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91.
GABAA receptors are heteromeric, ligand‐gated ion channels, built up with 19 different subunits. In the somatosensory cortex the most prevalent subunits are: alpha1, beta2 and gamma2. We showed that classical conditioning, in which stimulation of a row of vibrissae (CS) was paired with a tail shock (UCS), results in changes in cortical inhibitory system. Among others, GABAAalpha1 mRNA is up‐regulated in layer IV of cortical representation of trained row of vibrissae. We examined the mRNA expression level of beta2 and gamma2 subunits of GABAAR. For in situ hybridization, 35S‐labeled oligonucleotides were used as antisense probes. The effects were examined 1 h, 24 h and 5 days after the training lasting 3 days. There are no changes observed in beta2 subunits mRNA level. Expression of mRNA of gamma2 subunits increased 5 days after the training, but in contrast to other elements of gabaergic system we investigated, the change was observed in layers II/III. This indicates that learning‐dependent regulation of GABAA receptor phenotype is specific for given neuronal subtype. 相似文献
92.
93.
McGahan MC Harned J Mukunnemkeril M Goralska M Fleisher L Ferrell JB 《American journal of physiology. Cell physiology》2005,288(5):C1117-C1124
Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole. retinal pigment epithelial cells; lens epithelial cells 相似文献
94.
Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action 总被引:2,自引:0,他引:2
Zakrzewska M Krowarsch D Wiedlocha A Olsnes S Otlewski J 《Journal of molecular biology》2005,352(4):860-875
Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1. 相似文献
95.
The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient,
direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency,
ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants.
These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin
distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues
of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic
potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis. 相似文献
96.
In vitro study of alginate–chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure 总被引:7,自引:0,他引:7
Haque T Chen H Ouyang W Martoni C Lawuyi B Urbanska AM Prakash S 《Biotechnology letters》2005,27(5):317-322
The application of alginate–chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate–poly-L-lysine (APA) microcapsules for the application of cell therapy. 相似文献
97.
Abate C Kolanos R Dukat M Setola V Roth BL Glennon RA 《Bioorganic & medicinal chemistry letters》2005,15(15):3510-3513
Optically active pyrrolidinylmethylindole analogs related in structure to the benzenesulfonyltryptamine 5-HT(6) receptor antagonist MS-245 were evaluated and their R-isomers were found to bind with affinity higher than their S-enantiomers. 相似文献
98.
Palczewska M Batta G Groves P Linse S Kuznicki J 《Protein science : a publication of the Protein Society》2005,14(7):1879-1887
Calretinin, a neuronal protein with well-defined calcium-binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N-terminal (CR I-II), and C-terminal (CR III-VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium-bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The TNS data for the N- and C-terminal domains of CR are additive, providing further support for their independence within the full-length protein. Our work concentrated on CR I-II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I-II from a phenyl-Sepharose column was consistent with the TNS data. The pH-dependent structural changes were further localized to residues 13-28 and 44-51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N-terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer. 相似文献
99.
Schwimmer C Lefebvre-Legendre L Rak M Devin A Slonimski PP di Rago JP Rigoulet M 《The Journal of biological chemistry》2005,280(35):30751-30759
In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F0F1-ATP synthase at elevated temperatures. The deltafmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37 degrees C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the deltafmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports alpha-ketoglutarate and alpha-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the alpha-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system. 相似文献
100.
Zheng P Patel B McMenamin M Reddy SE Paprocki AM Schramm RD Latham KE 《Biology of reproduction》2004,70(5):1411-1418