Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

白地霉Cryytococcus neoformans脂肪酶的双水相萃取

本文研究了不同无机盐的双水相体系对白地霉脂肪酶的萃取分离效果,对PEG/（NH4）2SO4成相系统进行了系统的研究,通过考察体系PEG分子量、不同的无机盐、PEG浓度、（NH4）2SO4浓度、离子强度、pH值及（NH4）2SO4浓度对反萃取的影响,并通过正交实验进一步优化了实验条件,初步确定在PEG浓度15%,（NH4）2SO4浓度22.5%,pH8.0,不加NaCl的条件下进行双水相萃取,脂肪酶分离系数和纯化倍数分别为6.8和7.5,比活力达到40.3 U/mg蛋白。     

Petalonia improves glucose homeostasis in streptozotocin-induced diabetic mice

The anti-diabetic potential of Petalonia binghamiae extract (PBE) was evaluated in vivo. Dietary administration of PBE to streptozotocin (STZ)-induced diabetic mice significantly lowered blood glucose levels and improved glucose tolerance. The mode of action by which PBE attenuated diabetes was investigated in vitro using 3T3-L1 cells. PBE treatment stimulated 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation. At the molecular level, peroxisome proliferator-activated receptor γ (PPARγ) and terminal marker protein aP2, as well as the mRNA of GLUT4 were up-regulated by PBE. In mature adipocytes, PBE significantly stimulated the uptake of glucose and the expression of insulin receptor substrate-1 (IRS-1). Furthermore, PBE increased PPARγ luciferase reporter gene activity in COS-1 cells. Taken together, these results suggest that the in vivo anti-diabetic effect of PBE is mediated by both insulin-like and insulin-sensitizing actions in adipocytes.     

Cellular Uptake and Antitumor Activity of DOX-hyd-PEG-FA Nanoparticles

A PEG-based, folate mediated, active tumor targeting drug delivery system using DOX-hyd-PEG-FA nanoparticles (NPs) were prepared. DOX-hyd-PEG-FA NPs showed a significantly faster DOX release in pH 5.0 medium than in pH 7.4 medium. Compared with DOX-hyd-PEG NPs, DOX-hyd-PEG-FA NPs increased the intracellular accumulation of DOX and showed a DOX translocation from lysosomes to nucleus. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was much higher than that of free DOX, DOX-ami-PEG-FA NPs and DOX-hyd-PEG NPs. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was attenuated in the presence of exogenous folic acid. The IC₅₀ of DOX-hyd-PEG-FA NPs and DOX-hyd-PEG NPs on A549 cells showed no significant difference. After DOX-hyd-PEG-FA NPs were intravenously administered, the amount of DOX distributed in tumor tissue was significantly increased, while the amount of DOX distributed in heart was greatly decreased as compared with free DOX. Compared with free DOX, NPs yielded improved survival rate, prolonged life span, delayed tumor growth and reduced the cardiotoxicity in tumor bearing mice model. These results indicated that the acid sensitivity, passive and active tumor targeting abilities were likely to act synergistically to enhance the drug delivery efficiency of DOX-hyd-PEG-FA NPs. Therefore, DOX-hyd-PEG-FA NPs are a promising drug delivery system for targeted cancer therapy.     

Prognostic Impact of IPSS-R and Chromosomal Translocations in 751 Korean Patients with Primary Myelodysplastic Syndrome

Chromosomal translocations are rare in myelodysplastic syndrome (MDS) and their impact on overall survival (OS) and response to hypomethylating agents (HMA) is unknown. The prognostic impact of the revised International Prognostic Scoring System (IPSS-R) and for chromosomal translocations was assessed in 751 patients from the Korea MDS Registry. IPSS-R effectively discriminated patients according to leukaemia evolution risk and OS. We identified 40 patients (5.3%) carrying translocations, 30 (75%) of whom also fulfilled complex karyotype criteria. Translocation presence was associated with a shorter OS (median, 12.0 versus 79.7 months, P < 0.01). Multivariate analysis demonstrated that translocations (hazard ratio [HR] 1.64 [1.06–2.63]; P = 0.03) as well as age, sex, IPSS-R, and CK were independent predictors of OS. In the IPSS-R high and very high risk subgroup (n = 260), translocations remained independently associated with OS (HR 1.68 [1.06–2.69], P = 0.03) whereas HMA treatment was not associated with improved survival (median OS, 20.9 versus 21.2 months, P = 0.43). However, translocation carriers exhibited enhanced survival following HMA treatment (median 2.1 versus 12.4 months, P = 0.03). Our data suggest that chromosomal translocation is an independent predictor of adverse outcome and has an additional prognostic value in discriminating patients with MDS having higher risk IPSS-R who could benefit from HMA treatment.     

Effect of Bacillus mesonae H20-5 Treatment on Rhizospheric Bacterial Community of Tomato Plants under Salinity Stress

Plant growth-promoting bacteria improve plant growth under abiotic stress conditions. However, their effects on microbial succession in the rhizosphere are poorly understood. In this study, the inoculants of Bacillus mesonae strain H20-5 were administered to tomato plants grown in soils with different salinity levels (EC of 2, 4, and 6 dS/m). The bacterial communities in the bulk and rhizosphere soils were examined 14 days after H20-5 treatment using Illumina MiSeq sequencing of the bacterial 16S rRNA gene. Although the abundance of H20-5 rapidly decreased in the bulk and rhizosphere soils, a shift in the bacterial community was observed following H20-5 treatment. The variation in bacterial communities due to H20-5 treatment was higher in the rhizosphere than in the bulk soils. Additionally, the bacterial species richness and diversity were greater in the H20-5 treated rhizosphere than in the control. The composition and structure of the bacterial communities varied with soil salinity levels, and those in the H20-5 treated rhizosphere soil were clustered. The members of Actinobacteria genera, including Kineosporia, Virgisporangium, Actinoplanes, Gaiella, Blastococcus, and Solirubrobacter, were enriched in the H20-5 treated rhizosphere soils. The microbial co-occurrence network of the bacterial community in the H20-5 treated rhizosphere soils had more modules and keystone taxa compared to the control. These findings revealed that the strain H20-5 induced systemic tolerance in tomato plants and influenced the diversity, composition, structure, and network of bacterial communities. The bacterial community in the H20-5 treated rhizosphere soils also appeared to be relatively stable to soil salinity changes.     

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.