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.     

Comparative Analysis of Endogenous Hormones and Metabolite Profiles in Early-Spring Flowering Plants and Unflowered Plants Revealing the Strategy of Blossom

Journal of Plant Growth Regulation - Early-spring plants are a special type of plant that complete their life cycle promptly in cold, early spring. Very little effort has been made into researching...     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.     

Crystal structure of a novel ATPase RadD from Escherichia coli

The helicase superfamily 2 (SF2) proteins are involved in essentially every step in DNA and RNA metabolism. The radD (yejH) gene, which belongs to SF2, plays an important role in DNA repair. The RadD protein includes all seven conserved SF2 motifs and has shown ATPase activity. Here, we first reported the structure of RadD from Escherichia coli containing two RecA-like domains, a zinc finger motif, and a C-terminal domain. Based on the structure of RadD and other SF2 proteins, we then built a model of the RedD-ATP complex.     

Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free

Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1-1 kV/cm, 100 μs) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs.