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  • Further expansion of the phenotypic spectrum associated with mutations in ALDH18A1, encoding ?¹-pyrroline-5-carboxylate synthase (P5CS). 21739576

    We report on the third case of cutis laxa and progeroid features caused by a homozygous mutation in ALDH18A1 that encodes ?¹-pyrroline-5-carboxylate-synthase (P5CS). This severely affected child, born to consanguineous parents of Pakistani origin, presented with lax, wrinkled and thin skin with dilated and tortuous subcutaneous blood vessels, corneal clouding, and hypotonia. The child had severe global developmental delay and feeding difficulties and died in infancy for an unknown reason. The proband was homozygous for a mutation in ALDH18A1, c.1923?+?1G?>?A which results in the production of two anomalous transcripts that are predicted to encode proteins lacking the catalytic site for the enzyme. The cellular phenotype is characterized by diminished production of collagen types I and III, altered elastin ultrastructure, and diminished cell proliferation of cultured dermal fibroblasts. This severe clinical and cellular phenotype overlaps with a broad group of neurocutaneous syndromes that include cutis laxa type II, wrinkly skin syndrome, de Barsy syndrome, and gerodermia osteodysplastica. The findings presented here emphasize the pleiotropic presentation of this group of conditions and suggest that multiple components of the extracellular matrix are perturbed in these disorders.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1940
    Product Catalog Name:
    Anti-Fibronectin Antibody, cellular, clone DH1

  • Immuno-spin trapping analyses of DNA radicals. 17406615

    Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO-DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.
    Document Type:
    Reference
    Product Catalog Number:
    12-348
    Product Catalog Name:
    Goat Anti-Rabbit IgG Antibody, HRP-conjugate

  • Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitrite-mediated reperfusion injury. 8910581

    Altered nitric oxide (NO.) production is a critical factor in tissue reperfusion injury; however, controversy remains regarding these alterations and how they cause injury. Since superoxide (O-2) generation is triggered during the early period of reperfusion the cytotoxic oxidant peroxynitrite (ONOO-) could be formed, but it is not known if this occurs. Therefore electron paramagnetic resonance and chemiluminescence studies were performed of the magnitude and time course of NO., O-2, and ONOO- formation in the postischemic heart. Isolated rat hearts were subjected either to normal perfusion or to reperfusion after 30 min of ischemia in the presence of the NO. trap Fe2+-N-methyl-D-glucamine dithiocarbamate with electron paramagnetic resonance measurements performed on the effluent. Although only trace signals were present prior to ischemia, prominent NO. adduct signals were seen during the first 2 min of reflow which were abolished by nitric oxide synthase (NOS) inhibition. Similar studies with the O-2 trap 5, 5-dimethyl-1-pyrroline N-oxide demonstrated a burst of O-2 generation over the first 2 min of reflow. Chemiluminescence measurements using 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) demonstrated a similar marked increase in ONOO- which was blocked by NOS inhibitors or superoxide dismutase. NOS inhibition or superoxide dismutase greatly enhanced the recovery of contractile function in postischemic hearts. Immunohistology demonstrated that the ONOO--mediated nitration product nitrotyrosine was formed in postischemic hearts but not in normally perfused controls. Thus, NO. formation is increased during the early period of reflow and reacts with O-2 to form ONOO-, which results in amino acid nitration and cellular injury.
    Document Type:
    Reference
    Product Catalog Number:
    06-284
    Product Catalog Name:
    Anti-Nitrotyrosine Antibody

  • Immuno-spin trapping of protein and DNA radicals: tagging free radicals to locate and understand the redox process. 19159679

    Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.
    Document Type:
    Reference
    Product Catalog Number:
    12-348
    Product Catalog Name:
    Goat Anti-Rabbit IgG Antibody, HRP-conjugate

  • Huntingtin bodies sequester vesicle-associated proteins by a polyproline-dependent interaction. 14715959

    Polyglutamine expansion in the N terminus of huntingtin (htt) causes selective neuronal dysfunction and cell death by unknown mechanisms. Truncated htt expressed in vitro produced htt immunoreactive cytoplasmic bodies (htt bodies). The fibrillar core of the mutant htt body resisted protease treatment and contained cathepsin D, ubiquitin, and heat shock protein (HSP) 40. The shell of the htt body was composed of globules 14-34 nm in diameter and was protease sensitive. HSP70, proteasome, dynamin, and the htt binding partners htt interacting protein 1 (HIP1), SH3-containing Grb2-like protein (SH3GL3), and 14.7K-interacting protein were reduced in their normal location and redistributed to the shell. Removal of a series of prolines adjacent to the polyglutamine region in htt blocked formation of the shell of the htt body and redistribution of dynamin, HIP1, SH3GL3, and proteasome to it. Internalization of transferrin was impaired in cells that formed htt bodies. In cortical neurons of Huntington's disease patients with early stage pathology, dynamin immunoreactivity accumulated in cytoplasmic bodies. Results suggest that accumulation of a nonfibrillar form of mutant htt in the cytoplasm contributes to neuronal dysfunction by sequestering proteins involved in vesicle trafficking.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2166
    Product Catalog Name:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8
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