Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins

Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis, exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (-SH) were identified in 2-DE separations by labelling with 5-iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free -SH with N-ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In-gel tryptic digestion followed by LC/MS-MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone-like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.     

Historical reflections on the afterhyperpolarization–firing rate relation of vertebrate spinal neurons

In mammalian spinal motoneurons (MNs), the slow component of the afterhyperpolarization (AHP) that follows the spike of each action potential is a major but not the sole determinant of the cells' firing rate. In this brief historical review, we emphasize four points about the AHP-firing rate relation. (1) There is a relatively sparse literature across vertebrates that directly addresses this topic. (2) After the advent of intracellular recording in the early 1950s, there was evidence from mammals to the contrary of an idea that subsequently became prevalent: that the high-firing rates attainable by spinal interneurons (INs) and low-threshold MNs was attributable to their small AHP at rheobase. (3) Further work is needed to determine whether our present findings on the AHP-firing rate relation of turtle cells generalize to the spinal neurons of other vertebrate species. (4) Relevant to point 3, substantial in vivo and in vitro work is potentially available in raw data used in reports on several mammalian and non-mammalian vertebrates. In summary, the factors in addition to the slow AHP that help determine spinal INs and MN firing rate deserve further evaluation across vertebrates, with relevant data already potentially available in several laboratories.     

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice - a model

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversibly-acting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and general-kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcumin-administration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice increased serum free MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.     

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo

BACKGROUND: Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. METHODS: We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. RESULTS: In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. DISCUSSION: We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.     

Factor V is a substrate for the transamidase factor XIIIa

Coagulation Factor V (Mr = 330,000), upon cleavage by thrombin, produces Factor Va, which is composed of two subunits with Mr values of 94,000 and 74,000, along with two activation fragments possessing no known function. Studies were undertaken to assess the ability of the transamidase Factor XIIIa to covalently incorporate the lysine analogs [3H]putrescine and dansylcadaverine into the thrombin-cleaved (activated) and unactivated forms of human and bovine Factor V. The incorporation of either probe into thrombin-activated Factor V proceeded at an initial rate approximately twice that for unactivated Factor V. The extent of the incorporation of [3H]putrescine or dansylcadaverine into activated or unactivated human Factor V was identical; 4 mol of either probe per mol of Factor V. In the case of bovine Factor V, however, while 4 mol of probe were bound per mol of the unactivated pro-cofactor, 5 mol of either lysine analog were covalently linked to 1 mol of thrombin-cleaved Factor V. Polyacrylamide gel fluorography, immunoaffinity chromatography, and immunoprecipitation identified the largest activation fragment of human Factor V (Mr = 150,000) and bovine Factor V (Mr = 120,000) to contain the sites of incorporation of the covalently bound probes. High molecular weight, apparently covalent polymers of Factor V were produced by the action of Factor XIIIa on activated and unactivated human or bovine Factor V. The absence of either probe in the reaction mixtures did not appear to allow an enhancement of protein polymerization.