Analysis of gene expression profile in p130(Cas)-deficient fibroblasts

p130(Cas) (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 alpha 1, procollagen 3 alpha 1, procollagen 11 alpha 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression.     

Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.     

Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli

Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.     

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi’s sarcoma-associated herpesvirus latent replication

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/−) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3^low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.     

Development and validation of a high performance liquid chromatography-tandem mass spectrometry for the determination of etodolac in human plasma

A simple and specific method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C(18) column with 65% acetonitrile and 35% water containing 10mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99>172.23 for etodolac and m/z 357.92>139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1microg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.