treatment with the GABA(A) receptor antagonist bicuculline or picrotoxin, which prevented the depression of PS caused by ginsenoside Rb(3), also reduced the neuroprotection. CONCLUSION: The results indicate that the activation of the GABA(A) receptor is correlated with the neuroprotective mechanisms of ginsenoside Rb(3).
Asunto(s)
Antagonistas de Receptores de GABA-A/farmacología , Ginsenósidos/farmacología , Hipocalcina/efectos de los fármacos , Hipocalcina/fisiología , Fármacos Neuroprotectores/farmacología , Animales , Bicuculina/farmacología , China , Etnofarmacología , Agonistas de Receptores de GABA-A/farmacología , Glucosa/metabolismo , Hipoxia/tratamiento farmacológico , Hipoxia/fisiopatología , Técnicas In Vitro , Masculino , Medicina Tradicional China , Ratones , Panax/química , Picrotoxina/farmacología , Plantas Medicinales/química , Transmisión Sináptica/efectos de los fármacosRESUMEN
Hippocalcin is a member of the neuronal Ca(2+) sensor protein family. Among its many biochemical functions, its established physiological function is that via neuronal apoptosis inhibitory protein it protects the neurons from Ca(2+)-induced cell death. The precise biochemical mechanism/s, through which hippocalcin functions, is not clear. In the present study, a new mechanism by which it functions is defined. The bovine form of hippocalcin (BovHpca) native to the hippocampus has been purified, sequenced, cloned, and studied. The findings show that there is the evolutionary conservation of its structure. It is a Ca(2+)-sensor of a variant form of the ROS-GC subfamily of membrane guanylate cyclases, ONE-GC. It senses physiological increments of Ca(2+) with a K(1/2) of 0.5 microM and stimulates ONE-GC or ONE-GC-like membrane guanylate cyclase. The Hpca-modulated ONE-GC-like transduction system exists in the hippocampal neurons. And hippocalcin-modulated ONE-GC transduction system exists in the olfactory receptor neuroepithelium. The Hpca-gene knock out studies demonstrate that the portion of this is about 30% of the total membrane guanylate cyclase transduction system. The findings establish Hpca as a new Ca(2+) sensor modulator of the ROS-GC membrane guanylate cyclase transduction subfamily. They support the concept on universality of the presence and operation of the ROS-GC transduction system in the sensory and sensory-linked neurons. They validate that the ROS-GC transduction system exists in multiple forms. And they provide an additional mechanism by which ROS-GC subfamily acts as a transducer of the Ca(2+) signals originating in the neurons.
Asunto(s)
Guanilato Ciclasa/metabolismo , Hipocalcina/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía en Gel , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Hipocalcina/química , Hipocalcina/genética , Hipocalcina/aislamiento & purificación , Hipocampo/enzimología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (K(d) 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1-14)-ECFP (enhanced cyan fluorescent protein) or NCS-1-ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase delta1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.
Asunto(s)
Hipocalcina/química , Hipocalcina/metabolismo , Ácido Mirístico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/metabolismo , Supervivencia Celular , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Células HeLa , Hipocalcina/genética , Humanos , Liposomas/metabolismo , Proteínas Sensoras del Calcio Neuronal , Neuropéptidos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
We have recently identified PP7, a novel group of plant protein Ser/Thr phosphatases, and hypothesized that PP7 may possess a calmodulin-binding site. To test this hypothesis, we assessed the effect of calmodulin on the activity of recombinant Arabidopsis thaliana PP7 and directly tested interaction between PP7 and calmodulin using surface plasmon resonance. Calmodulin exerted a moderate inhibitory effect on the phosphatase activity of PP7 with submicromolar affinity. PP7 specifically interacted with immobilized calmodulin (but not with recoverin, another EF hand Ca(2+)-binding protein) in a strictly Ca(2+)-dependent manner with nanomolar affinity. Deletion of an insert in the catalytic domain of PP7, predicted to function as a calmodulin-binding site, greatly decreased PP7 binding to calmodulin. These findings provide the first evidence for a plant protein phosphatase directly interacting with calmodulin and indicate that PP7 might be regulated by Ca(2+) levels in vivo.
Asunto(s)
Proteínas de Arabidopsis , Calmodulina/metabolismo , Proteínas del Ojo , Lipoproteínas , Proteínas del Tejido Nervioso , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/metabolismo , Sitios de Unión , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calmodulina/química , Dominio Catalítico , Clonación Molecular , Citoplasma/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Hipocalcina , Cinética , Mutación , Fosfoproteínas Fosfatasas/química , Proteínas de Plantas/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Recoverina , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
PURPOSE: It was recently found that recoverin acts as an autoantigen recognized by sera of patients with cancer-associated retinopathy (CAR), and that CAR-like retinal dysfunction is produced by intravitreous administration of anti-recoverin antibody in Lewis rat eyes. To examine the pathologic molecular mechanism of CAR, and to elucidate an effective therapy for CAR, the function and morphology of CAR were compared with those of phototoxic retinal damage, another form of photoreceptor dysfunction, and the effect of nilvadipine, a Ca(2+) antagonist, on the retinal degenerations was studied, using these models. METHODS: Under different illumination conditions and/or medication with nilvadipine, the functional and morphologic properties of the retinas were evaluated after intravitreous injection of anti-recoverin antibody into Lewis rat eyes (six rats, 12 eyes in each experimental condition), using electroretinogram (ERG), rhodopsin phosphorylation, and light microscopy. RESULTS: Anti-recoverin antibody administered into the vitreous of Lewis rat eyes induced a significant decrease and increase of ERG responses and rhodopsin phosphorylation levels, respectively, under cyclic or continuous light. Similar changes were observed in eyes of rats bred under continuous illumination that did not receive anti-recoverin antibodies. However, anti-recoverin antibody-induced retinal dysfunctions were not observed in rat eyes under dark conditions. Administration of nilvadipine, a Ca(2+) antagonist, to the anti-recoverin antibody-treated rats and rats with phototoxic retinal dysfunction caused significant improvement of the deterioration of ERG and normalization of rhodopsin phosphorylation. CONCLUSIONS: The present data indicate that anti-recoverin antibody-induced retinal dysfunction was functionally similar to phototoxic retinal dysfunction and was markedly suppressed under dark conditions or by systemic administration of a Ca(2+) antagonist.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Adaptación a la Oscuridad , Proteínas del Ojo , Lipoproteínas , Proteínas del Tejido Nervioso , Nifedipino/uso terapéutico , Síndromes Paraneoplásicos/terapia , Enfermedades de la Retina/terapia , Animales , Anticuerpos/administración & dosificación , Antígenos de Neoplasias/inmunología , Proteínas de Unión al Calcio/inmunología , Electrorretinografía , Hipocalcina , Inyecciones , Inyecciones Intraperitoneales , Luz , Nifedipino/análogos & derivados , Síndromes Paraneoplásicos/metabolismo , Síndromes Paraneoplásicos/patología , Fosforilación , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Recoverina , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Rodopsina/metabolismo , Cuerpo VítreoAsunto(s)
Antígenos de Neoplasias/inmunología , Autoanticuerpos/análisis , Proteínas de Unión al Calcio/inmunología , Neoplasias Endometriales/inmunología , Proteínas del Ojo , Lipoproteínas , Proteínas del Tejido Nervioso , Síndromes Paraneoplásicos/inmunología , Enfermedades de la Retina/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Aminoácidos/uso terapéutico , Anticuerpos Antineoplásicos/análisis , Biomarcadores de Tumor/análisis , Carbohidratos/uso terapéutico , Terapia Combinada , Combinación de Medicamentos , Neoplasias Endometriales/complicaciones , Neoplasias Endometriales/terapia , Femenino , Glucocorticoides/uso terapéutico , Hipocalcina , Humanos , Sustancias Húmicas/uso terapéutico , Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Síndromes Paraneoplásicos/etiología , Síndromes Paraneoplásicos/terapia , Recoverina , Enfermedades de la Retina/etiología , Enfermedades de la Retina/terapia , Linfocitos T/inmunología , Ácidos Urónicos/uso terapéuticoRESUMEN
A hippocalcin cDNA from rat brain cDNA library was amplified by polymerase chain reation(PCR) and cloned using TA Cloning technique. For this PCR cloning, 29mer and 28mer oligonucleotide primers containing BamHl and EcoRl sites at the 5' end and 3' end, respectively were used. The nucleotide sequence of hippocalcin cDNA c1one was determined, and the complete amino acid sequence was deduced. Recombinant clone contained a cDNA insert of 610 base pairs with 582 nucleotides of open reading frame including the temination codon, 23 nucleotide of 5'-untranslated region, and 5nucleotides of 3'-nutran,slated region. The open reading frame encoded a polypepetid comprising 193 amino acids with molecular weight of 22kDa. The cDNA insert was subcloned into pVLI393 Baculovirus transfer vector. The recombinant hippocalcin was expressed in insect cell(Sf9 cell) using expression vector pVL1393. The hippocalcin expressed was purified as a single band on polyacrylamide gel electrophoresis following hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. Molecular size of rat brain hippocalcin protein expressed in this system was estimated to be 22kDa. Myristoylated hippocalcin migrated faster than nonmyristoyated form on SDS-polyacrylamide gel. Less than 10% of total hippocalcin expressed was myristoylated in this baculovirus expression system.
Asunto(s)
Animales , Ratas , Secuencia de Aminoácidos , Aminoácidos , Baculoviridae , Emparejamiento Base , Secuencia de Bases , Encéfalo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Células Clonales , Clonación de Organismos , Codón , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Biblioteca de Genes , Hipocalcina , Insectos , Peso Molecular , Nucleótidos , Sistemas de Lectura Abierta , Reacción en Cadena de la PolimerasaRESUMEN
A frog retinal protein named s26 is a 26-kDa protein found during purification of S-modulin in frog retina (Kawamura, S. (1992) Photochem. Photobiol. 56, 1173-1180). To identify its role in frog retina, first s26 was purified to nearly homogeneity with three chromatographical steps. Based on the partial amino acid sequences of the proteolysed fragments of s26, we isolated cDNAs that encode s26. The analysis of its amino acid sequence revealed that s26 is an S-modulin-like protein, while it shows higher homology to visinin. Visinin is a Ca2+-binding protein reported to be present in chicken cones, but its localization in the retina had been a subject in dispute. The present study showed that s26 is present in cone photoreceptors. The study also showed that s26 inhibits phosphorylation of rhodopsin after a light flash at high Ca2+ concentrations as S-modulin does. From these results, we concluded that s26 is a cone homologue of S-modulin. The result is consistent with the idea that each type of photoreceptors expresses each cell-type specific version of phototransduction proteins.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas del Ojo/genética , Lipoproteínas , Proteínas del Tejido Nervioso , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Proteínas del Ojo/aislamiento & purificación , Proteínas del Ojo/metabolismo , Hipocalcina , Datos de Secuencia Molecular , Fosforilación , Filogenia , Rana catesbeiana , Recoverina , Rodopsina/antagonistas & inhibidores , Rodopsina/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We examined the biologic properties of a small-cell-lung-carcinoma (SCLC) cell line (designated MN-1112) established from a patient with SCLC who showed paraneoplastic retinopathy syndrome. Morphologic and immunocytochemical analyses showed that MN-1112 cells possess features of the classic type of SCLC. MN-1112 cells grew in suspension forming relatively large clumps of cells with a doubling time of 72 hr. By light-microscope examination, the cells were relatively small and had scanty cytoplasm. The cells produced NSE, ACTH and CK (BB isozyme); they also expressed recoverin, a novel photoreceptor protein, detected by Northern-blot and Western-immunoblot analysis using human-recoverin-specific DNA probe and anti-bovine-recoverin polyclonal antibody. This report shows that human recoverin is expressed in cultured SCLC cells. Our results support the hypothesis that, in cancer-associated retinopathy (CAR) patients, auto-immune antibody targeting for ectopic recoverin in SCLC is initially produced and cross-reacts with the retinal protein, resulting in the retinal degeneration that occurs in CAR patients.
Asunto(s)
Carcinoma de Células Pequeñas/patología , Proteínas del Ojo , Lipoproteínas , Proteínas del Tejido Nervioso , Degeneración Retiniana/patología , Células Tumorales Cultivadas , Anciano , Aneuploidia , Secuencia de Bases , Biomarcadores de Tumor , Proteínas de Unión al Calcio/genética , División Celular , Cartilla de ADN/química , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Hipocalcina , Humanos , Cariotipificación , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , RecoverinaRESUMEN
The Myb-Ets oncoprotein encoded by the E26 avian leukaemia virus represents a fusion of two transcription factors which cooperate in transforming multipotent haematopoietic progenitors (MEPs) in vitro and in vivo. Previous studies with a temperature sensitive mutant in ets (ts1.1 E26) have suggested that the Ets part of the Myb-Ets fusion protein blocks multilineage differentiation of transformed MEPs, by regulating specific target genes. Using this system in a differential screening approach we have now identified a new gene, called rem-1, as a target for the E26 virus. Following shift of ts1.1 mutant transformed cells to the nonpermissive temperature a decreased expression of rem-1 was observed which increased upon downshift. The finding that this reexpression did not require new protein synthesis suggests that the Ets component of the fusion protein directly regulates rem-1 transcription. Rem-1 is related to a family of EF-hand-containing calcium-binding proteins that are predominantly expressed in the brain and in retinal cells. This family includes recoverin and visinin, proteins that have been implicated in regulating photoreception. Rem-1 is likewise expressed in these tissues but in addition in haematopoietic cells and in the gut. Enforced expression of rem-1 in ts1.1-transformed MEP cells, using a retroviral vector, showed that this gene is not sufficient to block their differentiation, but that it may provide them with a growth advantage.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas del Ojo , Células Madre Hematopoyéticas/metabolismo , Lipoproteínas , Proteínas del Tejido Nervioso , Proteínas de Fusión Oncogénica/genética , Proteínas Oncogénicas de Retroviridae/genética , Secuencia de Aminoácidos , Animales , Virus de la Mieloblastosis Aviar/genética , Línea Celular , ADN Complementario , Regulación de la Expresión Génica , Hipocalcina , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Oncogénicas v-myb , Recoverina , Homología de Secuencia de AminoácidoAsunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , Proteínas de Unión al Calcio/genética , Mapeo Cromosómico , Proteínas del Ojo/genética , Genes , Lipoproteínas , Ratones/genética , Proteínas del Tejido Nervioso , Retina/metabolismo , Animales , Calnexina , Cruzamientos Genéticos , ADN Complementario/genética , Expresión Génica , Hipocalcina , Muridae/genética , RecoverinaRESUMEN
PURPOSE: Recoverin, a calcium-binding protein that selectively localizes to the retina and pineal gland, has been identified as the target for the putative pathogenic autoimmune process of cancer-associated retinopathy (CAR). The present study was aimed at testing the capacity of recoverin to induce experimental autoimmune uveoretinitis and pinealitis in Lewis rats. METHODS: Lewis rats were immunized against recombinant myristoylated recoverin by a single footpad injection of the protein, at various doses, emulsified in complete Freund's adjuvant. Development of uveoretinitis was monitored by clinical and histologic examinations, whereas pinealitis was detected by histologic examination. RESULTS: Immunization with recoverin induced severe panuveitic changes that closely resemble those induced by S-antigen (arrestin). The effect was dose-dependent, with 10 micrograms/rat the lowest immunopathogenic dose. Rats immunized with recoverin also developed pineal inflammation. CONCLUSION: Recoverin is highly immunopathogenic in Lewis rats. Although the ocular changes induced in rats differ from those seen in CAR, the data recorded here are in line with the concept that recoverin can initiate pathogenic autoimmune processes in the eye.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Proteínas de Unión al Calcio/toxicidad , Proteínas del Ojo/toxicidad , Lipoproteínas , Proteínas del Tejido Nervioso , Retinitis/inmunología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/patología , Relación Dosis-Respuesta a Droga , Femenino , Hipocalcina , Masculino , Glándula Pineal/patología , Ratas , Ratas Endogámicas Lew , Recoverina , Retinitis/patología , Uveítis/patologíaRESUMEN
A cDNA clone (hHLP2) encoding a novel calcium-binding protein structurally related to hippocalcin has been isolated from the human hippocampus cDNA library. The primary structure consists of 193 amino acids, and contains three EF-hand structures and a possible NH2-terminal myristoylation site. A single transcript at a position corresponding to 1.7 kilobases was detected only in the brain. The hHLP2 gene was mapped to human chromosome 2.
Asunto(s)
Química Encefálica , Proteínas de Unión al Calcio/genética , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al Calcio/química , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Hipocalcina , Datos de Secuencia Molecular , Estructura Molecular , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
We have isolated a cDNA clone encoding human hippocalcin from a human hippocampus cDNA library. This clone (hHLP1) consists of 840 nucleotides, including the entire open reading frame of 582 nucleotides, 10 nucleotides of the 5' leader and 248 nucleotides of the 3' noncoding regions. Comparison of the human hippocalcin sequence with the corresponding rat sequence revealed an amino acid identity of 100% and nucleotide identity of 92%. Northern blot analysis showed that a single transcript at a position corresponding to 2.0 kb was detected only in the brain. The human hippocalcin gene was mapped to chromosome 1 by amplification of human hippocalcin-specific DNA fragment on DNA from human-rodent somatic cell hybrids by using the polymerase chain reaction.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Cromosomas Humanos Par 1 , ADN/genética , Hipocampo/metabolismo , Proteínas del Tejido Nervioso , ARN Mensajero/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , Cricetinae , Cartilla de ADN , ADN Complementario/metabolismo , Biblioteca de Genes , Hipocalcina , Humanos , Células Híbridas , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE: We have inquired into the reason why patients with cancer-associated retinopathy (CAR) produce antibody reactions with the 23-kd retinal CAR antigen. Possible reasons include the expression of this antigen in the related carcinoma. Previous studies have failed to identify any antigenic counterpart expressed by in vitro cultivated small-cell carcinoma of the lung. We, therefore, inquired into the effects of in vivo cultivation of the cancer cells and its influence on protein expression, with specific reference to the appearance of the 23-kd retinal CAR antigen. DESIGN: A complementary DNA library was prepared from small-cell carcinoma of the lung cells propagated intraperitoneally in Lewis rats and probed with antibodies reactive with the 23-kd retinal CAR antigen. RESULTS: We found evidence of the expression of a cancer-associated gene in ascites-propagated small-cell carcinoma of the lung that encodes for a protein antigenically similar to the 23-kd retinal CAR antigen. A complementary DNA encoding this protein revealed complete DNA sequence homology with the retinal CAR antigen showing the cancer cells are expressing this photoreceptor protein. CONCLUSIONS: We hypothesize that the carcinoma-retina immunologic cross-reaction is responsible for the induction of the unique antibody response encountered in patients with CAR with vision loss developing as a cancer-evoked autoimmune retinopathy.
Asunto(s)
Proteínas de Unión al Calcio/genética , Carcinoma de Células Pequeñas/inmunología , Proteínas del Ojo , Regulación Neoplásica de la Expresión Génica , Lipoproteínas , Neoplasias Pulmonares/inmunología , Proteínas del Tejido Nervioso , Animales , Anticuerpos Antineoplásicos/análisis , Biomarcadores de Tumor , Proteínas de Unión al Calcio/inmunología , Carcinoma de Células Pequeñas/patología , ADN de Neoplasias/genética , Femenino , Hipocalcina , Neoplasias Pulmonares/patología , ARN Neoplásico/aislamiento & purificación , Ratas , Ratas Endogámicas Lew , Recoverina , Enfermedades de la Retina/inmunologíaRESUMEN
Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.