Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling

Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD(P)H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD(P)H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD(P)H oxidase activation, in the form of phosphorylation of p47phox subunit of NAD(P)H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD(P)H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD(P)H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.     

Insulin action in intact mouse diaphragm I.

Summary The incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2–10 mm) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0-methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.Abbreviations GS Glycogen synthase - GS-I Glycogen synthase activity independent of G6P - GS-D Glycogen synthase activity dependent on G6P - G6P Glucose-6-phosphate - ATP Adenosine triphosphate - EDTA Ethylene diamine tetracetic acid - Mops Morpholinopropane sulfonic acid - 2DG 2-Deoxy glucose - 3-0-MG 3-0-Methyl glucose - tricine N-tris(Hydroxymethyl)methyl glycine Enzymes: Glycogen Synthase — UDPGlucose — Glycogen Glucosyl — Transferase (EC 2.4.1.11) J. Larner is an established investigator of the American Diabetes Association.     

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes

Summary Commercially available concanavalin A binds Ca²⁺ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca²⁺ uptake (defined as an increased association of ⁴⁵Ca²⁺ with cells) in rat splenocytes and Ca²⁺ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in ⁴⁵Ca²⁺ uptake over control. The concanavalin A stimulated uptake of ⁴⁵Ca²⁺ occurred within minutes, and required concentrations of concanavalin A which promoted [³H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca²⁺ uptake than concanavalin A. Sodium periodate inhibited Ca²⁺ uptake at concentrations which promoted ³H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca²⁺ uptake which is not due to binding of ⁴⁵Ca²⁺ to concanavalin A. Although the concanavalin A-promoted Ca²⁺ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by ³H-thymidine incorporation, the role of Ca²⁺ in this event remains unclear.     

Body size of the weasel Mustela nivalis and the stoat M. erminea in Sweden

In this study we examined temporal and geographical variations in a sample of 124 skulls of the weasel Mustela nivalis and 146 skulls of the stoat M. erminea, collected in Sweden between 1959-1992 and 1913-1990, respectively.We used Principal Component Analysis (PCA) to combine the effects of latitude, longitude, year of collection, mean ambient temperature and Net Primary Productivity (NPP). The first principal component (PC1) contained latitude, ambient temperature and NPP and was significantly and positively related to male (but not female) skull size of both stoats and weasels. None of the other factors or their interactions were significantly related to skull size.We conclude that ambient temperature, either directly through energy savings, or indirectly through improved food availability (increased NPP), had a significant effect on determining body size of male stoats and weasels in Sweden. Our results support the hypothesis that male and female of these species are affected by different selection pressures and thus react differently to changing environmental conditions.     

Cutting edge: MHC class I-Ly49 interaction regulates neuronal function

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.     

Mapping of sites on the surface membrane of mammalian cells

Summary Cells transformed by Simian Virus 40 have sites on the surface membrane for Concanavalin A (Con A) and a copolymer of ornithine, leucine (POL). The cells can be rapidly agglutinated by Con A, more slowly aggregated by POL, and they can be killed by both compounds. Treatment with Con A or POL has been used to select resistant cell variants from the transformed cells. Variants selected for resistance to Con A were also resistant to POL, but variants selected for resistance to POL were not resistant to Con A. The POL-selected variants showed less aggregation by POL but no decrease in agglutinability by Con A, whereas Con A-selected variants showed a decrease both in POL aggregation and Con A agglutination. The selection for both sites by Con A and only for POL sites by POL, can be explained in that the sites for POL are part of the sites for Con A and/or are included in clusters of Con A sites.Paper I in this series is Inbar, Ben-Bassat and Sachs (1971a).     

Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

Ubiquinone reduction and proton uptake by chromatophores of Rhodopseudomonas sphaeroides R-26. Periodicity of two in consecutive light flashes

Chromatophores of Rhodopseudomonas sphaeroides strain R-26 were subjected to a series of brief flashes of light in the presence of diaminodurene as an electron donor. Odd-numbered flashes induced the reduction of ubiquinone to the anionic semiquinone, as indicated by absorbance changes near 450 nm. This reaction was not attended by proton binding. Even-numbered flashes caused disappearance of the semiquinone, presumably by conversion to the fully reduced form. This reaction was attended by proton uptake.     

Prolonging the action of protein and peptide drugs by a novel approach of reversible polyethylene glycol modification

Polyethylene glycol (PEG)-conjugated therapeutic peptides/proteins have been shown to exhibit clinical properties superior to those of their corresponding unmodified parent molecules. However, the desirable pharmacological features gained by protein PEGylation become irrelevant if conjugates are inactivated or cannot reach their target tissues. Here we describe the design and synthesis of MAL-FMS-OSU. This bifunctional agent enables PEG chains to be linked to peptides and proteins through a slowly hydrolysable chemical bond. PEG-FMS-peptide/protein conjugates thus formed undergo spontaneous hydrolysis at a slow rate upon incubation at pH 8.5, 37 degrees C with a t(1/2) value of 8-14 +/- 2 h, generating the unmodified parent molecule. The validity of this approach was studied with exendin-4 and human growth hormone. A single subcutaneous administration of PEG(40,000)-FMS-exendin-4 facilitated a prolonged and stable reduction in glucose levels in mice (t(1/2) = 30 +/- 2 h) and exceeded the effect obtained by the same dose of the native hormone by 7-8 times.