Differential degradation of bicyclics with aromatic and alicyclic rings by Rhodococcus sp. strain DK17

The metabolically versatile Rhodococcus sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the meta-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in Escherichia coli, the DK17 o-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin cis-dihydrodiol, indan-1,2-diol, and cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17 o-xylene dioxygenase to perform distinct regioselective hydroxylations.     

Plasmid typing and resistance profiling of Escherichia fergusonii and other Enterobacteriaceae isolates from South Korean farm animals

In this study, we focused on determining the distribution and prevalence of major plasmid replicons in β-lactam-resistant Escherichia fergusonii and Enterobacteriaceae of animal and human origin. A high degree of plasmid variability and multiple plasmid replicons were observed among the isolates. The IncF and IncI1 replicons were the most prevalent in E. fergusonii and Salmonella enterica serovar Indiana isolated from swine and poultry in South Korea, respectively. The presence of broad-host-range plasmid replicons such as IncN, IncA/C, IncHI1, and IncHI2 that are associated with important virulence genes and toxins as well as antimicrobial resistance determinants indicates that E. fergusonii has the potential to become an important pig pathogen and possible emerging opportunistic zoonotic pathogen.     

A stress-responsive caleosin-like protein, AtCLO4, acts as a negative regulator of ABA responses in Arabidopsis

Caleosins or related sequences have been found in a wide range of higher plants. In Arabidopsis, seed-specific caleosins are viewed as oil-body (OB)-associated proteins that possess Ca(2+)-dependent peroxygenase activity and are involved in processes of lipid degradation. Recent experimental evidence suggests that one of the Arabidopsis non-seed caleosins, AtCLO3, is involved in controlling stomatal aperture during the drought response; the roles of the other caleosin-like proteins in Arabidopsis remain largely uncharacterized. We have demonstrated that a novel stress-responsive and OB-associated Ca(2+)-binding caleosin-like protein, AtCLO4, is expressed in non-seed tissues of Arabidopsis, including guard cells, and down-regulated following exposure to exogenous ABA and salt stress. At the seed germination stage, a loss-of-function mutant (atclo4) was hypersensitive to ABA, salt and mannitol stresses, whereas AtCLO4-overexpressing (Ox) lines were more hyposensitive to those stresses than the wild type. In adult stage, atclo4 mutant and AtCLO4-Ox plants showed enhanced and decreased drought tolerance, respectively. Following exposure to exogenous ABA, the expression of key ABA-dependent regulatory genes, such as ABF3 and ABF4, was up-regulated in the atclo4 mutant, while it was down-regulated in AtCLO4-Ox lines. Based on these results, we propose that the OB-associated Ca(2+)-binding AtCLO4 protein acts as a negative regulator of ABA responses in Arabidopsis.     

Small heat shock protein Hsp17.8 functions as an AKR2A cofactor in the targeting of chloroplast outer membrane proteins in Arabidopsis

Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids. Ankyrin repeat protein 2A (AKR2A) and AKR2B were recently shown to be involved in the targeting of proteins to the plastid outer envelope. However, it remains unknown whether other factors are involved in this process. In this study, we investigated a factor involved in AKR2A-mediated protein targeting to chloroplasts in Arabidopsis (Arabidopsis thaliana). Hsp17.8, a member of the class I (CI) cytosolic small heat shock proteins (sHsps), was identified in interactions with AKR2A. The interaction between Hsp17.8 and AKR2A was further confirmed by coimmunoprecipitation experiments. The carboxyl-terminal ankyrin repeat domain of AKR2A was responsible for AKR2A binding to Hsp17.8. Other CI cytosolic sHsps also interact with AKR2A to varying degrees. Additionally, Hsp17.8 binds to chloroplasts in vitro and enhances AKR2A binding to chloroplasts. HSP17.8 was expressed under normal growth conditions, and its expression increased after heat shock. Hsp17.8 exists as a dimer under normal physiological conditions, and it is converted to high oligomeric complexes, ranging from 240 kD to greater than 480 kD, after heat shock. High levels of Hsp17.8 together with AKR2A resulted in increased plastid targeting of Outer Envelope Protein7 (OEP7), a plastid outer envelope protein expressed as a green fluorescent protein fusion protein. In contrast, artificial microRNA suppression of HSP17.8 and closely related CI cytosolic sHSPs in protoplasts resulted in a reduction of OEP7:green fluorescent protein targeting to plastids. Based on these data, we propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions.     

An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis

The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.     

Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by macrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of copper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch.     

A distinct role of neutrophil lactoferrin in RelA/p65 phosphorylation on Ser536 by recruiting TNF receptor-associated factors to IkappaB kinase signaling complex

The activation of NF-kappaB by neutrophil lactoferrin (Lf) is regulated via the IkappaB kinase (IKK) signaling cascade, resulting in the sequential phosphorylation and degradation of IkappaB. In this study, we observed that Lf protein augmented p65 phosphorylation at the Ser(536), but not the Ser(276) residue, and stimulated the translocation of p65 into the nucleus. Lf was also shown to enhance the association between p65 and CREB-binding protein/p300 in vivo. To elucidate the mechanism by which Lf triggers these signaling pathways, we attempted to delineate the roles of the upstream components of the IKK complex, using their dominant-negative mutants and IKKalpha(-/-) and IKKbeta(-/-) mouse embryonic cells. We demonstrated that both IKKalpha and IKKbeta as well as NF-kappaB-inducing kinase are indispensable for Lf-induced p65 phosphorylation. However, MAPK kinase kinase 1 is not essentially required for this activation. We also observed that Lf-induced p65 phosphorylation was either partially or completely abrogated as the result of treatment with the mutant forms of TNFR-associated factor (TRAF) 2, TRAF5, or TRAF6. Moreover, we demonstrated that Lf directly interacted with TRAF5. Expression of the dominant-negative mutant of TRAF5 or its small interfering RNA almost completely abrogated the Lf-induced p65 phosphorylation. These results suggest that signaling pathways, including TRAFs/NF-kappaB-inducing kinase/IKKs, may be involved in the regulation of Lf-induced p65 activation, thereby resulting in the activation of members of the NF-kappaB family.     

Triamcinolone acetonide protects the rat retina from STZ-induced acute inflammation and early vascular leakage

Streptozotocin (STZ) has been commonly used to induce in vivo and in vitro hyperglycemic diabetes and its toxicity leads to inflammation and vascular injury. Triamcinolone acetonide (TA), as an anti-angiogenic/anti-inflammatory drug, is clinically used to improve the visual acuity in neovascular and edematous ocular diseases. The aim of this study was to investigate the effect of TA on early inflammation and vascular leakage in the retina of STZ-induced hyperglycemic rats. Hyperglycemia was induced in 8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of STZ (65 mg/kg); only rats with blood glucose levels >13.9 mmol/l 1 day after STZ injection were included in STZ-hyperglycemic group. Sex- and age-matched SD rats injected with buffer were used as the control group. One day before STZ and buffer injection, 2 microl TA (4 mg/ml in saline) and 2 microl saline were intravitreal-injected into the right and the left eyes of rats, respectively. Retinal vascular leakage was measured using the Evans-blue method. Changes in pro-inflammatory target genes, such as tumor necrotic factor (TNF)-alpha, intracellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were assessed by immunoblottings, immunostaining, and ELISA analyses. Vascular hyperleakage and up-regulation of most pro-inflammatory genes peaked within a few days after STZ injection and had recovered. However, these changes were blocked by TA pretreatment. Our data suggest that TA controls STZ-induced early vascular leakage and temporary pro-inflammatory signals in the rat retina.     

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.