Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids.

Coenzyme Q (ubiquinone or Q) plays a well known electron transport function in the respiratory chain, and recent evidence suggests that the reduced form of ubiquinone (QH2) may play a second role as a potent lipid-soluble antioxidant. To probe the function of QH2 as an antioxidant in vivo, we have made use of a Q-deficient strain of Saccharomyces cerevisiae harboring a deletion in the COQ3 gene [Clarke, C. F., Williams, W. & Teruya, J. H. (1991) J. Biol. Chem. 266, 16636-16644]. Q-deficient yeast and the wild-type parental strain were subjected to treatment with polyunsaturated fatty acids, which are prone to autoxidation and breakdown into toxic products. In this study we find that Q-deficient yeast are hypersensitive to the autoxidation products of linolenic acid and other polyunsaturated fatty acids. In contrast, the monounsaturated oleic acid, which is resistant to autoxidative breakdown, has no effect. The hypersensitivity of the coq3delta strains can be prevented by the presence of the COQ3 gene on a single copy plasmid, indicating that the sensitive phenotype results solely from the inability to produce Q. As a result of polyunsaturated fatty acid treatment, there is a marked elevation of lipid hydroperoxides in the coq3 mutant as compared with either wild-type or respiratory-deficient control strains. The hypersensitivity of the Q-deficient mutant can be rescued by the addition of butylated hydroxytoluene, alpha-tocopherol, or trolox, an aqueous soluble vitamin E analog. The results indicate that autoxidation products of polyunsaturated fatty acids mediate the cell killing and that QH2 plays an important role in vivo in protecting eukaryotic cells from these products.

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.

Enhanced and accelerated lymphoproliferation in Fas-null mice.

Fas is a 45-kDa membrane protein that transduces an apoptotic signal. The mouse lymphoproliferation (lpr) mutation is a leaky mutation of Fas. In this study, we examined lymphocyte development in Fas-null mice generated by gene targeting. The Fas-/- mice progressively accumulated abnormal T cells (Thy1+, B220+, CD4-, and CD8-) and developed lymphadenopathy and splenomegaly, which were much more accelerated and pronounced than those in lpr mice. In addition, the Fas-null mice showed lymphocytosis, accompanied by lymphocytic infiltration in the lungs and liver. The number of apparently normal B cells also increased, and large amounts of immunoglobulins, including anti-DNA antibodies, were produced. Thymic clonal deletion, assessed by deletion of T cells reactive to mouse endogenous superantigens, was apparently normal in the Fas-/- mice, whereas the peripheral clonal deletion of mature T cells against a bacterial superantigen was impaired. These results suggested that Fas plays a decisive role in peripheral clonal deletion but not in negative selection in the thymus.

Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.

Recent studies have demonstrated that the overexpression of the c-myc gene in the liver of transgenic mice leads to an increase in both utilization and accumulation of glucose in the liver, suggesting that c-Myc transcription factor is involved in the control of liver carbohydrate metabolism in vivo. To determine whether the increase in c-Myc might control glucose homeostasis, an intraperitoneal glucose tolerance test was performed. Transgenic mice showed lower levels of blood glucose than control animals, indicating that the overexpression of c-Myc led to an increase of blood glucose disposal by the liver. Thus, the increase in c-Myc might counteract diabetic hyperglycemia. In contrast to control mice, transgenic mice treated with streptozotocin showed normalization of concentrations of blood glucose, ketone bodies, triacylglycerols and free fatty acids in the absence of insulin. These findings resulted from the normalization of liver metabolism in these animals. While low glucokinase activity was detected in the liver of diabetic control mice, high levels of both glucokinase mRNA and enzyme activity were noted in the liver of streptozotocin-treated transgenic mice, which led to an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in the control of gluconeogenesis and ketogenesis and the production of glucose and ketone bodies was observed in streptozotocin-treated transgenic mice. Thus, these results suggested that c-Myc counteracted diabetic alterations through its ability to induce hepatic glucose uptake and utilization and to block the activation of gluconeogenesis and ketogenesis.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Enhanced conformational sampling in Monte Carlo simulations of proteins: application to a constrained peptide.

A Monte Carlo simulation method for globular proteins, called extended-scaled-collective-variable (ESCV) Monte Carlo, is proposed. This method combines two Monte Carlo algorithms known as entropy-sampling and scaled-collective-variable algorithms. Entropy-sampling Monte Carlo is able to sample a large configurational space even in a disordered system that has a large number of potential barriers. In contrast, scaled-collective-variable Monte Carlo provides an efficient sampling for a system whose dynamics is highly cooperative. Because a globular protein is a disordered system whose dynamics is characterized by collective motions, a combination of these two algorithms could provide an optimal Monte Carlo simulation for a globular protein. As a test case, we have carried out an ESCV Monte Carlo simulation for a cell adhesive Arg-Gly-Asp-containing peptide, Lys-Arg-Cys-Arg-Gly-Asp-Cys-Met-Asp, and determined the conformational distribution at 300 K. The peptide contains a disulfide bridge between the two cysteine residues. This bond mimics the strong geometrical constraints that result from a protein's globular nature and give rise to highly cooperative dynamics. Computation results show that the ESCV Monte Carlo was not trapped at any local minimum and that the canonical distribution was correctly determined.

Three-dimensional tracking of motile bacteria near a solid planar surface.

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.

Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.

Deformable templates for plaque thickness estimation of intravascular ultrasound sequences

Comunicación presentada en el VII Symposium Nacional de Reconocimiento de Formas y Análisis de Imágenes, SNRFAI, Barcelona, abril 1997.

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ12,14-prostaglandin J2 through oxidative stress in human neutrophils

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.