Human immunodeficiency virus protein gp120 interferes with β-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes

Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of³²P into a soluble acidic protein of 80,000M _r , which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to³H-myristic acid indicated that the acidic 80,000M _r protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M _r MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.     

Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus

To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped with the 6xHis tag, a simple colony-blot procedure can be performed and a colony giving strong and efficient expression can easily be selected for high range protein expression. We utilized this procedure to produce one of potato mop-top virus (PMTV) movement proteins, namely triple gene block protein 1 (TGBp1) which was very hard to express in bacteria in its original length. The TGBp1 gene was digested with exonuclease III and nuclease S1 from its 5' terminus, leaving 6xHis tag intact. The clone that showed the strongest signal with anti-His antibodies in colony-blot procedure was found to have 44 amino acids (of total 463) deleted. The SDS-PAGE and Western blot of high range bacterial culture lysate confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.     

Pharmacogenomic profiling of an oxidative stress-mediated spongiform encephalopathy

The majority of cellular superoxide is generated in the mitochondria as a by-product of normal oxidative metabolism. In the mitochondria, superoxide is detoxified by manganese superoxide dismutase (SOD2). Mice lacking SOD2 demonstrate a multifaceted neonatal lethal phenotype, including a spongiform encephalopathy that is preventable through antioxidant treatment. The molecular events behind the observed pathology in the cortex of these mice are unknown. We hypothesized that the lack of SOD2 would result in significant changes in cortical gene expression and that therapeutically beneficial antioxidant treatment would normalize the expression of some genes, providing insight into the mechanism by which mitochondrial oxidative stress results in neurodegeneration. We report the identification of gene expression profiles associated with this paradigm, which characterize the degree of response to the pharmacologic intervention. We have identified specific pathways targeted by endogenous oxidative stress, including glutathione metabolism, iron metabolism, and cell-survival pathways centering on the kinase AKT. The normalization of expression of some of these pathways by antioxidant treatment suggests approaches to treating disease in which endogenous oxidative stress plays a role.     

Synthesis and anti-viral activity of a series of d- and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2'-deoxy-2'-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2'-fluoro group, was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.     

Synthesis and anti-hepatitis C virus activity of nucleoside derivatives of N3, 5'-anhydro-4-(beta-D-ribofuranosyl)-8-aza-purin-2-ones

A series of N3, 5-Anhydro-4-(beta-D-ribofuranosyl)-8-azapurin-2-ones were prepared in multistep reactions from uridine as potential anti-hepatitis C virus (HCV) agents. The synthetic details as well as biological evaluations are discussed.     

Bifidobacterium breve and Streptococcus thermophilus secretion products enhance T helper 1 immune response and intestinal barrier in mice

Lactic acid bacteria or their secretion products can modulate immune responses differently in normal and inflammatory conditions. This comparative study analyzes the effect of oral administration of living lactic acid bacteria, or their conditioned media, on the epithelial and immune functions of colitis-prone C57BL/6 IL-10-deficient mice. Mice were untreated (control) or infected with Helicobacter hepaticus with or without oral treatment with living bacteria, Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LB), or their culture-conditioned media (CM). Histology, cytokine mRNA, electrical resistance, and barrier capacity of colonic samples as well as cytokine secretion by mesenteric lymph node (MLN) cells were studied. Helicobacter hepaticus mice developed only mild colitis, which was not modified in LB or CM groups. In the CM (but not the LB) group, the colonic barrier was reinforced as compared to the other groups, as evidenced by decreased horseradish peroxidase (HRP) transcytosis and mannitol fluxes and increased electrical resistance. In MLN, the percentage of CD4+ and CD8+ T cells secreting IFNgamma was significantly higher in CM (2.06% and 1.98%, respectively) mice than in H. hepaticus (1.1% and 0.47%, P < 0.05) or control mice. In addition, the nonspecific stimulation of IFNgamma, TNFalpha, and IL-12 secretion by MLN cells was significantly higher in the CM group as compared to the other groups. In the absence of severe colitis, Bifidobacterium breve C50- and Streptococcus thermophilus 065-conditioned media can reinforce intestinal barrier capacity and stimulate Th1 immune response, highlighting the involvement of lactic acid bacteria-derived components in host defense.     

Only seed size matters for germination in different populations of the dimorphic Tragopogon pratensis subsp. pratensis (Asteraceae)

Many studies have focused on the ecology of seed dimorphism, the production of two seed types by a single plant. Morphology and seed size are usually correlated, but how morphology affects germination percentage and seedling growth is poorly understood. Here we explicitly separate these effects for nine populations of the dimorphic species Tragopogon pratensis subsp. pratensis. Larger seeds yielded higher germination percentages, yet seed morphology had no additional direct effect on germination. Neither seed size nor seed morphology affected seedling growth. Neither germination nor seedling growth varied among populations, but seed head varied significantly. Results show that germination is mainly controlled by seed size rather than by seed morphology. This study is one of the few to distinguish explicitly between seed size and seed morphology effects on ecological characteristics and suggests that seed dimorphism may exert its ecological effects predominantly through its correlated size.     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.     

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

In vivo growth of bacterial flagellar filaments by self-assembly of flagellin is promoted by a capping structure composed of a pentameric assembly of hook associated protein 2 (HAP2). Isolated native filaments with intact HAP2 cap exhibited higher melting temperature (deltaTm = 4 degrees C) and significantly increased resistance against heat-induced depolymerization than non-capped ones. Reconstituted filaments were also stabilized by HAP2 binding, but the obtained filament-HAP2 complexes were less stable than native assemblies. Their fast depolymerization at elevated temperatures and sensitivity to proteolysis indicated that native-like filament-HAP2 complexes are rarely obtained by in vitro reconstitution. A procedure was developed to isolate perfectly capped native filaments to facilitate high-resolution structural analysis.     

The brain is getting ready for dinner

Every evening, as we get ready for dinner, in addition to the routine behaviors of preparing the meal itself, we also prepare our bodies to cope with the upcoming meal. This could take the form of making restaurant reservations, changing into appropriate attire, washing hands, priming ourselves with an aperitif, or even consciously avoiding snacks as the meal approaches. A study by Johnstone and colleagues in this issue of Cell Metabolism (Johnstone et al., 2006) provides evidence that in parallel to our learned preparatory behaviors, our central nervous system is going through comparable motions as it gets ready for the anticipated meal.