Extended Anti-inflammatory Action of a Degradation-resistant Mutant of Cell-penetrating Suppressor of Cytokine Signaling 3

Suppressor of cytokine signaling 3 (SOCS3) regulates the proinflammatory cytokine signaling mediated by the JAK/STAT signaling pathway. SOCS3 is rapidly induced and then targeted to the ubiquitin-proteasome pathway via a mechanism that requires the C-terminal SOCS box. Due to its rapid turnover, the intracellular stores of SOCS3 seem insufficient to control acute or protracted inflammatory diseases. Previously, we developed an intracellular protein therapy that uses a recombinant cell-penetrating form of SOCS3 (CP-SOCS3) to inhibit the JAK/STAT pathway and prevent cytokine-mediated lethal inflammation and apoptosis of the liver (Jo, D., Liu, D., Yao, S., Collins, R. D., and Hawiger, J. (2005) Nat. Med. 11, 892–898). The potent anti-inflammatory and cytoprotective activity of CP-SOCS3 prompted us to analyze its intracellular turnover, as compared with that of endogenous SOCS3 protein induced in macrophages by the proinflammatory agonists, interferon-γ and lipopolysaccharide. We found that the half-life (t½) of endogenous SOCS3 is 0.7 h in activated macrophages, compared with a t½ of 6.2 h for recombinant CP-SOCS3. Deletion of the SOCS box in CP-SOCS3 renders it more resistant to proteasomal degradation, extending its t½ to 29 h. Consequently, this SOCS box-deleted form of CP-SOCS3 displays persistent inhibitory activity for 24 h toward interferon-γ- and lipopolysaccharide-induced cytokine and chemokine production. Compared with the wild-type suppressor, this gain-of-function CP-SOCS3 mutant provides a longer acting inhibitor of cytokine signaling, a feature that offers a clear advantage for the intracellular delivery of proteins to treat acute or protracted inflammatory diseases.     

Long-term cyclosporine treatment in non-transplanted rats and metabolic risk factors of vascular diseases

Cyclosporine (CsA) is an immunosuppressive agent frequently used in the clinic for prevention of allograft rejection and for the treatment of autoimmune diseases. Despite its desired action on the immune system, CsA treatment may present serious adverse effects, which are masked by the concomitant use of other drugs. The search for effective immunosuppression protocols which does not affect the quality of life of patients is driving research to investigate the CsA involvement in vascular diseases, frequent in patients under immunosuppression. Thus, 45 non-transplanted Wistar rats were treated for 8 weeks with vehicle or 5 or 15 mg/kg CsA (n = 15/group) by gavage administration to evaluate the specific influence of cyclosporine on the levels of risk factors (metabolic and inflammatory) of vascular disease and its mechanism of action. Therefore, serum insulin levels, glucose tolerance test, serum lipids profile, total homocysteine and fibrinogen levels were assessed. The biochemical alterations reported here suggest the development of a framework straight to diabetes. Glucose homeostasis was affected as indicated by decreased insulin levels and altered glucose tolerance test in CsA 15 mg/kg group compared to other groups. Serum insulin and total homocysteine levels presented a significant negative correlation (R = ? 0.76, P < 0.0001). Fibrinogen and serum lipids profiles were significantly increased in CsA 15 mg/kg group compared to other groups and correlated positively with total homocysteine levels. Considering the well-established correlation among insulin resistance, lipid and total homocysteine levels, hypercoagulability and atherosclerosis, we can assume that this protocol of long-term CsA treatment in non-transplanted rats alter biochemical parameters related to cardiovascular and cerebrovascular risk, mainly in CsA 15 mg/kg group. Insulin and tHcy serum levels appear to be central in this process.     

Predicting the timing and duration of sleep in an operational setting using social factors

In recent years, there has been increasing interest in the use of bio-mathematical models to predict alertness, performance, and/or fatigue in operational settings. Current models use only biological factors to make their estimations, which can be limited in operational settings where social and geo-physical factors also dictate when sleep occurs. The interaction between social and biological factors that help determine the timing and duration of sleep during layover periods have been investigated in order to create and initially validate a mathematical model that may better predict sleep in the field. Participants were 32 male transmeridian airline pilots (17 captains, 10 first officers, and 5 second officers) flying the Sydney-Bangkok-London-Singapore-Sydney (SYD-LHR) pattern. Participants continued their regular schedule while wearing activity monitors and completing sleep and work diaries. The theoretical sleep timing model underpinning this analysis consists of separate formulations for short (<32 h) and long (>32 h) break periods. Longer break periods are split into three distinct phases-recovery (break start until first local night), personal (first local night until last local night), and preparation phases (last local night until break end)-in order to exploit potential differences specific to each. Furthermore, an iterative procedure combining prediction and retrodiction (i.e., using future duty timing information to predict current sleep timing) was developed to optimize predictive ability. Analysis found an interaction between the social and circadian sleep pressures that changed over the break period. Correlation analysis indicated a strong relationship between the actual sleep and new model's predictions (r = 0.7-0.9), a significant improvement when compared to existing models (r = 0.1-0.4). Social and circadian pressures play important roles in regulating sleep for international flight crews. An initial model has been developed in order to regulate sleep in these crews. The initial results have shown promise when applied to small sets of data; however, more rigorous validation must be carried out.     

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.     

Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.     

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.