Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Ralpha or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Ralpha- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Ralpha gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Ralpha-deficient mice. While levels of IL-5 production in IL-4Ralpha- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides

We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.     

LacSwitchTM Inducible Mammalian Expression System in mouse Swiss 3T3 fibroblasts

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells

The objective ofthis study was to assess the effects of two structurally distinct yetselective proteasome inhibitors (PS-341 and lactacystin) on leukocyteadhesion, endothelial cell adhesion molecule (ECAM) expression, andnuclear factor-B (NF-B) activation in tumor necrosisfactor (TNF)--stimulated human umbilical vein endothelial cells(HUVEC) and the transformed, HUVEC-derived, ECV cell line. We foundthat TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclearneutrophil (PMN) adhesion to HUVEC but not to ECV; TNF alsosignificantly enhanced surface expression of vascular cell adhesionmolecule 1 and E-selectin (in HUVEC only), as well as intercellularadhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVECwith lactacystin completely blocked TNF-stimulated PMN adhesion,partially blocked U-937 adhesion, and completely blocked TNF-stimulatedECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1expression in ECV. Pretreatment of HUVEC with PS-341 partially blockedTNF-stimulated leukocyte adhesion and ECAM expression. These effects oflactacystin and PS-341 were associated with inhibitory effects onTNF-stimulated NF-B activation in both HUVEC and ECV. Our resultsdemonstrate the importance of the 26S proteasome in TNF-inducedactivation of NF-B, ECAM expression, and leukocyte-endothelialadhesive interactions in vitro.     

[3H]taurine and D-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

Volume-dependent anion channels permeable forCl and amino acids arethought to play an important role in the homeostasis of cell volume.Astrocytes are the main cell type in the mammalian brain showing volumeperturbations under physiological and pathophysiological conditions. Weinvestigated the involvement of tyrosine phosphorylation in hyposmoticmedium-induced[³H]taurine andD-[³H]aspartaterelease from primary astrocyte cultures. The tyrosine kinase inhibitorstyrphostin 23 and tyrphostin A51 partially suppressed thevolume-dependent release of[³H]taurine in adose-dependent manner with half-maximal effects at ~40 and 1 µM,respectively. In contrast, the release ofD-[³H]aspartatewas not significantly affected by these agents in the sameconcentration range. The inactive analog tyrphostin 1 hadno significant effect on the release of both amino acids. The dataobtained suggest the existence of at least two volume-dependent anionchannels permeable to amino acids in astrocyte cultures. One of thesechannels is permeable to taurine and is under the control of tyrosinekinase(s). The other is permeable to both taurine and aspartate, butits volume-dependent regulation does not require tyrosine phosphorylation.     

Preservation of murine embryos in a state of dormancy at 4C

With the aim ofimproving preservation of blood products and organs fortransplantation, we designed solutions to induce a state of dormancy incells and tissues at 4°C. The solutions were devoid of combinationsof ions (e.g., K⁺,Rb⁺,Cs⁺, andNH⁺₄ withHCO₃,H₂PO₄, andCl) that are believed tobreak down low-density water in the entrance compartments of ionchannels, resulting in cyclical open states (normal water) and closedstates (low-density water). The total osmolality was always0.29-0.3 osmol/kgH₂O, made upof combinations of a di- or trisaccharide, a compatible solute, sodiumsulfate, citrate, or chloride, and 1.75 mMCaCl₂. The end point was the ability of murine embryos to progress to hatching in culture after preservation in such a solution at 4°C. Embryos hatched after 5 or6 days in some preservative solutions compared with 1-3 days inmost saline solutions; survival was improved by pretreatment withsodium butyrate.