Higher respiratory rates and improved creatine stimulation in brain mitochondria isolated with anti-oxidants

We tested the effect of an anti-oxidant mixture on respiration in isolated rat brain mitochondria. Mitochondria were isolated in mannitol/sucrose/EGTA/BSA +/- SCAVEGR anti-oxidants (SOD, catalase, vitamin E, vitamin E acetate, and glutathione reduced). TBARS were reduced by greater than 40% with SCAVEGR. Respiration driven by ADP showed a two-fold higher V(max) and a 15% higher respiratory control ratio when mitochondria were prepared with SCAVEGR. SCAVEGR also stabilized the octameric state of mitochondrial creatine kinase and thus improved creatine-stimulated respiration. These results suggest that significant improvements in brain mitochondrial function are obtained by isolation in the presence of an anti-oxidants mixture.     

Tetrathiomolybdate therapy protects against concanavalin a and carbon tetrachloride hepatic damage in mice

Tetrathiomolybdate, an anticopper drug, has been shown to protect mice against pulmonary fibrosis from bleomycin. Our hypothesis is that it does so by inhibiting fibrosis-inducing cytokines. Indeed, we have good evidence, not yet published, that tetrathiomolybdate inhibits pulmonary levels of transforming growth factor-beta and tumor necrosis factor-alpha expression in these bleomycin experiments. Herein, we evaluate tetrathiomolybdate's effectiveness in mitigating hepatitis and fibrosis in mice from the hepatotoxins, concanavalin A and carbon tetrachloride, and its inhibition of cytokines as a possible mechanism. In short-term experiments, concanavalin A elevated serum amino leucine transferase levels several fold, and tetrathiomolybdate completely prevented this increase. In additional experiments, tetrathiomolybdate therapy reversed the elevated serum transaminase levels despite continued concanavalin A injections, with nearly significant serum interleukin-1beta inhibition. Concanavalin A given for 12 weeks produced mild fibrosis, whereas concomitant tetrathiomolybdate treatment resulted in normal histology. Carbon tetrachloride given for 12 weeks resulted in very high serum amino leucine transferase levels, high serum transforming growth factor-beta levels, cirrhosis as seen histologically, and increase in liver hydroxyproline, a measure of fibrosis. Concomitant tetrathiomolybdate partially and significantly protected against increases in amino leucine transferase and transforming growth factor-beta, fully protected against the increase in hydroxyproline, and resulted in normal histology. In conclusion, tetrathiomolybdate protects against the hepatitis and fibrosis produced by these hepatotoxins, probably by inhibiting the excessive increase in inflammatory and fibrotic cytokines.     

Inducible costimulatory molecule-B7-related protein 1 interactions are important for the clonal expansion and B cell helper functions of naive,Th1, and Th2 T cells

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

Endocrine PBL in the year 2000

The Southern Illinois University School of Medicine (SIU-SOM) has utilized problem-based learning (PBL) in its curriculum since 1981, when Dr. Howard Barrows joined the faculty. From 1989 to 2000, SIU-SOM implemented two parallel curricula for the basic science years (years I and II), one curriculum being a problem-based learning curriculum (PBLC). An executive decision to design and implement a single curriculum, to begin in Fall 2000, fostered a review of existing pedagogy upon which to base this new curriculum: C2000. The results of this review, which considered formal outcome measures as well as internal review in consideration of the institutional mission, led to PBL becoming the predominant pedagogy for C2000, albeit with some modifications from its PBLC predecessor. C2000, then, represents the third iteration of PBL in use at SIU-SOM, and its design and rationale offer insight for the teaching of Endocrine Physiology in a PBLC.     

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.