Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.     

db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响

We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.     

Fractionation and quantitation of egg antigens from Schistosoma japonicum by the single-tube kinetic-dependent enzyme-linked immunosorbent assay (k-ELISA): higher antigenic activity in urea-soluble than in aqueous-soluble fractions

To identify sources of high potency antigens for use in serodiagnosis, aqueous-soluble egg antigens from Schistosoma japonicum were extracted with Dulbecco's phosphate-buffered saline. Residual particulates were solubilized with Tris-buffered 8 M urea, yielding a urea-soluble egg antigen fraction. The urea-soluble fraction was further fractionated with Bio Gel A50m and QAE-Sephadex. All fractions were quantitatively assayed for their specific antigenic activities against serum specimens from infected rabbits by the single-tube enzyme-linked immunosorbent assay (k-ELISA). In antigen rate-limiting conditions, the urea-soluble particulate fractions were more antigenically active than the aqueous-soluble fraction. In antigen-excess and antibody-limiting assay conditions, the ideal conditions for serologic assays, the urea-derived antigens also showed superior activities against sera from infected humans. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on gradient gels revealed numerous low molecular weight protein bands in the aqueous-soluble fraction, whereas the urea-soluble fractions appeared to be much simpler with the majority of their proteins concentrated in one or two high molecular weight bands (greater than or equal to 200 kdaltons). Electro-transfer blots of the SDS-PAGE onto nitrocellulose papers and subsequent visualization of antigens by enzyme-linked immunoabsorbence confirmed these findings. The above data suggest that the urea-soluble fraction of S. japonicum eggs is antigenically active and has potential use in the development of a diagnostic reagent.     

Modeling the Transmission of Middle East Respirator Syndrome Corona Virus in the Republic of Korea

The 2015 epidemic of Middle East respiratory syndrome (MERS) in the Republic of Korea has been the largest outbreak outside Middle East. This epidemic had caused 185 laboratory-confirmed cases and 36 deaths in the Republic of Korea until September 2, 2015, which attracted public’s attention. Based on the detailed data of patients released by World Health Organization (WHO) and actual propagation of the epidemic, we construct two dynamical models to simulate the propagation processes from May 20 to June 8 and from June 9 to July 10, 2015, respectively and find that the basic reproduction number R ₀ reaches up to 4.422. The numerical analysis shows that the reasons of the outbreak spread quickly are lack of self-protection sense and targeted control measures. Through partial correction analysis, the parameters β ₁ and γ have strong correlations with R ₀, i.e., the infectivity and proportion of the asymptomatic infected cases have much influence on the spread of disease. By sensitivity analysis, strengthening self-protection ability of susceptible and quickly isolating or monitoring close contacts are effective measures to control the disease.     

YAP regulates periodontal ligament cell differentiation into myofibroblast interacted with RhoA/ROCK pathway

During orthodontic tooth movement (OTM), periodontal ligament cells (PDLCs) receive the mechanical stimuli and transform it into myofibroblasts (Mfbs). Indeed, previous studies have demonstrated that mechanical stimuli can promote the expression of Mfb marker α-smooth muscle actin (α-SMA) in PDLCs. Transforming growth factor β1 (TGF-β1), as the target gene of yes-associated protein (YAP), has been proven to be involved in this process. Here, we sought to assess the role of YAP in Mfbs differentiation from PDLCs. The time-course expression of YAP and α-SMA was manifested in OTM model in vivo as well as under tensional stimuli in vitro. Inhibition of RhoA/Rho-associated kinase (ROCK) pathway using Y27632 significantly reduced tension-induced Mfb differentiation and YAP expression. Moreover, overexpression of YAP with lentiviral transfection in PDLCs rescued the repression effect of Mfb differentiation induced by Y27632. These data together suggest a crucial role of YAP in regulating tension-induced Mfb differentiation from PDLC interacted with RhoA/ROCK pathway.     

Kinase–substrate Edge Biomarkers Provide A More Accurate Prognostic Prediction in ER-negative Breast Cancer

The estrogen receptor (ER)-negative breast cancer subtype is aggressive with few treatment options available. To identify specific prognostic factors for ER-negative breast cancer, this study included 705,729 and 1034 breast invasive cancer patients from the Surveillance, Epidemiology, and End Results (SEER) and The Cancer Genome Atlas (TCGA) databases, respectively. To identify key differential kinase–substrate node and edge biomarkers between ER-negative and ER-positive breast cancer patients, we adopted a network-based method using correlation coefficients between molecular pairs in the kinase regulatory network. Integrated analysis of the clinical and molecular data revealed the significant prognostic power of kinase–substrate node and edge features for both subtypes of breast cancer. Two promising kinase–substrate edge features, CSNK1A1–NFATC3 and SRC–OCLN, were identified for more accurate prognostic prediction in ER-negative breast cancer patients.     

FHA2 is a plant-specific ISWI subunit responsible for stamen development and plant fertility

Imitation Switch (ISWI) chromatin remodelers are known to function in diverse multi‐subunit complexes in yeast and animals. However, the constitution and function of ISWI complexes in Arabidopsis thaliana remain unclear. In this study, we identified forkhead‐associated domain 2 (FHA2) as a plant‐specific subunit of an ISWI chromatin‐remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. The stamen filament is shorter in the fha2 and rlt1/2 mutants than in the wild type, whereas their pistil lengths are comparable. The shorter filament, which is due to reduced cell size, results in insufficient pollination and reduced fertility. The rlt1/2 mutant shows an early‐flowering phenotype, whereas the phenotype is not shared by the fha2 mutant. Consistent with the functional specificity of FHA2, our RNA‐seq analysis indicated that the fha2 mutant affects a subset of RLT1/2‐regulated genes that does not include genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex and is exclusively involved in regulating stamen development and plant fertility.