No abstract

The lipid peroxidation product 4-hydroxynonenal (4-HNE) is a signaling mediator with wide-ranging biological effects. In this paper, we report that disruption of mGsta4, a gene encoding the 4-HNE-conjugating enzyme mGSTA4-4, causes increased 4-HNE tissue levels and is accompanied by age-dependent development of obesity which precedes the onset of insulin resistance in 129/sv mice. In contrast, mGsta4 null animals in the C57BL/6 genetic background have normal 4-HNE levels and remain lean, indicating a role of 4-HNE in triggering or maintaining obesity. In mGsta4 null 129/sv mice, the expression of the acetyl-CoA carboxylase (ACC) transcript is enhanced several-fold with a concomitant increase in the tissue level of malonyl-CoA. Also, mitochondrial aconitase is partially inhibited, and tissue citrate levels are increased. Accumulation of citrate could lead to allosteric activation of ACC, further augmenting malonyl-CoA levels. Aconitase may be inhibited by 4-HNE or by peroxynitrite generated by macrophages which are enriched in white adipose tissue of middle-aged mGsta4 null 129/sv mice and, upon lipopolysaccharide stimulation, produce more reactive oxygen species and nitric oxide than macrophages from wild-type mice. Excessive malonyl-CoA synthesized by the more abundant and/or allosterically activated ACC in mGsta4 null mice leads to fat accumulation by the well-known mechanisms of promoting fatty acid synthesis and inhibiting fatty acid beta-oxidation. Our findings complement the recent report that obesity causes both a loss of mGSTA4-4 and an increase in the level of 4-HNE [Grimsrud, P. A., et al. (2007) Mol. Cell. Proteomics 6, 624-637]. The two reciprocal processes are likely to establish a positive feedback loop that would promote and perpetuate the obese state.

We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein γ subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion.G protein-coupled receptors | live cell imaging | signaling | insulin

Nanozymes have drawn significant scientific interest due to their high practical importance in terms of overcoming the instability, complicated synthesis, and high cost of protein enzymes. However, their activity is generally limited to particular pHs, especially acidic ones. Herein, we report that luminescent N, S, and P-co-doped carbon quantum dots (NSP-CQDs) act as attractive peroxidase mimetics in a wide pH range, even at neutral pH, for the peroxidase substrate 2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the presence of H2O2. The synergistic effects of multiple heteroatoms doping in CQDs boost the catalytic activity in a wide pH range attributed to the presence of high density of active sites for enzymatic-like catalysis and accelerated electron transfer during the peroxidase-like reactions. A possible reaction mechanism for the peroxidase-like activity of CQDs is investigated based on the radical trapping experiments. Moreover, the multifunctional activity of NSP-CQDs was further utilized for antibacterial assays for both Gram-negative and Gram-positive model species, including Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. The growths of the employed E. coli and S. aureus were found to be significantly inhibited due to the peroxidase-mediated perturbation of cell walls. The present work signifies the current advance in the rational design of N, S, and P-co-doped CQDs as highly active peroxidase mimics for novel applications in diverse fields, including catalysis, medical diagnostics, environmental chemistry, and biotechnology.

Cr(VI) is a known toxic and non-biodegradable pollutant that results from multiple industrial processes, and can cause significant environmental damage if it is not removed from wastewater. However, it can be reduced to Cr(III), which is less toxic and can be readily precipitated out and removed. Here, a fast and facile single-step technique is reported for the synthesis of nitrogen-phosphorus doped fluorescent carbon dots (NP-CD) using a domestic microwave, as a potential photocatalytic material. Under natural sunlight, a simple photocatalytic experiment reveals that the NP-CD are highly efficient for the quantitative reduction of Cr(VI) to Cr(III) in synthetic contaminated water, in a linear range from 10 ppm (in approximately 10 min) to 2000 ppm (in approximately 320 min) by increasing the sunlight irradiation time followed by its removal by precipitation. NP-CD exhibit high recyclability of up to six cycles without any apparent loss in photocatalytic activity, demonstrating NP-CD as a potential photocatalyst material for Cr(VI) water treatment.

The lipid peroxidation product 4-hydroxynonenal (4-HNE) forms as a consequence of oxidative stress. By electrophilic attack on biological macromolecules, 4-HNE mediates signaling or may cause toxicity. A major route of 4-HNE disposal is via glutathione conjugation, in the mouse catalyzed primarily by glutathione transferase mGSTA4-4. Unexpectedly, mGsta4-null mice, in which 4-HNE detoxification is impaired, have an extended life span. This finding could be explained by the observed activation of the transcription factor Nrf2 in the knockout mice, which in turn leads to an induction of antioxidant and antielectrophilic defenses. Especially, the latter could provide a detoxification mechanism that contributes to enhanced longevity. We propose that disruption of 4-HNE conjugation elicits a hormetic response in which an initially increased supply of 4-HNE is translated into activation of Nrf2, leading to a new steady state in which the rise of 4-HNE concentrations is dampened, but life-extending detoxification mechanisms are concomitantly induced.

The present finding describes an efficient facile approach for the fabrication of nitrogen-doped carbon dots (N-CDs) as a "fluorescent nanoswitch". Highly fluorescent blue-light-emitting N-CDs have been synthesized via a simpler hydrothermal method using 2,2′-(ethylenedioxy)-bis(ethylamine) and malic acid as the precursors. N-CDs showed excitation-dependent and pH-independent emission along with a quantum yield of ∼25%. The blue fluorescent emission of N-CDs has been selectively "turned off" (quenching of fluorescence (FL)) during the sensing of Cr(VI) with 0.02 μM limit of detection and further been selectively "turned on" (restoration of FL) on sensing ascorbic acid, compared with other metal cations and biomolecules tested. For testing the practical applicability of N-CDs, the switchable reversibility of the fluorescent nanoswitch has been tested for up to four cycles on the basis of FL "on−off−on". Furthermore, the toxicological test showed the antibacterial effect of the N-CDs on the tested Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli cells. Additionally, these N-CDs can also be used as a fluorescent ink for imaging purposes.

Background Lower respiratory viral infections predispose to Bronchiolitis Obliterans Syndrome (BOS). In addition, there is emerging evidence to support the role of autoimmunity in the pathogenesis of BOS. Since CD4+CD25+Foxp3+ T-cell (Treg) control autoimmunity, we tested the hypothesis that respiratory virus-induced Treg dysfunction leads to BOS. Methodology Treg frequency was monitored using flow-cytometry. Apoptosis, cytokines, and antibodies were analyzed using annexin V assay, LUMINEX, and ELISA, respectively. Murine studies were performed using orthotopic tracheal transplant model. Results A) Human studies: Treg troughs (decrease >50% of baseline) were found in 13 (43.3%) of 30 lung transplant recipients. Treg isolated during troughs revealed increased apoptosis (37.8%). Patients with Treg troughs had increased prevalence of antibodies to self antigens collagen type I (23.1% Vs 5.8% pre-trough), collagen V (7.7% Vs 0%), and k-alpha tubulin (30.7% Vs 11.7%, p<0.01) at 6-months. Increased number of Treg troughs correlated with more rapid onset of BOS. B) Murine studies: Infection of tracheal transplant recipients with murine parainfleunza sendai virus led to increased Treg apoptosis (50.5%) in the draining lymph nodes. Vaccination against sendai virus prior to transplant abrogated apoptosis of Treg. In vitro, sendai virus infected, but not naive, tracheal epithelial cells demonstrated upregulation of FasL (>3.5 fold) and induction of co-cultured Treg apoptosis (5.6 fold increase). Conclusions RVI cause Treg apoptosis which leads to the development of de novo autoimmunity that may play a role in the pathogenesis of BOS.