Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.

Glucan-rich polysaccharides obtained from split gill mushroom [Schizophyllum commune (Fr.)] ameliorate hyperglycemia by enhancing insulin and GLUT2 pancreas in type 2 diabetic rats.

AIMS: Abnormalities in the secretion of insulin are the cause of pathology and complications in diabetic patients. The aim of this study was to investigate the anti-diabetic effect of polysaccharide extracts from the split gill mushroom in type 2 diabetes rats administered a low dose of streptozotocin (STZ) in combination with a high-fat diet. METHODS: The rats were divided into 6 groups: the control group (ND), the control group fed with polysaccharide extract from split gill (ND240), the diabetes group (HFD+DM), the diabetic group fed 120 (HFD+S120) and 240 mg/kg BW polysaccharide extract (HFD+S240), and the diabetic group receiving metformin (HFD+Met). Subsequently, the Islets of Langerhans of pancreatic tissue were studied using a light microscope and transmission electron microscopy (TEM). Immunofluorescence for the detection of insulin and glucose transporter 2 (GLUT2) proteins, and malondialdehyde (MDA) were also detected in pancreatic tissue. RESULTS: In the diabetic and HFD+120 groups, the tissues harbored various pathologies. The HFD+S240 and HFD+Met groups were found to have lower blood sugar levels. The levels of insulin and GLUT2 increased compared with the diabetic group. Additionally, the levels of MDA were reduced. CONCLUSIONS: The use of polysaccharide extract from split gill mushrooms (240 mg/kg BW) is an alternative to treating various pathologies in the relief or treatment of diabetes mellitus.

Fas-associated death domain (FADD) and the E3 ubiquitin-protein ligase TRIM21 interact to negatively regulate virus-induced interferon production.

The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKε and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.

Mechanism of the fungal-like particles in the inhibition of adipogenesis in 3T3-L1 adipocytes.

The dynamic ability of adipocytes in adipose tissue to store lipid in response to changes in the nutritional input and inflammatory elicitors has a major impact on human health. Previously, we established laminarin-coated beads or LCB as an inflammatory elicitor for adipocytes. However, it was not clear whether LCB inhibits lipid accumulation in adipocytes. Here, we show that LCB acts in the early stage of adipogenesis through both interleukin-1 receptor-associated kinases (IRAK) and spleen tyrosine kinase (SYK) pathways, resulting in the activation of the AMP-activated protein kinase (AMPK) and nuclear factor-κB (NF-κB) complexes, which subsequently cause cell cycle arrest, downregulation of the key transcription factors and enzymes responsible for adipogenesis, inhibition of adipogenesis, and stimulation of an inflammatory response. While LCB could effectively block lipid accumulation during the early stage of adipogenesis, it could stimulate an inflammatory response at any stage of differentiation. Additionally, our results raise a possibility that toll-like receptor 2 (TLR2) and C-type lectin domain family 7 member A (CLEC7A/Dectin-1) might be potential ß-glucan receptors on the fat cells. Together, we present the mechanism of LCB, as fungal-like particles, that elicits an inflammatory response and inhibits adipogenesis at the early stage of differentiation.

Fungal-like particles and macrophage-conditioned medium are inflammatory elicitors for 3T3-L1 adipocytes.

Adipocytes from white-adipose tissue are known to produce inflammatory cytokines, which play a major role in energy balance and metabolism. While they can respond to pathogen-associated molecular pattern (PAMPs) such as lipopolysaccharide (LPS) from bacteria, it is not known whether adipocytes can be stimulated by fungal cells. Previously, adipocytes were shown to produce toll-like receptor 2 (TLR2), a ß-glucan receptor, suggesting that they could respond to ß-glucan on the fungal cell wall. In this study, we show that heat-killed yeast induce an inflammatory response in adipocytes. Using fungal-like particles, namely laminarin-coated beads (LCB), we find that these particles trigger the expression of many key inflammatory genes in dose- and time-dependent fashions in adipocytes. These results suggest that ß-glucan on the fungal cell wall is sufficient to elicit an inflammatory response in adipocytes. In addition, we show that both LCB and LCB-treated conditioned medium from RAW 264.7 murine macrophages (LCB-RM) induce the expression of those inflammatory genes through IKKß-IκBα proteins. Together, we conclude that the fungal-like particles and the conditioned medium elicit an inflammatory response in adipocytes through the canonical or classical NF-κB pathway.

The Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex requires the Enhancer of Polycomb A domain and chromodomain to acetylate nucleosomes.

Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

Rhinacanthins-rich Extract Enhances Glucose Uptake and Inhibits Adipogenesis in 3T3-L1 Adipocytes and L6 Myotubes.

BACKGROUND: Obesity is one of the imperative dynamics in the incidence and intensification of type 2 diabetes mellitus (T2DM). Rhinacanthus nasutus leaf extracts are previously reported for their antidiabetic and antiobesity potential. OBJECTIVE: The present study was performed to evaluate glucose uptake stimulatory and antiadipogenic activities of a standardized rhinacanthins-rich extract (RRE) and its marker compounds namely rhinacanthin-C (RC), rhinacanthin-D (RD), and rhinacanthin-N (RN) in 3T3-L1 and L6 cells. MATERIALS AND METHODS: RRE was prepared by a green extraction process, and the marker compounds (RC, RD, and RN) were isolated from the RRE using a silica gel column chromatography. Glucose uptake stimulation in both 3T3-L1 and L6 cells was performed by quantification of residual glucose in the media using glucose oxidase kit. Antiadipogenic activity in 3T3-L1 adipocytes was performed by intracellular lipids quantification using oil red O dye. RESULTS: At the highest effective dose, RRE (20 µg/mL) exhibited satisfactory glucose uptake stimulatory effect in 3T3-L1 adipocytes that equivalent to RN (20 µg/mL) and the positive control insulin (0.58 µg/mL) but higher than RC (20 µg/mL) and RD (20 µg/mL). In addition, treatments of L6 myotubes showed that RRE (2.5 µg/mL) exhibited potent and equivalent glucose uptake stimulation (>80%) to RC (2.5 µg/mL) and the standard drugs, insulin (2.90 µg/mL) and metformin (219.5 µg/mL), but higher than RD (2.5 µg/mL) and RN (2.5 µg/mL). Furthermore, RRE (20 µg/mL) exhibited potent antiadipogenic effect in 3T3-L1 adipocytes, which equivalent to RC (20 µg/mL) but higher than RD (20 µg/mL) and RN (20 µg/mL). CONCLUSIONS: The undertaken study suggests that RRE could be used as an effective remedy in the treatment of obesity-associated T2DM. SUMMARY: Rhinacanthins-rich extract and its marker compounds showed potent glucose uptake stimulatory activity in 3T3-L1 adipocytes and L6 myotubesRhinacanthins-rich extract and rhinacanthin-C showed comparable antiadipogenic effect in 3T3-L1 adipocytesRRE could be used as an effective remedy in the treatment of obesity-associated T2DM. Abbreviations used: T2DM: Type-2 diabetes mellitus; RRE: Rhinacanthins-rich extract; RC: Rhinacanthin-C; RD: Rhinacanthin-D; RN: Rhinacanthin-N; α-MEM: α-Minimum essential medium; DMEM: Dulbecco's modified Eagle's medium; HS: Horse serum; FBS: Fetal bovine serum; BSA: Bovine serum albumin; IBMX: 3-isobutyl-1-methylxanthine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GO: Glucose oxidase; NMR: Nuclear magnetic resonance; HPLC: High-performance liquid chromatography.

Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes.

Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gcn5 subcomplex of SAGA which acetylates histones H3 and H2B on nucleosomes, and the Piccolo NuA4 complex which acetylates histones H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify Escherichia coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes.

SAGA binds TBP via its Spt8 subunit in competition with DNA: implications for TBP recruitment.

In yeast, the multisubunit SAGA (Spt-Ada-Gcn5-acetyltransferase) complex acts as a coactivator to recruit the TATA-binding protein (TBP) to the TATA box, a critical step in eukaryotic gene regulation. However, it is unclear which SAGA subunits are responsible for SAGA's direct interactions with TBP and precisely how SAGA recruits TBP to the promoter. We have used chemical crosslinking to identify Spt8 and Ada1 as potential SAGA subunits that interact with TBP, and we find that both Spt8 and SAGA bind directly to TBP monomer in competition with TBP dimer. We further find that Spt8 and SAGA compete with DNA to bind TBP rather than forming a triple complex. Our results suggest a handoff model for SAGA recruitment of TBP: instead of binding together with TBP at the TATA box, activator-recruited SAGA transfers TBP to the TATA box. This simple model can explain SAGA's observed ability to both activate and repress transcription.