Acquired mitochondrial impairment as a cause of optic nerve disease.
(9/1174)
BACKGROUND: Blindness from an optic neuropathy recently occurred as an epidemic affecting 50,000 patients in Cuba (CEON) and had clinical features reminiscent of both tobacco-alcohol amblyopia (TAA) and Leber's hereditary optic neuropathy (Leber's; LHON). Selective damage to the papillomacular bundle was characteristic, and many patients also developed a peripheral neuropathy. Identified risk factors included vitamin deficiencies as well as exposure to methanol and cyanide. In all 3 syndromes, there is evidence that singular or combined insults to mitochondrial oxidative phosphorylation are associated with a clinically characteristic optic neuropathy. PURPOSE: First, to test the hypothesis that a common pathophysiologic mechanism involving impairment of mitochondria function and, consequently, axonal transport underlies both genetic optic nerve diseases such as Leber's and acquired toxic and nutritional deficiency optic neuropathies. According to this hypothesis, ATP depletion below a certain threshold leads to a blockage of orthograde axonal transport of mitochondria, which, in turn, leads to total ATP depletion and subsequent cell death. Second, to address several related questions, including (1) How does impaired energy production lead to optic neuropathy, particularly since it seems to relatively spare other metabolically active tissues, such as liver and heart? (2) Within the nervous system, why is the optic nerve, and most particularly the papillomacular bundle, so highly sensitive? Although there have been previous publications on the clinical features of the Cuban epidemic of blindness, the present hypothesis and the subsequent questions have not been previously addressed. METHODS: Patients in Cuba with epidemic optic neuropathy were personally evaluated through a comprehensive neuro-ophthalmologic examination. In addition, serum, lymphocytes for DNA analysis, cerebrospinal fluid (CSF), sural nerves, and eyes with attached optic nerves were obtained from Cuban patients, as well as from Leber's patients, for study. Finally, we developed an animal model to match the low serum folic acid and high serum formate levels found in the CEON patients, by administering to rats low doses of methanol after several months of a folic acid-deficient diet. Optic nerves and other tissues obtained from these rats were analyzed and compared with those from the Cuban patients. RESULTS: Patients from the Cuban epidemic of optic neuropathy with clinical evidence of a selective loss of the papillomacular bundle did much better once their nutritional status was corrected and exposure to toxins ceased. Patients with CEON often demonstrated low levels of folic acid and high levels of formate in their blood. Histopathologic studies demonstrated losses of the longest fibers (in the sural nerve) and those of smallest caliber (papillomacular bundle) in the optic nerve, with intra-axonal accumulations just anterior to the lamina cribrosa. Our animal model duplicated the serologic changes (low folic acid, high formate) as well as these histopathologic changes. Furthermore, ultrastructural examination of rat tissues demonstrated mitochondrial changes that further matched those seen on ultrastructural examination of tissues from patients with Leber's. CONCLUSION: Mitochondria can be impaired either genetically (as in Leber's) or through acquired insults (such as nutritional or toxic factors). Either may challenge energy production in all cells of the body. While this challenge may be met through certain compensatory mechanisms (such as in the size, shape, or number of the mitochondria), there exists in neurons a threshold which, once passed, leads to catastrophic changes. This threshold may be that point at which mitochondrial derangement leads to such ATP depletion that axonal transport is compromised, and decreased mitochondrial transport results in even further ATP depletion. Neurons are singularly dependent on the axonal transport of mitochondria. ( (+info)
Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm.
(10/1174)
Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin. (+info)
Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish.
(11/1174)
We produced monoclonal antibodies (MAbs) to the extracellular proteins of Listeria monocytogenes EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from Listeria ivanovii. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of L. monocytogenes. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from L. ivanovii and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between L. monocytogenes PC-PLC and L. ivanovii sphingomyelinase. The reactions of the MAbs with catfish isolates of L. monocytogenes suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of L. monocytogenes in food and clinical samples and for detecting L. ivanovii in veterinary clinical samples. (+info)
NSP4 elicits age-dependent diarrhea and Ca(2+)mediated I(-) influx into intestinal crypts of CF mice.
(12/1174)
Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4(114-135) peptide, can overcome these gastrointestinal complications in CF (CFTR(m3Bay) null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR(+/+)), heterozygous (CFTR(+/-)), or homozygous (CFTR(-/-)) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca(2+) mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I(-) influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR(-/-) background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca(2+) or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca(2+)-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4(114-135) to counteract chronic constipation/obstructive bowel syndrome in CF patients. (+info)
Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.
(13/1174)
Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains. (+info)
Diarrhea induction by rotavirus NSP4 in the homologous mouse model system.
(14/1174)
Comparison of the NSP4 amino acid sequences from 31 strains of mammalian rotaviruses revealed the presence of four distinct NSP4 alleles; i.e., the Wa, KUN, AU-1, and EW alleles. The EW allele consists only of NSP4s from murine rotavirus strains and is divergent from other NSP4 alleles from the evolutionary perspective. There have been conflicting reports regarding the enterotoxigenic activity of NSP4 in the mouse model system; heterologous simian and porcine rotavirus NSP4s function as an enterotoxin in mice, while a homologous EC NSP4 does not play a dominant role as an enterotoxin in the cystic fibrosis conductance regulator knockout mice. To further examine the enterotoxigenic activity of NSP4, we expressed in Escherichia coli a recombinant protein consisting of glutathione S-transferase and amino acid residues 86-175 of the EW NSP4. We found that this fusion protein caused diarrhea in the majority (8/14) of 5- to 6-day-old CD1 mice. This study confirmed and extended that group A rotavirus NSP4s were able to induce diarrhea in neonatal mice and had an enterotoxigenic activity. (+info)
Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.
(15/1174)
Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations. (+info)
Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis.
(16/1174)
Streptococcal and staphylococcal superantigens (SAg's) have been implicated in the pathogenesis of inflammatory skin diseases, but the mechanisms by which these toxins act are unknown. The present study assessed the ability of nanogram quantities of topically applied purified toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin type B, and streptococcal pyrogenic enterotoxin types A and C to induce inflammatory reactions in clinically uninvolved skin of normal controls and subjects with psoriasis, atopic dermatitis, and lichen planus. These SAg's triggered a significantly greater inflammatory skin response in psoriatics than in normal control subjects or in subjects with atopic dermatitis or lichen planus. Surprisingly, skin biopsies did not exhibit the T-cell receptor Vbeta stimulatory properties predicted for SAg-induced skin reactions. By 6 hours after patch testing with SAg's, TNF-alpha mRNA had increased in the epidermis (but not the dermis) in biopsies from psoriatics, compared with controls. Immunohistochemical studies revealed significantly higher HLA-DR expression in keratinocytes from psoriatics than from controls. However, a mutant TSST-1 protein that fails to bind HLA-DR did not elicit an inflammatory skin reaction. These results indicate that keratinocyte expression of HLA-DR enhances inflammatory skin responses to SAg's. They may also account for previous studies failing to demonstrate selective expansion of T-cell receptor Vbetas in psoriatics colonized with SAg-producing Staphylococcus aureus, and they identify a novel T cell-independent mechanism by which SAg's contribute to the pathogenesis of inflammatory skin diseases. (+info)