Antibodies to Cholinergic Neurons in Alzheimer''s Disease

Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross-react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilodaltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.     

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein-bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.     

Effect of Nitrogen on Polysaccharide Production in a Porphyridium sp

Porphyridium cultures grown on either nitrate or ammonium as the nitrogen source showed similar patterns of growth and cell wall polysaccharide production. The effect of nitrogen on growth and cell wall polysaccharide production was studied by applying three regimens of supply: batch mode, in which nitrate was supplied at the beginning of the experiment and became depleted at day 6; continual mode, in which nitrate was added daily; and deficient mode, in which the cells were cultured in a nitrate-free medium. Growth was similar in the batch- and continual-mode cultures, whereas it was totally inhibited in the deficient-mode culture. Polysaccharide content (per volume) was highest in the batch-mode culture and lowest in the deficient-mode culture. However, polysaccharide production per cell was similar in the continual- and deficient-mode cultures, the highest value being found in the batch-mode culture. In addition to its effect on polysaccharide content, nitrogen affected the polysaccharide distribution between soluble and bound polysaccharides. In the deficientmode culture, most of the cell wall polysaccharide was dissolved in the medium.     

Ultraviolet B radiation converts Langerhans cells from immunogenic to tolerogenic antigen-presenting cells. Induction of specific clonal anergy in CD4+ T helper 1 cells.

We have recently demonstrated that a single dose (200 J/m2) of UVB radiation abrogates the capacity of mouse epidermal Langerhans cells (LC) or splenic adherent cells (SAC) to present keyhole limpet hemocyanin (KLH) to Ag-specific, MHC-restricted CD4+ Th1 cells. In the present study we determined whether such Th1 unresponsiveness represented long-lasting immunologic tolerance. To address this question, Th1 were preincubated with KLH-pulsed UVB-LC or UVB-SAC, then isolated and restimulated with unirradiated APC (LC or SAC) plus KLH or with exogenous rIL-2 in the absence of APC. Preincubation with KLH and UVB-LC or UVB-SAC rendered Th1 unresponsive to subsequent restimulation with APC and KLH. In addition, such Th1 were defective in their autocrine IL-2 production, but could respond normally to exogenous rIL-2, indicating that unresponsiveness was due to functional inactivation and not to cell death. Th1 unresponsiveness was Ag-specific, MHC-restricted, and long lasting (greater than 16 days). In addition, it appears that Th1 unresponsiveness is not due to the release of soluble suppressor factors from UVB-LC or UVB-SAC because supernatants from such cells had no effect on Th1 proliferation. Addition of unirradiated allogeneic SAC during preincubation prevented the induction of unresponsiveness by UVB-LC or UVB-SAC, suggesting that UVB interferes with the capacity of LC or SAC to deliver a costimulatory signal(s) that can be provided by allogeneic SAC. We conclude that UVB can convert LC or SAC from immunogenic to tolerogenic APC.     

In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein

In several pathological conditions, when conversion of Protoporphyrin (PP)IX into heme is impaired, a toxic accumulation of PPIX might occur. PPIX has been found to have affinity to the mitochondrial Translocator Protein 18 kDa. Since it is known that TSPO is abundant in human osteoblast cells, thus we assumed that PPIX can affect cellular functions via interactions with TSPO in these cells. Therefore we aimed to study the metabolic responses of human osteoblast to a high (10^?5M) concentration of PPIX in vitro. We found that in primary culture of human osteoblast-like cells cell numbers decreased following exposure to PPIX(10^?5M). Cellular [¹⁸F]-FDG incorporation, mitochondrial mass, ATP content were suppressed, and ΔΨm collapsed. Lactate dehydrogenase activity was enhanced in culture media, indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation was not affected. Protein expression of TSPO, VDAC 1, and hexokinase 2 decreased, although the synthesis of mRNA for hexokinase 2 increased. Thus, PPIX(10^?5M) has a cytotoxic effect on human osteoblast-like cell in vitro. Since these cells remain viable following exposure to another TSPO ligand, PK 11195 (10^?5M), as observed previously by us, the mode of action of PPIX on osteoblast-like cells is not identical to that of PK 11195. Accordingly pathological accumulation of PPIX may cause necrosis of osteoblasts leading to bone mass loss. We show that this phenomenon is unrelated to iron overload.     

ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor

Na⁺/H⁺exchange is a passive process not requiring expenditure of metabolicenergy. Nevertheless, depletion of cellular ATP produces a markedinhibition of the antiport. No evidence has been found for directbinding of nucleotide to exchangers or alteration in their state ofphosphorylation, suggesting ancillary factors may be involved. Thispossibility was tested by comparing the activity of dog red blood cells(RBC) and their resealed ghosts. Immunoblotting experiments usingisoform-specific polyclonal and monoclonal antibodies indicated RBCmembranes expressNa⁺/H⁺exchanger isoform 1 (NHE1). In intact RBC, uptake ofNa⁺ was greatly stimulated whenthe cytosol was acidified. The stimulated uptake was largely eliminatedby amiloride and by submicromolar concentrations of the benzoylguanidinium compound HOE-694, consistent with mediation by NHE1.Although exchange activity could also be elicited by acidification inresealed ghosts containing ATP, the absolute rate of transport wasmarkedly diminished at comparable pH. Dissipation of the pH gradientwas ruled out as the cause of diminished transport rate in ghosts. Thiswas accomplished by a "pH clamping" procedure based on continuedexport of base equivalents by the endogenous anion exchanger. Theseobservations suggest a critical factor required to maintain optimalNa⁺/H⁺exchange activity is lost or inactivated during preparation of ghosts.Depletion of ATP, achieved by incubation with2-deoxy-D-glucose, inhibitedNa⁺/H⁺exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate ofNa⁺/H⁺exchange in ATP-depleted but otherwise intact cells was similar to thetransport rate of ghosts. Therefore, we tentatively conclude that fullactivation of NHE1 requires both ATP and an additional regulatoryfactor, which may mediate the action of the nucleotide. Ancillaryphosphoproteins or phospholipids or the kinases that mediate theirphosphorylation are likely candidates for the regulatory factor(s) thatis inactivated or missing in ghosts.

     

The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant phenotype

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1^w (hp-1^w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2^j (hp-2^j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1^w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A⁹³¹-to-T⁹³¹ base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1^w mutant, on the other hand, a single G²³⁹²-to-A²³⁹² transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F₂ population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R²=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1^w mutant phenotypes.Communicated by R. Hagemann     

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.     

Contrasting impacts of immunosuppressive agents (rapamycin,FK506, cyclosporin A,and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation

Rapamycin (RAP), tacrolimus (FK506), cyclosporin A, and glucocorticoids represent modern and classic immunosuppressive agents being used clinically. Although these agents have distinct molecular mechanisms of action and exhibit different immunoregulatory profiles, their direct influences on Ag presentation processes remain relatively unknown. Here we report quantitative and qualitative differences among the above four immunosuppressants in their impact on Ag-specific, bidirectional interaction between dendritic cells (DC) and CD4(+) T cells. In the presence of relevant Ag, bone marrow-derived DC delivered activation signals to CD4(+) T cells isolated from the DO11.10 TCR transgenic mice, leading to clonal expansion; secretion of IFN-gamma, IL-2, and IL-4; and surface expression of CD69. Conversely, DO11.10 T cells delivered maturation signals to DC, leading to IL-6 and IL-12 production and CD40 up-regulation. FK506 (10(-10)-10(-8) M) and cyclosporin A (10(-9)-10(-7) M) each blocked efficiently and uniformly all the changes resulting from intercellular signaling in both DC-->T cell and T cell-->DC directions. Dexamethasone (10(-9)-10(-6) M) suppressed all changes, except for CD69 up-regulation, rather incompletely. Remarkably, RAP (10(-10)-10(-8) M) efficiently inhibited DC-induced T cell proliferation and T cell-mediated CD40 up-regulation by DC without abrogating other changes. Interestingly, T cell-independent DC maturation triggered by LPS stimulation was inhibited by dexamethasone, but not by other agents. Our results demonstrate contrasting pharmacological effects of RAP vs calcineurin inhibitors on Ag presentation, thus forming a conceptual framework for rationale-based selection (and combination) of immunosuppressive agents for clinical application.