RESUMO
BACKGROUND: Many users in Japan of skin brightening/lightening cosmetics containing rhododendrol (RD) have developed leucoderma. Leucoderma appears on skin areas repeatedly treated with RD-containing cosmetics. RD-induced leucoderma (RDIL) presents different degrees of well-defined hypopigmentation. It is crucial to determine the degree of hypopigmentation to differentiate RDIL from vitiligo vulgaris (VV). OBJECTIVES: To quantitatively evaluate hypopigmentation of RDIL lesions and the recovery of pigmentation, and to compare the hypopigmentation with VV and normal skin. MATERIALS AND METHODS: Sixteen cases of RDIL, nine cases of VV and 15 healthy controls were examined using a novel multispectral camera (MSC) that can simultaneously obtain the reflection intensity at 10-nm wavelength intervals from 400 to 760 nm of the photographed area. ∆Absorbance was calculated by subtracting the log of reflection intensity of the target area from that of a white reflection standard. RESULTS: Most RDIL lesions showed lower ∆Absorbance than healthy skin and higher ∆Absorbance than VV lesions between 400 and 550 nm. Statistical comparison of the maximum ∆Absorbance from 420 to 460 nm (Max∆Absorbance) for VV, RDIL and control skin showed that the Max∆Absorbance of RDIL was significantly higher than that of VV and lower than that of control skin. The comparison of ∆Absorbance of the same sites in RDIL lesions between the initial visit and 6 months later showed significant improvement after 6 months. CONCLUSIONS: These studies demonstrated quantitative changes in RDIL and its recovery phase and suggested the utility of a MSC in obtaining objective colour information of skin disorders.
Assuntos
Butanóis/efeitos adversos , Hipopigmentação/induzido quimicamente , Preparações Clareadoras de Pele/efeitos adversos , Adulto , Idoso , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Humanos , Hipopigmentação/diagnóstico , Japão , Pessoa de Meia-Idade , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Vitiligo/diagnósticoRESUMO
Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and alphav integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and alphav integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while alphav integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as alphav integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with alphav integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with alphav integrin is likely to be crucial to the development of targeted Ad vectors.
Assuntos
Adenoviridae/genética , Deleção de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Receptores Virais/genética , Receptores de Vitronectina/genética , Animais , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Engenharia Genética , Vetores Genéticos/genética , Humanos , Injeções Intravenosas , Fígado/metabolismo , Camundongos , Oligopeptídeos/genética , Transdução GenéticaRESUMO
One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Oligopeptídeos/genética , Animais , Linhagem Celular , Terapia Genética , Vetores Genéticos , Humanos , Integrinas/análise , Camundongos , Receptores Virais/análise , Receptores de Vitronectina/análise , Células Tumorais CultivadasRESUMO
Plasmin-induced platelet activation is considered to be a cause of reocclusion after thrombolytic therapy with plasminogen activators. However, little is known regarding its mechanism and regulation, particularly with respect to the initial step shape change. We here demonstrate that a Ca2+-independent pathway is involved in plasmin-induced human platelet shape change, and that Rho-kinase plays an important role in this pathway. When the increase in cytosolic Ca2+ was prevented by an intracellular Ca2+ chelator, 5,5'-dimethyl-BAPTA, plasmin-induced platelet shape change was partially inhibited but still occurred. In the presence of 5,5'-dimethyl-BAPTA, a specific Rho-kinase inhibitor, Y-27632, completely inhibited the shape change. Phosphorylation of myosin light chain, a key regulator of platelet shape change, was completely inhibited by Y-27632 in 5,5'-dimethyl-BAPTA-treated platelets. Although plasmin caused tyrosine phosphorylation of the 80 kDa protein during the shape change, it did not seem to have a critical role. cAMP-elevating agents inhibited plasmin-induced shape change in 5,5'-dimethyl-BAPTA- or Y-27632-treated platelets with similar efficiency. These results indicated that plasmin causes platelet shape change by activating Ca2+-dependent and Ca2+-independent-Rho-kinase-dependent pathways, both of which are sensitive to cAMP.
Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Fibrinolisina/farmacologia , Amidas/farmacologia , Animais , Plaquetas/metabolismo , Células Cultivadas , AMP Cíclico/farmacologia , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , Ovinos , Quinases Associadas a rhoRESUMO
In studies regarding both gene therapy and gene function, transgene expression by plasmid vectors benefits from the use of transcriptional regulatory elements which permit high-level gene expression. Therefore, with respect to transgene (luciferase) expression activity both in vitro (using HeLa, HepG2, and ECV304 cells) and in vivo (mouse liver and skeletal muscle), we investigated the effective combination of commonly-used regulatory elements, such as the promoter/enhancer, intron, and polyadenylation signal (P(A)) sequence by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoter/enhancers that were tested, hybrid CA promoter/enhancer containing human cytomegalovirus immediate-early 1 gene (CMV) enhancer and chicken beta-actin promoter with the beta-actin intron sequence, and the improved CMV promoter/enhancer containing the largest intron of CMV (intron A) produced the highest levels of expression both in vitro and in vivo. P(A) sequences were found to have significant effects on transgene expression. The effect of a multiple enhancer was also examined. Optimized plasmids of this study were pCASL3 (composed of CMV enhancer, beta-actin promoter, beta-actin intron, Simian virus (SV40) P(A) sequence and SV40 enhancer) and pCMVSL3 (composed of CMV enhancer, CMV promoter, intron A, SV40 P(A) sequence and SV40 enhancer). These comparative analyses could provide a systematic reference for the development of vector construction for gene therapy, vaccine development, and gene transfer experiments.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Citomegalovirus/genética , Elementos Facilitadores Genéticos/genética , Feminino , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Poli A/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The internal ribosome entry site (IRES) has been widely used to coexpress heterologous gene products by a message from a single promoter. However, little is known about the efficiency of IRES-dependent second gene expression in comparison with that of first gene expression. This study was undertaken to characterize the relative expression of IRES-dependent second gene in a bicistronic vector, which was derived from the 5' untranslated regions of the encephalomyocarditis virus (EMCV). IRES-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines and in mouse liver in vivo. The expression of the IRES-dependent second gene ranged from 6 to 100% (in most cases between 20 and 50%) that of the first gene. Second gene expression in a plasmid without the IRES was 0.1-0.8% (with some exceptions) that of the first gene. These findings have important implications for the use of IRES, i.e., care should be taken regarding the decreased capacity of IRES-dependent downstream gene expression as well as in determining which gene should be positioned as the first or second gene in a bicistronic vector.
Assuntos
Expressão Gênica , Vetores Genéticos , Regiões 5' não Traduzidas , Fosfatase Alcalina/genética , Animais , Células CHO , Cloranfenicol O-Acetiltransferase/genética , Cricetinae , Vírus da Encefalomiocardite/genética , Técnicas de Transferência de Genes , Genes/genética , Células HeLa , Humanos , Células L , Fígado/metabolismo , Luciferases/genética , Camundongos , Plasmídeos/genética , Capuzes de RNA/genéticaRESUMO
Interactions between noradrenergic and cholinergic receptor signaling may be important in some forms of learning. To investigate whether noradrenergic and cholinergic receptor interactions regulate forms of synaptic plasticity thought to be involved in memory formation, we examined the effects of concurrent beta-adrenergic and cholinergic receptor activation on the induction of long-term potentiation (LTP) in the hippocampal CA1 region. Low concentrations of the beta-adrenergic receptor agonist isoproterenol (ISO) and the cholinergic receptor agonist carbachol had no effect on the induction of LTP by a brief train of 5 Hz stimulation when applied individually but dramatically facilitated LTP induction when coapplied. Although carbachol did not enhance ISO-induced increases in cAMP, coapplication of ISO and carbachol synergistically activated p42 mitogen-activated protein kinase (p42 MAPK). This suggests that concurrent beta-adrenergic and cholinergic receptor activation enhances LTP induction by activating MAPK and not by additive or synergistic effects on adenylyl cyclase. Consistent with this, blocking MAPK activation with MEK inhibitors suppressed the facilitation of LTP induction produced by concurrent beta-adrenergic and cholinergic receptor activation. Although MEK inhibitors also suppressed the induction of LTP by a stronger 5 Hz stimulation protocol that induced LTP in the absence of ISO and carbachol, they had no effect on LTP induced by high-frequency synaptic stimulation or low-frequency synaptic stimulation paired with postsynaptic depolarization. Our results indicate that MAPK activation has an important, modulatory role in the induction of LTP and suggest that coactivation of noradrenergic and cholinergic receptors regulates LTP induction via convergent effects on MAPK.
Assuntos
Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Colinérgicos/efeitos dos fármacos , Animais , Carbacol/farmacologia , AMP Cíclico/metabolismo , Estimulação Elétrica , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , Isoproterenol/farmacologia , Potenciação de Longa Duração/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Colinérgicos/metabolismoAssuntos
Autoanticorpos/sangue , Dermatoses Faciais/complicações , Sarcoidose/complicações , Vitiligo/complicações , Braço , Diabetes Mellitus Tipo 2/complicações , Diagnóstico Diferencial , Dermatoses Faciais/diagnóstico , Feminino , Humanos , Hipertensão/complicações , Pessoa de Meia-Idade , Sarcoidose/diagnóstico , Sarcoidose/patologiaRESUMO
In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.
Assuntos
Fusão de Membrana/fisiologia , Respirovirus/fisiologia , Animais , Antígenos Virais/análise , Células COS , Linhagem Celular , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Toxina Diftérica/análise , Toxina Diftérica/farmacocinética , Técnica Indireta de Fluorescência para Anticorpo , Genoma Viral , Células HeLa , Humanos , Cinética , Lipossomos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/farmacocinética , Respirovirus/genética , Transfecção , Replicação ViralRESUMO
Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.
Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/efeitos dos fármacos , Fibrinolisina/farmacologia , Proteínas de Membrana , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Apirase/farmacologia , Plaquetas/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Interações Medicamentosas , Epinefrina/farmacologia , Fibrinolíticos/farmacologia , Humanos , Técnicas In Vitro , Ligantes , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12RESUMO
Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.
Assuntos
Terapia Genética , Vetores Genéticos , Lipossomos , Respirovirus/genética , Humanos , Lipossomos/ultraestrutura , Fusão de Membrana , Modelos Biológicos , Respirovirus/ultraestruturaRESUMO
Fusogenic liposome, a unique vector prepared by fusing ultraviolet-inactivated Sendai virus and liposome, is known to efficiently deliver content into various animal cells through membrane fusion. In this study, we examined the target-cell specificity of fusogenic liposome (FL)-mediated macromolecule delivery into human blood cells using diphtheria toxin fragment A (DTA) as a probe. Among the peripheral blood mononuclear cells (PBMC), FL was able to deliver its encapsulates into CD14+ monocytes and CD4-/CD8- T-cells, but not into CD19+ B-lymphocytes, CD4+ T-cells or CD8+ T-cells. The susceptibility of human leukemia cell lines to FL was similar to that of PBMC; the order of the reactivity was U937 (monoblastic leukemia)>MOLT4, Jurkat (T-lymphoma)>Daudi, BALL1 (B-lymphoma)>K562 (erythroblastic leukemia). Interestingly, FL showed similar binding activity to all of these leukemia cell lines. These findings indicate that, among blood cells, monocytes, monoblastic leukemia cells, CD4-/CD8- T-cells and T-lymphoma cells are preferable targets for FL-mediated macromolecule delivery. This is the first demonstration of the existence of non-permissive cells against FL. Our results also suggest that some molecules on target-cells other than the binding targets of SV-derived protein may participate in fusion between FL and cells.
Assuntos
Membranas Intracelulares/fisiologia , Fusão de Membrana , Monócitos/fisiologia , Toxina Diftérica/farmacologia , Portadores de Fármacos , Células HeLa , Humanos , Lipossomos , Monócitos/ultraestrutura , Fragmentos de Peptídeos/farmacologia , Respirovirus/fisiologia , Linfócitos T , Células Tumorais Cultivadas , Proteínas Virais/fisiologiaRESUMO
Specific patterns of neuronal firing induce changes in synaptic strength that may contribute to learning and memory. If the postsynaptic NMDA (N-methyl-D-aspartate) receptors are blocked, long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and the learning of spatial information are prevented. The NMDA receptor can bind a protein known as postsynaptic density-95 (PSD-95), which may regulate the localization of and/or signalling by the receptor. In mutant mice lacking PSD-95, the frequency function of NMDA-dependent LTP and LTD is shifted to produce strikingly enhanced LTP at different frequencies of synaptic stimulation. In keeping with neural-network models that incorporate bidirectional learning rules, this frequency shift is accompanied by severely impaired spatial learning. Synaptic NMDA-receptor currents, subunit expression, localization and synaptic morphology are all unaffected in the mutant mice. PSD-95 thus appears to be important in coupling the NMDA receptor to pathways that control bidirectional synaptic plasticity and learning.
Assuntos
Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transdução de Sinais , Animais , Proteína 4 Homóloga a Disks-Large , Eletrofisiologia , Marcação de Genes , Guanilato Quinases , Hipocampo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Deficiências da Aprendizagem/fisiopatologia , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Mutação , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/fisiologiaRESUMO
Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr492-Thr512), corresponding to the beta 8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu513-Lys542), corresponding to alpha-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of alpha-amylase at non-permissive temperature.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/genética , Mutação , Ligação Proteica , Deleção de Sequência , alfa-Amilases/metabolismoRESUMO
A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Respirovirus/genética , Núcleo Celular/metabolismo , DNA/administração & dosagem , DNA/metabolismo , Terapia Genética , Humanos , Microscopia Eletrônica , Sinais de Localização NuclearRESUMO
Long-term potentiation (LTP), a persistent enhancement of synaptic transmission that may be involved in some forms of learning and memory, is induced at excitatory synapses in the CA1 region of the hippocampus by coincident presynaptic and postsynaptic activity. Although action potentials back-propagating into dendrites of hippocampal pyramidal cells provide sufficient postsynaptic activity to induce LTP under some in vitro conditions, it is not known whether LTP can be induced by patterns of postsynaptic action potential firing that occur in these cells in vivo. Here we report that a characteristic in vivo pattern of action potential generation in CA1 pyramidal cells known as the complex spike burst enables the induction of LTP during theta frequency synaptic stimulation in the CA1 region of hippocampal slices maintained in vitro. Our results suggest that complex spike bursting may have an important role in synaptic processes involved in learning and memory formation, perhaps by producing a highly sensitive postsynaptic state during which even low frequencies of presynaptic activity can induce LTP.
Assuntos
Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Transmissão Sináptica/fisiologia , Ritmo Teta , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Periodicidade , Células Piramidais/química , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse alpha-amylase. The mutation site was identified as a point mutation at t1337 to c1337 resulting in the Ile-404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) of the putative peptide-binding domain. This isoleucine is highly conserved in the Hsp70 family. By pulse-chase experiments, no obvious difference was detected in the intracellular secretion rate of MF alpha 1-prepro-signal-mouse-alpha-amylase between the wild type and the kar2-404 mutant. However, only about half the amount of secreted heterologous protein, mouse alpha-amylase, was detected in the mutant culture medium compared with wild type. A smaller amount of homologous protein, alpha-factor, was also detected and decreased faster in the mutant culture medium than in wild type. Kar2-404p was expressed about 3-fold more than wild type Kar2p, probably to cover its defective functions, and the turnover rates of Kar2p and Kar2-404p were about the same in vivo. The purified Kar2-404p was slightly more sensitive to chymotryptic digestion than Kar2p in vitro.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Meios de Cultura , Proteínas Fúngicas/isolamento & purificação , Proteínas de Choque Térmico HSP70/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas , Engenharia de Proteínas/métodos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
This study was undertaken to determine if reocclusion after treatment of myocardial infarction with a tissue-plasminogen activator (t-PA) may be due to plasmin-induced platelet aggregation. t-PA caused platelet aggregation by conversion of plasminogen to plasmin under certain conditions. Plasmin-induced platelet aggregation was inhibited by serine protease inhibitors, aprotinin and bdellin, and a lysine binding site inhibitor, epsilon-aminocaproic acid (EACA). Extracellular [Ca2+], and RGDS sequence-dependent steps were involved in the aggregation process. The action of plasmin was inhibited by large thrombin antagonistic molecules such as argatroban-inactivated thrombin or anti-thrombin receptor peptide antibodies but not by small molecules like thrombin receptor antagonist peptides. This suggests that target molecules of plasmin on the surface of platelets may not be thrombin receptors but may exist very close to thrombin receptors. Binding experiments using FITC-labeled plasmin showed that plasmin has its binding sites on platelets. Flow cytometric analyses with four types of anti-plasmin(ogen) monoclonal antibodies suggested that plasmin might bind to platelets through the N-terminal region. The binding of plasmin to platelets was suppressed by aprotinin and EACA, furthermore indicating that protease catalytic sites and lysine binding regions are involved in interaction of plasmin to platelet.
Assuntos
Fibrinolisina/farmacologia , Compostos Orgânicos , Agregação Plaquetária/efeitos dos fármacos , Ácido Aminocaproico/farmacologia , Aprotinina/farmacologia , Plaquetas/metabolismo , Membrana Celular/metabolismo , Fibrinolisina/metabolismo , Hirudinas/farmacologia , Humanos , Técnicas In Vitro , Inibidores da Agregação Plaquetária/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.
Assuntos
Comportamento Animal/fisiologia , Síndrome de Down/genética , Aprendizagem/fisiologia , Camundongos Transgênicos/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Encéfalo/patologia , Cromossomos Artificiais de Levedura , Eletrofisiologia , Olho/patologia , Dosagem de Genes , Humanos , Aprendizagem em Labirinto/fisiologia , Camundongos , Dados de Sequência Molecular , Atividade Motora/genética , Proteínas Tirosina Quinases , Homologia de Sequência do Ácido Nucleico , Transgenes , Quinases DyrkRESUMO
The linear double stranded DNA plasmid pGKL1 encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Saccharomyces gene library, and isolated three DNA fragments that complement the iki1, iki3, and iki4 mutations, respectively.