Astrocytic glutamate uptake and prion protein expression

Factors influencing glutamate uptake by astrocytes may indirectly influence neuronal survival. Elevated extracellular glutamate may be excitotoxic or may exacerbate neurodegeneration in various neurological diseases. By using a cell culture model, we have investigated the influence of astrocytic prion protein (PrPc) expression on glutamate uptake. Type 1 astrocytes expressing PrPc have a higher rate of Na+-dependent glutamate uptake than PrPc-deficient type 1 astrocytes. This difference is exacerbated when serum free media is used to culture the astrocytes. Further analysis suggested that a decrease in substrate affinity is responsible for the sensitivity of PrP-deficient astrocytic glutamate uptake to culture conditions. PrPc has been shown to bind copper. Greater sensitivity of cells to copper concentrations may be responsible for the decreased substrate affinity observed. PrPc-deficient cerebellar cells are more sensitive to glutamate toxicity in the presence of copper. These results show that glutamate uptake from astrocytes is dependent on PrPc expression which in turn may be related to copper metabolism.     

Homologues of the Cf-9 disease resistance gene (Hcr9s) are present at multiple loci on the short arm of tomato chromosome 1

The tomato Cf-4 and Cf-9 genes map at a genetically complex locus on the short arm of chromosome 1 and confer resistance against Cladosporium fulvum through recognition of different pathogen-encoded avirulence determinants. Cf-4 and Cf-9 are members of a large gene family (Hcr9s, Homologues of Cladosporium fulvum resistance gene Cf-9), some of which encode additional distinct recognition specificities. A genetic analysis of the majority of Hcr9s suggests that their distribution is spatially restricted to the short arm of chromosome 1. Two loci of clustered Hcr9 genes have been analyzed physically that mapped distal (Northern Lights) and proximal (Southern Cross) to the Cf-4/9 locus (Milky Way). Sequence homologies between intergenic regions at Southern Cross and Milky Way indicate local Hcr9 duplication preceded cluster multiplication. The multiplication of clusters involved DNA flanking Hcr9 sequences as indicated by conserved lipoxygenase sequences at Southern Cross and Milky Way. The similar spatial distribution of Hcr9 clusters in different Lycopersicon spp. suggests Hcr9 cluster multiplication preceded speciation.     

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.     

Persistent transgene expression and normal differentiation of immortalized human keratinocytes in vivo

The homeobox gene otx2 is a key regulator for specifying the rostral part of the vertebrate head. In Xenopus, otx2 directly controls the differentiation of the cement gland, the anterior-most organ formed in the tadpole. Since embryos of a direct developing frog, Eleutherodactylus coqui, lack a cement gland, we are interested in whether altered expression of the otx2 gene is involved in this evolutionary change. We have cloned the E. coqui homologue of otx2, Ecotx2. The homeodomain of the Ecotx2 protein is identical to the mouse and zebrafish Otx2 proteins and differs by a single amino acid from the Xenopus Otx2 protein. Study of the spatiotemporal expression pattern shows that Ecotx2 RNA is progressively restricted to the anterior region of the embryo during gastrulation and becomes further restricted to the future forebrain and midbrain during neural development. In Xenopus, in addition to the conserved expression in the anterior neuroectoderm, the expression in ectoderm expands more anteriorly to the cement gland primordium. This anterior expansion of otx2 expression is not found in E. coqui, correlating with the loss of a cement gland. When misexpressed in Xenopus laevis ectoderm, Ecotx2 can activate expression of the cement-gland-specific genes XCG and XAG1, indicating that the function of activating the pathway of cement gland formation is retained by the Ecotx2 protein. Our results indicate that there are modifications in the pathway of cement gland formation, upstream of otx2 expression, in the development of E. coqui.     

Studies on enzymatic activity and conformational stability of muscle acylphosphatase mutated at conserved lysine residues

An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.      

A role for perforin in downregulating T-cell responses during chronic viral infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf -/-) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf -/- mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral "exhaustion" of activated CD8 T cells occurred less efficiently in perf -/- mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf -/- mice; approximately 50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

Inflammatory cell distribution within and along asthmatic airways

Asthmatic airways are infiltrated with inflammatory cells that release mediators and cytokines into the microenvironment. In this study, we evaluated the distribution of CD45-positive leukocytes and eosinophils in lung tissue from five patients who died with severe asthma compared with five patients with cystic fibrosis. For morphometric analysis, the airway wall was partitioned into an "inner" area (between basement membrane and smooth muscle) and an "outer" area (between smooth muscle and alveolar attachments). Large airways (with a perimeter greater than 3.0 mm) from patients with asthma or cystic fibrosis had a greater density of CD45-positive cells (p < 0.05) and eosinophils (p < 0.001) in the inner airway region compared with the same airway region in small airways. Furthermore, in small airways, asthmatic lungs showed a greater density of CD45-positive cells (p < 0.01) and eosinophils (p < 0.01) in the outer compared with the inner airway wall region. These observations indicate that there are regional variations in inflammatory cell distribution within the airway wall in patients with asthma that are not observed in airways from patients with cystic fibrosis. We speculate that this inflammatory cell density in peripheral airways in severe asthma may relate to the peripheral airway obstruction characteristic of this condition.     

The beta protein of phage lambda binds preferentially to an intermediate in DNA renaturation

Phage lambda encodes two recombination proteins that are required for homologous recombination in a recA- host strain. Of these two recombination proteins, one is an exonuclease whose action on double-stranded DNA produces 3' single-stranded ends; the other, called beta protein, is a DNA binding protein that promotes the renaturation of complementary single strands. The enzymes of phage lambda provide a model for understanding a recombination pathway called "single-strand annealing". Further investigation of the binding of beta protein to DNA has revealed a new mechanism of renaturation. As reported before, beta protein binds directly to single-stranded DNA, but not to double-stranded DNA. However, in the experiments reported here, we observed that beta protein bound more strongly to a presumed intermediate in the renaturation reaction that beta itself catalyzed, and beta thereby protected all of a renatured duplex 83-mer oligonucleotide from nuclease digestion.