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At least 1,099 records · Page 61Linked to original sources
PubMed1993

Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride.

A simple method for the transfer of 32P-labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS-buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.

Amino Acids↗
PubMed1988

p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix.

Protein I is a hetero-tetramer which contains two copies each of p11 and p36. p36 (calpactin I, lipocortin II) is a major substrate of retrovirally encoded tyrosine protein kinases, while p11 modulates several Ca2+-induced properties also displayed by p36 alone. Here we have characterized the p11 binding site on p36 by fluorescence spectroscopy using porcine p36 labelled at cysteine 8 with the fluorophore Prodan (6-proprionyl-2-dimethylamino-naphthalene). We have used peptides of differing length from the amino-terminal domain of p36 to restrict the major binding site to the first 12 residues. Noticeable binding is still observed with a peptide containing only the first nine residues. Interestingly the N-terminal acetyl group of p36 forms a functional part of the p11 binding site. CD studies indicate that the binding region can form an alpha-helix, which seems to have amphiphatic properties when projected on a helical wheel. This structural element is also known for a calmodulin binding protein. Thus the question is raised whether other p11/calmodulin-related proteins interact with their target proteins via a similar mechanism. We also discuss how p11 could modulate p36 associated properties.

Amino Acid Sequence↗
PubMed2003

Phosphorylation of proteins in neuron terminals: specificity depends on coincidental signaling.

We investigate the role of neuronal coincidental signaling mediated by the second messengers, on phosphorylation of three major proteins of neurosecretory vesicles. Our data show that different combinations of coincidental signaling generate specific pattern of phosphoproteins and not strictly additional effects. This suggests that an added phosphate on a site might 'mask' or 'unmask' the next sites for specific kinases and phosphatases action by inducing conformation change or protein association. We show that a function of vesicles such as the uptake of glutamate is highly regulated by coincidental signaling.

Animals↗
PubMed2006

Amyloid beta up-regulates brain-derived neurotrophic factor production from astrocytes: rescue from amyloid beta-related neuritic degeneration.

Astrocytes, the most abundant type of glia in the brain, are considered to play a key role in Alzheimer's disease (AD) pathologies. In a cell culture study, we have previously shown that astroglial responses against amyloid beta (Abeta) occur before obvious neuronal damage could be detected, suggesting the possibility that astrocytes might be an attractive therapeutic target for treating AD. In the present study, we investigated astroglial gene expression changes in response to Abeta to elucidate further the role of astrocytes in Abeta toxicity. By using real-time PCR and ELISA analyses, we found that Abeta rapidly induced astrocytes to produce brain-derived neurotrophic factor (BDNF). Abeta42 was more effective than Abeta40 in increasing astroglial BDNF production. Moreover, BDNF treatment rescued the neuronally differentiated human neuroblastoma cells from neuritic degeneration caused by Abeta toxicity. This is the first study to demonstrate that astrocytes are capable of increasing the production of a particular neurotrophic factor in response to Abeta. Our findings also identify BDNF as a potential therapeutic agent for preventing Abeta-related neuritic degeneration.

Amyloid↗
PubMed2005

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta.

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non-neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7-day-old cultures a subgroup of small non-neural cells with processes expressed ChAT activity, and in 14-day-old cultures non-neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non-neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1-day-old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non-neural cells during the development of Manduca antenna.

Animals↗
PubMed1996

Ultrastructural localization of a synaptic ribbon protein recognized by antibody B16.

This study examines the immunolabelled structures in the mouse retina following incubation with a monoclonal antibody (B16) that recognizes a highly conserved antigen found in retinas from lizards, frogs, fish, birds, mice, rats, rabbits, cats, and monkeys. This paper focuses on observations in the murine retina. The B16 labelling pattern in the retina was compared with that of two synaptic vesicle antigens: SV2 and anti-synaptophysin in the outer plexiform layer were more diffuse and apparently filled the entire presynaptic terminal whereas B16 labelling was more restricted and labelled a discrete structure resembling a semi-ellipse or an arc with the ends pointing to the inner nuclear layer and the middle curve facing the outer nuclear layer (1-2 microns long by < 0.05 micron in width). The structure labelled in the inner plexiform layer resembles a short bar (0.8 micron long by < 0.05 micron in width) that is confined to the inner half the inner plexiform layer. Cryo-ultra microtomy was used to examine the ultrastructural distribution of the labelling, because the B16 epitope is sensitive to fixation and plastic embedding. The tissue was incubated with the B16 antibody and visualized with goat-anti-mouse 10 nm gold particles. In all cases label was found to be confined to the ribbon structure in the photoreceptor terminal. This is consistent with previous evidence linking B16 to an epitope associated with the synaptic ribbon. The labelling is confined to the ribbon structure and does not appear to be associated with synaptic vesicles.

Animals↗
PubMed2001

Expression of connexin 43 and connexin 32 gap-junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex.

The expression of the gap-junction proteins connexin (CX) 43 and 32 was evaluated in surgical specimens of brain tumors and perilesional cortex from patients with chronic medically intractable epilepsy. In human normal brain CX32 was expressed in neurons and oligodendrocytes. CX32 immunoreactivity (IR) was observed in the neuronal component of glioneuronal tumors and in all oligodendrogliomas, 50% of which showed strong labeling, independent of the grade of differentiation. CX43, normally expressed in astrocytes, was also detected in most of the human astrocytomas and in the astroglial component of glioneuronal tumors. Whereas most of the low-grade gliomas (>60%) showed strong membranous staining, most high-grade astrocytomas exhibited a reduction of the typical plasma membrane CX43-IR and intracytoplasmic localization. Immunoblot analysis showed different CX43 isoforms in control cortex and in low-grade gliomas. However, only one single isoform (corresponding to the non-phosphorylated form of CX43) appeared to be present in most high-grade gliomas. Increased expression of CX43 protein was present in reactive astrocytes in the epileptic cortex surrounding low-grade tumors as compared to control cortex, indicating the existence of a regulatory pathway involving CX43 in the reorganization of the astrocytic syncytium in regions undergoing reactive gliosis. The high expression of connexin proteins in low-grade tumors and in the peritumoral reactive astrocytes suggests that they could contribute to tumor-related seizures.

Astrocytes↗
PubMed1984

A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.

A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.

Alkaline Phosphatase↗
PubMed1986

A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters.

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.

Animals↗
PubMed1986

Phosphorylation of p36 in vitro by protein kinase C.

The 36kDa subunit of protein I (p36) is a major substrate of several tyrosine protein kinases. Here we demonstrate that protein kinase C catalyzes the incorporation of 1.7 moles of phosphate per mole of protein I. Phosphorylation is absolutely dependent on the presence of both calcium and phospholipid, and is specific for serine and threonine residues. Phosphorylation of protein I by the c-AMP dependent protein kinase, phosphorylase kinase, casein kinase I, and casein kinase II was not observed. The in vivo significance of protein kinase C dependent phosphorylation of p36 is discussed.

Animals↗
PubMed1987

Development of spontaneous and glutamate-evoked activity is altered by chronic ethanol in cultured cerebellar Purkinje neurons.

The effects of continuous exposure to ethanol on the cytological and physiological development of a central nervous system (CNS) neuron were studied using the cultured Purkinje neuron of the rat cerebellar cortex. Purkinje neurons in fetal rat brain cultures which are established at one day before birth show development comparable to that described in vivo in other studies. In culture, Purkinje neurons progress from immature rounded cells with fine neurites to mature neurons with a branched dendritic structure. These structural changes are accompanied by an increase in the duration and complexity of the excitatory response to glutamate, by transitions in the patterns of spontaneous activity, and by an increase in mean firing rate. Our results demonstrate that chronic exposure to a low concentration of ethanol (90 mg%; 19.5 mM) during development selectively alters the electrophysiological but not the morphological properties of Purkinje neurons. Specifically, ethanol treatment reduces the responsiveness of these neurons to glutamate, delays the expected developmental transitions in patterns of spontaneous activity, and induces increased spontaneous bursting activity, particularly at the stage of dendritic formation. Impairment of responsiveness to glutamate is significant in that it may reflect the compromise by ethanol of a major excitatory pathway in the cerebellar cortex, resulting from the decreased efficacy of glutamatergic input from parallel fibers. In contrast to the results of other studies using adult neurons as a model for the effects of ethanol, our work suggests that the developing CNS neuron does not become tolerant; that is, in the continuing presence of ethanol, it does not express physiological function equivalent to that of the control.

Action Potentials↗
PubMed1994

Synapse alterations in the hippocampal-entorhinal formation in Alzheimer's disease with and without Lewy body disease.

We quantified by microdensitometry the immunoreactivity (IR) to monoclonal antibodies (SP6, SP12, SP15 and SP18) against various synaptic proteins in the molecular layers of the dentate gyrus, CA4, CA3, CA1, subiculum and entorhinal cortex in Alzheimer's disease (AD), Lewy body variant of AD (LBV) and diffuse Lewy body disease (DLBD). A significant decrease in SP6 IR was observed in almost all regions in AD (28.4-70.1%, mean 41.3%), LBV (19.0-42.5%, mean 26.8%) and DLBD (19.9-31.7%, mean 27.1%) compared to controls. In addition, SP6 IR in the outer molecular layer of the dentate gyrus was strongly correlated with tangle count in the entorhinal cortex (r = -0.70, P < 0.002), suggesting loss of perforant pathway projection. Although the decrease in SP12 and SP15 IR was less pronounced, the mean values were decreased in dementia. Furthermore, SP12 and SP15 labeled a large number of neuritic plaques, and SP15 occasionally stained cortical LBs. The present findings indicate (i) that in the hippocampal-entorhinal formation, the decrease of synapse protein IR in AD is more severe than that in LBV and DLBD, (ii) that synaptic markers detect a subset of dystrophic neurites in the plaques and (iii) that synapse proteins are involved in the formation of cortical LBs.

Aged↗
PubMed1988

Morphological and physiological properties of rat cerebellar neurons in mature and developing cultures.

Immunohistochemical techniques and antibodies to gamma-aminobutyric acid (GABA), parvalbumin, and cyclic guanosine monophosphate-dependent protein kinase were used to identify populations of cerebellar neurons in culture that exhibit morphological features and immunoreactivity characteristic of neuronal types present in the cortical region of the cerebellum in vivo. The cultures were examined at 3 culture ages: 6-9, 12-15 and greater than 15 days in vitro, reflecting early, intermediate and late periods in cerebellar development. Neurons identified as Purkinje neurons (PNs), granule cells or inhibitory interneurons (stellate, basket, Golgi and Lugaro cells) were present at all culture ages. The granule cells (GCs) and inhibitory interneurons (INs) were morphologically well developed at the youngest culture age studied; morphological features did not change dramatically during the culture period. In contrast, the PNs were morphologically immature at 6-9 DIV (DIV = days in vitro) and exhibited dramatic changes in morphological structure with culture age. Extracellular recordings from PNs. GCs and INs in living cultures revealed that all classes of neurons exhibited spontaneous activity, but that only a portion of the GCs and INs were spontaneously active. The spontaneously active GCs and INs exhibited variable patterns of activity and low firing rates (approximately 2-6 Hz) at all culture ages studied. At 6 DIV, PNs exhibited firing rates and patterns similar to that of the interneurons. At older culture ages, the firing rate and pattern of PNs was significantly different from the GCs and INs and was characterized by high frequency (greater than 10 Hz) spike activity usually in a regular pattern. All cerebellar neurons by excited by the transmitter glutamate (Glu). The Glu response in the GCs and INs consisted of a brief burst of single spikes; in PNs, the response to Glu was prolonged and multiphasic. These data indicate that the cerebellar GCs and INs express morphological, physiological and developmental properties that are significantly different from the PN.

Animals↗