IL-6 regulates neutrophil trafficking during acute inflammation via STAT3

The successful resolution of inflammation is dependent upon the coordinated transition from the initial recruitment of neutrophils to a more sustained population of mononuclear cells. IL-6, which signals via the common receptor subunit gp130, represents a crucial checkpoint regulator of neutrophil trafficking during the inflammatory response by orchestrating chemokine production and leukocyte apoptosis. However, the relative contribution of specific IL-6-dependent signaling pathways to these processes remains unresolved. To define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we used a series of gp130 knockin mutant mice displaying altered IL-6-signaling capacities in an experimental model of acute peritoneal inflammation. Hyperactivation of STAT1 and STAT3 in gp130(Y757F/Y757F) mice led to a more rapid clearance of neutrophils, and this coincided with a pronounced down-modulation in production of the neutrophil-attracting chemokine CXCL1/KC. By contrast, the proportion of apoptotic neutrophils in the inflammatory infiltrate remained unaffected. In gp130(Y757F/Y757F) mice lacking IL-6, neutrophil trafficking and CXCL1/KC levels were normal, and this corresponded with a reduction in the level of STAT1/3 activity. Furthermore, monoallelic ablation of Stat3 in gp130(Y757F/Y757F) mice specifically reduced STAT3 activity and corrected both the rapid clearance of neutrophils and impaired CXCL1/KC production. Conversely, genetic deletion of Stat1 in gp130(Y757F/Y757F) mice failed to rescue the altered responses observed in gp130(Y757F/Y757F) mice. Collectively, these data genetically define that IL-6-driven signaling via STAT3, but not STAT1, limits the inflammatory recruitment of neutrophils, and therefore represents a critical event for the termination of the innate immune response.     

Nitrolinoleate inhibits platelet activation by attenuating calcium mobilization and inducing phosphorylation of vasodilator-stimulated phosphoprotein through elevation of cAMP.

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO(2)). The effect of LNO(2) on human platelets was examined to define how nitrated lipids might behave in vivo. LNO(2), but not LA or 3-nitrotyrosine, dose dependently (0.5-10 microm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO(2) caused slight mobilization of calcium (Ca(2+)) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca(2+) elevations. LNO(2) did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO(2) treatment of platelets, and the adenylyl cyclase inhibitors 2',5'-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO(2) significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO(2). The platelet inhibitory actions of LNO(2) indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.     

Growth of synthetic stromatolites and wrinkle structures in the absence of microbes - implications for the early fossil record

Stromatolites and wrinkle structures are often taken to be an important indicator for early life. While both may be shaped by microbial mat growth, this can be open to doubt, so that the contribution of abiotic processes in their construction always needs to be established (Grotzinger & Knoll, 1999). We here report laboratory spray deposition experiments that can generate stromatolites and wrinkle structures in the absence of microbes. These minicolumnar and sometimes branched stromatolites are produced artificially by the aggregation of a synthetic colloid in a turbulent flow regime. They self-organize at the relatively low particle concentrations found in the outer parts of a spray beam. This contrasts with adjacent stratiform deposits that are produced by high rates of colloid deposition and relatively low sediment viscosities found in the centre of a spray beam. These stratiform laminae become subsequently wrinkled during hardening of the colloid. These results support numerical models that together suggest that physicochemical processes are capable of generating laminated sedimentary structures without the direct participation of biology. Geological environments where comparable abiogenic stromatolites and wrinkle structures may be found include: splash-zone silica sinters, desert varnish crusts and early Archean cherts formed from silica gel precursors.     

Population parameters and harvest risks for polar bears (<Emphasis Type="Italic">Ursus maritimus</Emphasis>) of Kane Basin,Canada and Greenland

We estimated demographic parameters and current harvest risks for a population of polar bears (Ursus maritimus Phipps) inhabiting northern Smith Sound and Kane Basin, Canada and Greenland. Our demographic analysis included a detailed assessment of age- and sex-specific survival and recruitment from 141 marked polar bears, using information contained within the standing age distribution of captures and mark-recapture analysis. Total survival rates for females were: 0.374 ± 0.180 (cubs), 0.686 ± 0.157 (ages 1–4), and 0.967 ± 0.043 (ages 5+). Mean litter size was 1.67 ± 0.08 cubs. Females did not reproduce until at least age 6, which is late compared to other populations of polar bears. The model-averaged, mark–recapture estimate of mean abundance (±1 SE) for years 1994–1997 was 164 ± 35 bears. We incorporated demographic parameters and their variances into a harvest risk analysis (i.e., a stochastic, harvested population viability analysis, PVA). Results suggest that polar bears in the region were severely over-harvested during the mark–recapture interval (1992–1997). The current status of the population is unknown.     

Beta2-integrins contribute to skeletal muscle hypertrophy in mice

We tested the contribution of β₂-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common β-subunit of β₂-integrins (CD18^–/–), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18^–/– mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in β₂-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18^–/– mice. In conclusion, our findings indicate that β₂-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling. skeletal muscle growth; neutrophils; macrophages; compensatory hypertrophy     

Pilot-scale production of intracellular and extracellular enzymes

Optimization of pilot-scale enzyme production is described for the case of an extracellular protease and an intracellular esterase. Media optimization was conducted to reduce medium costs and to determine the effect of various defined ingredients as well as complex nitrogen sources on enzyme production. Fermentation conditions such as inoculum transfer timing, agitation rate, and cultivation temperature were evaluated for their effect on enzyme production. Broths were harvested via microfiltration, diafiltered, and in the case of the extracellular enzyme, lysed via homogenization. An improvement in enzyme titer and reduction in medium costs for the extracellular protease was realized through replacement of Sabouraud dextrose broth medium with more reasonably priced complex nitrogen sources such as N-Z-amine A. An improvement in enzyme titer and reduction in medium costs for the intracellular esterase was realized by decreasing the amount of malt extract and omitting glycerol from the medium. An improvement in the harvest conditions for both enzymes was realized by using large-lumen-diameter hollow-fiber membranes (2.7 mm) which seemed wide enough to pass clumps of fungal and filamentous bacterial fermentation broth without clogging.     

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.