Polyphenol (-)-epigallocatechin gallate inhibits apoptosis induced by irradiation in human HaCaT keratinocytes

Green tea is a rich source of polyphenols, and (-)-epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. In the present study, we investigated the effect of EGCG on apoptosis induced by irradiation in the human keratinocytic cell line HaCaT. Irradiation by gamma-ray induced apoptosis with concomitant cleavage of caspase-3 and its in vivo substrate poly(ADP-ribose) polymerase. Treatment of cells with EGCG inhibited irradiation-induced apoptosis as detected by Hoechst staining and internucleosomal cleavage of DNA, and prevented the cleavage of these proteins by irradiation. We also found that the treatment of cells with EGCG alone suppressed cell growth and induced apoptosis in these cells. Our results suggest that EGCG inhibits irradiation-induced apoptosis by inactivating the caspase pathway in HaCaT cells. Our study also indicates that EGCG has a dual effect on the survival of these keratinocytes.     

CD40 ligand rescues inhibitor of differentiation 3-mediated G1 arrest induced by anti-IgM in WEHI-231 B lymphoma cells

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G(1) phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G(1) phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27(Kip1). Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G(1) arrest in WEHI-231 cells.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.     

Kinetics of glial glutamine efflux and the mechanism of neuronal uptake studied in vivo in mildly hyperammonemic rat brain

Kinetics of glial glutamine (GLN) transport to the extracellular fluid (ECF) and the mechanism of GLN(ECF) transport into the neuron--crucial pathways in the glutamine-glutamate cycle--were studied in vivo in mildly hyperammonemic rat brain, by NMR and microdialysis to monitor intra- and extracellular GLN. The minimum rate of glial GLN efflux, determined from the rate of GLN(ECF) increase during perfusion of alpha-(methylamino)isobutyrate (MeAIB), which inhibits neuronal GLN(ECF) uptake by sodium-coupled amino-acid transporter (SAT), was 2.88 +/- 0.22 micromol/g/h at steady-state brain [GLN] of 8.5 +/- 0.8 micromol/g. Our previous study showed that the rate of glutamine synthesis under identical experimental conditions was 3.3 +/- 0.3 micromol/g/h. At steady-state glial [GLN], this is equal to its efflux rate to the ECF. Comparison of the two rates suggests that SAT mediates at least 87 +/- 8% (= 2.88/3.3 x 100%) of neuronal GLN(ECF) uptake. While MeAIB induced > 2-fold elevation of GLN(ECF), no sustained elevation was observed during perfusion of the selective inhibitor of LAT, 2-amino-bicyclo[1,1,2]heptane-2-carboxylic acid (BCH), or of d-threonine, a putative selective inhibitor of ASCT2-mediated GLN uptake. The results strongly suggest that SAT is the predominant mediator of neuronal GLN(ECF) uptake in adult rat brain in vivo.     

Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin in vivo: sensitivity analysis with glycosylation-defective mutant mice

Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.