eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

A New Pathway for Senescence Regulation

<正>Research concerning senescence has become a hotspot since the conception of‘cellular senescence’was put forward by Drs.Hayflick and Moorhead over five decades ago[1].Recently,a paper published in Science by Kang and colleagues,which this article aims to comment on,provides evidence of a new pathway involved in senescence[2].Senescence is a physiological and pathological process induced by numerous factors,during which cell growth ceases     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Mapping QTLs and meta-QTLs for two inflorescence architecture traits in multiple maize populations under different watering environments

Drought significantly affects the architectural development of maize inflorescence, which leads to massive losses in grain yield. However, the genetic mechanism for traits involved in inflorescence architecture in different watering environments, remains poorly understood in maize. In this study, 19 QTLs for tassel primary branch number (TBN) and ear number per plant (EN) were detected in 2 F_2:3 populations under both well-watered and water-stressed environments by single environment mapping with composite interval mapping (CIM); 11/19 QTLs were detected under water-stressed environments. Moreover, 21 QTLs were identified in the 2 F_2:3 populations by joint analysis of all environments with a mixed linear model based on composite interval mapping (MCIM), 11 QTLs were involved in QTL × environment interactions, seven epistatic interactions were identified with additive by additive/dominance effects. Remarkably, 12 stable QTLs (sQTLs) were simultaneously detected by single environment mapping with CIM and joint analysis through MCIM, which were concentrated in ten bins across the chromosomes: 1.05_1.07, 1.08_1.10, 2.01_2.04, 3.01, 4.06, 4.09, 5.06_5.07, 6.05, 7.00, and 7.04 regions. Twenty meta-QTLs (mQTLs) were detected across 19 populations under 51 watering environments using a meta-analysis, and 34 candidate genes were predicted in corresponding mQTLs regions to be involved in the regulation of inflorescence development and drought resistance. Therefore, these results provide valuable information for finding quantitative trait genes and to reveal the genetic mechanisms responsible for TBN and EN under different watering environments. Furthermore, alleles for TBN and EN provide useful targets for marker-assisted selection to generate high-yielding maize varieties.     

High-speed broadband monitoring of cell viscoelasticity in real time shows myosin-dependent oscillations

Study of the dynamic evolutions of cell viscoelasticity is important as during cell activities such as cell metastasis and invasion, the rheological behaviors of the cells also change dynamically, reflecting the biophysical and biochemical connections between the outer cortex and the intracellular structures. Although the time variations of the static modulus of cells have been investigated, few studies have been reported on the dynamic variations of the frequency-dependent viscoelasticity of cells. Measuring and monitoring such dynamic evolutions of cells at nanoscale can be challenging as the measurement needs to meet two objectives inherently contradictory to each other—the measurement must be broadband (to cover a large frequency spectrum) but also rapid (to capture the time-elapsed changes). In this study, we exploited a recently developed control-based nanomechanical protocol of atomic force microscope to monitor in real time the dynamic evolutions of the viscoelasticity of live human prostate cancer cells (PC-3 cells) and study its dependence on myosin activities. We found that the viscoelasticity of PC-3 cells, followed the power law, and oscillated at a period of about 200 s. Both the amplitude and the frequency of the oscillation strongly depended on the intracellular calcium and blebbistatin-sensitive motor proteins.     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).