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  • Hyaline protoplasmic astrocytopathy of neocortex. 19151628

    Eosinophilic inclusions in the cytoplasm of protoplasmic astrocytes of the neocortex, usually in the clinical setting of epilepsy and/or psychomotor retardation, were first recognized and illustrated by Alois Alzheimer in 1910. Traditional special stains have failed to elucidate the specific nature of these inclusions. Ultrastructurally, the material was composed predominantly of highly electron-dense, non-membrane-bound, granular material distinct from Rosenthal fibers. Immunohistochemical examination has been informative but also sometimes inconsistent; it has recently been suggested that they may represent a filaminopathy (filamin A). We examined 5 cases with neocortical eosinophilic inclusions (3 autopsies, 2 surgical resections) using a standardized immunohistochemical protocol at a single institution. The specimens were immunostained with 32 antibodies to 30 potentially relevant proteins using several antigen retrieval protocols. We confirmed the presence of filamin A in these inclusions, but several additional proteins, particularly cytoglobin and glutamate transporter 1, were also identified. By electron microscopy in 2 cases, the granular fine structure of the inclusions was confirmed; mitochondria adjacent to, and perhaps within, the inclusions that contained many pleomorphic vesicular and membranous elements were also noted in 1 case. The pathophysiologic relevance of these proteins and the clinical significance of the hyaline inclusions are discussed.,
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5320
    Nombre del producto:
    Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody

  • Isolation and characterization of a native placental basement-membrane collagen and its component alpha chains. 456357

    Native type IV collagen was isolated from human placenta using pepsin solubilisation followed by fractional salt precipitation and chromatogarphic purification. The native preparation was characterised using amino acid analyses, disc gel electrophoresis, segment-long-spacing crystallites and immunological methods. Two component alpha chains were isolated with molecular weights of approximately 95000 and 70000. Cyanogen bromide digests of these chains indicated that they are not related to any of the known alpha chains of interstitial collagens or to the recently described collagen containing alphaA and alphaB chains. They are also not related to one another and are therefore probably fragments of two genetically distinct type IV collagen alpha chains.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Characterization of a novel collagen chain in human placenta and its relation to AB collagen. 224919

    A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Type VI collagen. Studies on its localization, structure, and biosynthetic form with monoclonal antibodies. 6368554

    Monoclonal antibodies were prepared by immunization with whole tissue and were selected for their reactivity with extracellular matrices in tissue immunofluorescence. Two such antibodies were used to isolate the corresponding antigen from pepsin extracts of human placental tissue by immunochromatography. In each case, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the isolated material was composed of four polypeptides of Mr between 57,000 and 85,000 that were disulfide-bonded into a high molecular weight aggregate. Amino acid analyses showed that the isolated material was partly collagenous. The material was shown to be antigenically related to previously isolated peptic fragments of type VI collagen and it shared their unique structure as revealed by electron microscopy. Based on these findings, it was concluded that the isolated material was a form of type VI collagen. In immunofluorescence, the monoclonal antibodies localized type VI collagen throughout the connective tissue and in the extracellular matrix of cultured fibroblasts. Polypeptides presumably comprising the intact form of this collagen were isolated from cultures of metabolically radiolabeled fibroblast cell cultures using the two monoclonal antibodies. The isolated material consisted of two polypeptides of Mr 240,000 and 140,000 that were extensively disulfide cross-linked. Four additional monoclonal antibodies bound the same radioactive polypeptides from fibroblast cultures, but only one of them reacted with the fragments isolated from pepsin-digested placenta. Since all six antibodies were originally selected based on tissue immunofluorescence, and therefore react with the tissue form of the protein, the tissue form appears to be more similar to the polypeptides detected in fibroblast cultures than to the pepsin-resistant fragments. Since these monoclonal antibodies apparently recognize different parts of the molecule, they will be useful for further study of the structure and function of the intact form of type VI collagen.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3303
    Nombre del producto:
    Anti-Collagen Type VI Antibody, clone VI-26

  • Expression of AMPA receptor subunits at synapses in laminae I-III of the rodent spinal dorsal horn. 18215271

    Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I-III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining.As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I-II, approximately 65% were GluR1-positive and approximately 60% were GluR3-positive, while in lamina III the corresponding values were 34% (GluR1) and 80% (GluR3). Puncta stained with antibody against the C-terminus of GluR4 (which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, approximately 8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses.Our results suggest that virtually all glutamatergic synapses in laminae I-III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I-II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Structure and activity dependence of recombinant human insulin-like growth factor II on disulfide bond pairing. 2722836

    The complete peptide map of purified folded recombinant human insulin-like growth factor II (rhIGF-II) was determined to verify its sequence and disulfide bonding scheme. Each peptide generated by digestion with pepsin was purified and characterized by amino acid analysis, amino acid sequence analysis, and fast atom bombardment/mass spectrometry. Some peptides were also sequenced using tandem mass spectrometry. The rhIGF-II peptide map was compared to that of rat insulin-like growth factor II and to that of a disulfide-bonded isomer of rhIGF-II. The data obtained in these studies are consistent with the conclusion that the rhIGF-II obtained from Escherichia coli has the correct amino acid composition, sequence, and the native disulfide-bonded structure. The binding affinities of these forms of recombinant IGF-II for IGF carrier proteins were measured in an IGF binding protein assay. The disulfide isomer of rhIGF-II was 160-fold less potent than native rhIGF-II in the competitive protein binding assay. These studies illustrate the need to characterize recombinant polypeptides containing disulfide bonds to allow the native structure to be verified before characterizing the biological properties of such molecules in hopes of elucidating their physiologic functions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    01-142

  • Feasibility of Pressurization To Speed Up Enzymatic Hydrolysis of Biological Materials for Multielement Determinations 17269790

    The feasibility of pressurized solvents (liquids at a high pressure and/or high temperature without the subcritical point being reached) has been newly investigated to accelerate enzymatic hydrolysis processes of mussel tissue for multielement determinations. The target elements (Al, As, Cd, Co, Cu, Fe, Hg, Li, Mn, Pb, Se, Sr, V, and Zn) were released from dried mussel tissue by action of two proteases (pepsin and pancreatin), and they have been evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES). Variables inherent to the enzymatic activity (pH, ionic strength, temperature, and enzyme mass) and factors affecting pressurization (static time, pressure, and number of cycles) were simultaneously studied by applying a Plackett-Burman design (PBD) as the screening method. Results showed that pH, ionic strength, and temperature were the most statistically significant factors (confidence interval of 95%) under pressurized conditions for pepsin, while pH and ionic strength affected pancreatin activity. This means that metal extraction is mostly attributed to enzymatic activity. The static time (enzymatic hydrolysis time) was found statistically nonsignificant for most of the elements, meaning that the hydrolysis procedure can be finished within a 2-15 min range. For pepsin, optimized conditions (pH 1.0, temperature 40 °C, pressure 1500 psi, static time 2 min, and number of cycles 3) gave quantitative extractions for As, Cd, Co, Cu, Hg, Li, Mn, Pb, Se, Sr, V, and Zn. The pepsin mass was 0.05 g, and the solution was Milli-Q water at pH 1.0 (adjusted with hydrochloric acid). For pancreatin, quantitative recoveries were only reached for As, Cd, Cu, Li, Pb, and Sr at room temperature, at a pressure of 1500 psi, for a static time of 2 min and a number of cycles of 3. The extraction solution was a 0.3 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer at a pH of 7.5 working at room temperature. Around 0.5 g of diatomaceous earth was used as dispersing agent for hydrolyses with either enzyme. Analytical performances, such as limits of detection and quantification and repeatability of the overall procedure, have been established. Finally, accuracy of the methods was assessed by analyzing seafood certified reference materials (GBW-08571, DORM-2, DOLT-3, TORT-2), fatty tissues certified reference materials (BCR 185, NIST 1577b), and fibrous certified reference materials (BCR 62, GBW-08501).
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo

  • Neurokinin 1 receptor-expressing projection neurons in laminae III and IV of the rat spinal cord have synaptic AMPA receptors that contain GluR2, GluR3 and GluR4 subunits ... 19200070

    alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPArs), which mediate fast excitatory glutamatergic transmission, are tetramers made from four subunits (GluR1-4 or GluRA-D). Although synaptic AMPArs are not normally detected by immunocytochemistry in perfusion-fixed tissue, they can be revealed by using antigen retrieval with pepsin. All AMPAr-positive synapses in spinal cord are thought to contain GluR2, while the other subunits have specific laminar distributions. GluR4 can be alternatively spliced such that it has a long or short cytoplasmic tail. We have reported that less than 10% of AMPAr-containing synapses in lamina II have the long form of GluR4, and that these are often arranged in dorsoventrally orientated clusters. In this study, we test the hypothesis that GluR4-containing receptors are associated with dorsal dendrites of projection neurons in laminae III and IV that express the neurokinin 1 receptor (NK1r). Immunostaining for NK1r was carried out before antigen retrieval, and sections were then reacted to reveal GluR2 and either GluR4 (long form), GluR3 or GluR1. All NK1r-positive lamina III/IV neurons had numerous GluR2-immunoreactive puncta in their dendritic plasma membranes, and virtually all (97%) of the puncta tested were labelled (usually strongly) with the GluR4 antibody. Sizes of puncta varied, but many were elongated and they were significantly larger than nearby puncta that were not associated with the NK1r cells. None of the GluR2 puncta on these cells was positive for GluR1, while 85% were GluR3-immunoreactive. These results show that synaptic AMPArs on the dendrites of the lamina III/IV NK1r projection neurons contain GluR2, GluR3 and GluR4, but not GluR1 subunits.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1504
    Nombre del producto:
    Anti-Glutamate receptor 1 Antibody

  • Widespread expression of the AMPA receptor GluR2 subunit at glutamatergic synapses in the rat spinal cord and phosphorylation of GluR1 in response to noxious stimulation ... 15215299

    Glutamate, the principal excitatory neurotransmitter in the spinal cord, acts primarily through AMPA receptors. Although all four AMPA subunits are expressed by spinal neurons, we know little about their distribution at glutamatergic synapses. We used an antigen-unmasking technique to reveal the synaptic distribution of glutamate receptor (GluR) 1-4 subunits with confocal microscopy. After pepsin treatment, punctate staining was seen with antibodies against each subunit: GluR2-immunoreactive puncta were distributed throughout the gray matter, whereas GluR1-immunoreactive puncta were restricted to the dorsal horn and were most numerous in laminas I-II. Punctate staining for GluR3 and GluR4 was found in all laminas but was weak in superficial dorsal horn. Colocalization studies showed that GluR2 was present at virtually all (98%) puncta that were GluR1, GluR3, or GluR4 immunoreactive and that most (>90%) immunoreactive puncta in laminas IV, V, and IX showed GluR2, GluR3, and GluR4 immunoreactivity. Evidence that these puncta represented synaptic receptors was obtained with electron microscopy and by examining the association of GluR2- and GluR1-immunoreactive puncta with glutamatergic boutons (identified with vesicular glutamate transporters or markers for unmyelinated afferents). The great majority (96%) of these boutons were associated with GluR2-immunoreactive puncta. Our findings suggest that GluR2 is almost universally present at AMPA-containing synapses, whereas GluR1 is preferentially associated with primary afferent terminals. We also found a substantial, rapid increase in staining for synaptic GluR1 subunits phosphorylated on the S845 residue in the ipsilateral dorsal horn after peripheral noxious stimulation. This finding demonstrates plastic changes, presumably contributing to central sensitization, at the synaptic level.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB397
    Nombre del producto:
    Anti-Glutamate Receptor 2 Antibody, extracellular, clone 6C4