Targeting palmitoyl acyltransferase ZDHHC21 improves gut epithelial barrier dysfunction resulting from burn-induced systemic inflammation.

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domain-containing protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-α-IFN-γ in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-α-IFN-γ-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acyl-biotin exchange assay and click chemistry, we show that TNF-α-IFN-γ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.NEW & NOTEWORTHY Increased mucosal permeability in the gut is one of the major complications following severe burn. Here we report the novel finding that zinc finger DHHC domain-containing protein-21 (ZDHHC21) mediates gut epithelial hyperpermeability resulting from an experimental model of thermal injury. The hyperpermeability response was significantly attenuated with a pharmacological inhibitor of palmitoyl acyltransferases and in mice with genetic ablation of ZDHHC21. These findings suggest that ZDHHC21 may serve as a novel therapeutic target for treating burn-induced intestinal barrier dysfunction.

Interleukin-1ß Mediates ß-Catenin-Driven Downregulation of Claudin-3 and Barrier Dysfunction in Caco2 Cells.

BACKGROUND: IL-1ß is a cytokine involved in mediating epithelial barrier dysfunction in the gut. It is known that IL-1ß mediates activation of non-muscle myosin light chain kinase in epithelial cells, but the precise mechanism by which epithelial barrier dysfunction is induced by IL-1ß is not understood. METHODS AND RESULTS: Using a Caco2 cell model, we show that the expression of the tight junction protein, claudin-3, is transcriptionally downregulated by IL-1ß treatment. In addition, after assessing protein and mRNA expression, and protein localization, we show that inhibition of nmMLCK rescues IL-1ß-mediated decrease in claudin-3 expression as well as junction protein redistribution. Using chromatin immunoprecipitation assays, we also show that ß-catenin targeting of the claudin-3 promoter occurs as a consequence of IL-1ß-mediated epithelial barrier dysfunction, and inhibition of nmMLCK interferes with this interaction. CONCLUSIONS: Taken together, these data represent the first line of evidence demonstrating nmMLCK regulation of claudin-3 expression in response to IL-1ß-treated epithelial cells.

Protein tyrosine kinase 6 mediates TNFα-induced endothelial barrier dysfunction.

A key event in the progression of systemic inflammation resulting from severe trauma or shock involves microvascular hyperpermeability, which leads to excessive plasma fluid and proteins accumulating in extravascular space resulting in tissue edema. The precise molecular mechanism of the hyperpermeability response is not completely understood. Protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) is a non-receptor tyrosine kinase related to Src-family proteins. Although it has also been shown that PTK6 participates in regulating epithelial barrier function, the role of PTK6 in endothelial barrier function has not been reported. In this study, we hypothesized that PTK6 is (1) expressed in vascular endothelial cells, and (2) contributes to vascular endothelial hyperpermeability in response to TNFα. Results showed that PTK6 was detected in mouse endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated TNFα induced decrease in endothelial barrier function as measured by electric cell-substrate impedance sensing (ECIS) and in vitro transwell albumin-flux assays. Furthermore, we showed that TNFα treatment of endothelial cells increased active PTK6 association with p120-catenin at endothelial cell-cell junctions. Further analysis using immunocytochemistry and immunoprecipitation demonstrated that PTK6 knockdown attenuated TNFα induced VE-cadherin internalization as well as promoting its association with p120-catenin. Our study demonstrates a novel role of PTK6 in mediating endothelial barrier dysfunction.

The social context of smoking: the next frontier in tobacco control?

A better understanding of the social context of smoking may help to enhance tobacco control research and practice.

Long-lived emissions from 4'-substituted Pt(trpy)Cl+ complexes bearing aryl groups. Influence of orbital parentage.

Pt(trpy)Cl+, where trpy denotes 2,2':6',2' '-terpyridine, is a versatile binding agent but has a limited photochemistry due to a short excited-state lifetime. However, this work shows that the introduction of aryl substituents at the 4' position of the trpy ligand drastically alters the picture. For the substituents phenyl, p-methoxyphenyl, 1-naphthyl, 2-naphthyl, 9-phenanthrenyl, and 1-pyrenyl, the ligand abbrevations are 4'-Ph-T, 4'-pMeOPh-T, 4'-Npl-T, 4'-Np2-T, 4'-Phe9-T, and 4'-Pyre1-T, respectively. Techniques utilized include electrochemistry as well as absorption and emission spectroscopies. While the lowest energy excited states of Pt(4'-Ph-T)Cl+ and the parent complex Pt(trpy)Cl+ exhibit mainly metal-to-ligand charge-transfer (MLCT) character, the emitting state takes on aryl-to-trpy intraligand charge-transfer (ILCT) character as the substituents become more electron-donating. Studies of Zn(trpy)Cl2, its aryl-substituted analogues, and the free ligands themselves provide information about the relative energies of participating ILCT and intraligand 3pi-pi excited states. Even though the emission energy decreases when larger aryl groups are present, the emission lifetime increases all the way from 85 ns for Pt(4'-Ph-T)Cl+ to 64 micros for Pt(4'-Pyre1-T)Cl+. (Data from deoxygenated, room-temperature dichloromethane solution.) Intraligand character appears to dominate in the case of Pt(4'-Pyre1-T)Cl+, which is unique in the series in that it exhibits singlet and triplet emissions in solution. In aerated solution the complex shows prompt as well as delayed fluorescence. Finally, studies in donor media establish that the introduction of intraligand character inhibits solvent-induced exciplex quenching.

Carbon isotope discrimination and oxygen isotope composition in clones of the F(1) hybrid between slash pine and Caribbean pine in relation to tree growth, water-use efficiency and foliar nutrient concentration.

The objectives of this study were: (1) to examine how foliar carbon isotope discrimination (Delta) and oxygen isotope composition (delta(18)O) are related to tree growth, ash mineral nutrient concentration and foliar nutrient concentration in 7-year-old clones of the F(1) hybrid between slash pine (Pinus elliottii Engelm.) and Caribbean pine (P. caribaea var. hondurensis Barr. et Golf.) in subtropical Australia; and (2) to evaluate the potential of using foliar Delta, ash mineral nutrient concentration and delta(18)O measurements for selecting F(1) hybrid pine clones with high water-use efficiency (WUE) and growth potential. There were significant differences in tree growth, foliar Delta, delta(18)O and ash mineral nutrient concentration among the eight clones tested. Significant negative linear relationships existed between tree growth and Delta, extrapolating to zero growth at Delta = 24-30 per thousand. There were strong genetic correlations (r = -0.83 to -0.96) between Delta and tree growth, particularly tree height. Significant non-genetic correlations (r = -0.62 to -0.80) existed between Delta and foliar K concentration. Foliar delta(18)O, ash mineral nutrient concentration and foliar nutrient concentration were unrelated to tree growth. In the F(1) hybrid pine clones, variation in tree WUE, as reflected by Delta, was largely attributed to a genetic effect on leaf photosynthetic capacity rather than on stomatal conductance, as reflected by foliar delta(18)O.

Acute soman effects in the juvenile baboon: effects on a match-to-sample discrimination task and on total blood acetylcholinesterase.

Male juvenile baboons, trained on a match-to-sample operant discrimination task, were given acute intramuscular injections of soman (methyl pinacolyl phosphonofluoridate) at 1.0, 2.0, 3.0, 4.0, and 5.0 micrograms/kg. The different doses were given in a mixed order just before a behavioral test session. Just prior to administration of each soman dose and immediately following the 2-hr behavioral test session, a sample of blood (0.5 ml) was drawn from the baboon and analyzed for inhibition of acetylcholinesterase activity. Thereafter, blood sampling was accomplished at weekly intervals and soman was administered again only when whole blood acetylcholinesterase reached at least 80% of pre-soman control level. Behavioral effects of soman included a slowing of response times, a decrease in extra inconsequential responses, a decrease in responsiveness to the visual stimuli and an increase in errors. These effects were observed when acetylcholinesterase (AChE) levels fell to 25 mumoles/hr/ml blood or less. The threshold dose for behavioral effects was very close to the dose of soman which induced seizures.