A low-density cDNA microarray with a unique reference RNA: pattern recognition analysis for IFN efficacy prediction to HCV as a model

We have designed and established a low-density (295 genes) cDNA microarray for the prediction of IFN efficacy in hepatitis C patients. To obtain a precise and consistent microarray data, we collected a data set from three spots for each gene (mRNA) and using three different scanning conditions. We also established an artificial reference RNA representing pseudo-inflammatory conditions from established hepatocyte cell lines supplemented with synthetic RNAs to 48 inflammatory genes. We also developed a novel algorithm that replaces the standard hierarchical-clustering method and allows handling of the large data set with ease. This algorithm utilizes a standard space database (SSDB) as a key scale to calculate the Mahalanobis distance (MD) from the center of gravity in the SSDB. We further utilized sMD (divided by parameter k: MD/k) to reduce MD number as a predictive value. The efficacy prediction of conventional IFN mono-therapy was 100% for non-responder (NR) vs. transient responder (TR)/sustained responder (SR) (P < 0.0005). Finally, we show that this method is acceptable for clinical application.     

Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages

The ATP-binding-cassette transporter A1 (ABCA1) plays an essential role in cellular cholesterol efflux and helps prevent macrophages from becoming foam cells. The statins are widely used as cholesterol-lowering agents and have other anti-atherogenic actions. We tested the effects of four different statins (fluvastatin, atorvastatin, simvastatin, and lovastatin) on ABCA1 expression in macrophages in vitro. The statins suppressed ABCA1 mRNA expression in RAW246.7 and THP-1 macrophage cell lines and in mouse peritoneal macrophages. The effect was time- and dose-dependent and was abolished by the addition of the post-reductase product, mevalonate. These findings imply that there is a possible modulation of the well-known beneficial effects of the statins on the reverse cholesterol transport pathway.     

Conditional activation of RhoA suppresses the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation

Gastrulation is a pivotal event of mouse early embryogenesis. In telencephalin (TLCN)-Cre mice carrying the Cre recombinase gene inserted into the translational initiation site of the TLCN gene, Cre-mediated recombination took place at the postimplantation stage. To examine the role of RhoA signaling in early embryogenesis, we produced Rho36 mice carrying constitutively active RhoA(G14V) gene inducible by Cre recombinase and crossed with TLCN-Cre mice. In doubly transgenic embryos at the gastrulation stage, there appeared an abnormal bulge of cells protruded from the primitive streak region into the amniotic cavity. The bulged cell mass expressed the epiblast marker gene Oct3 and E-cadherin, but not the primitive streak marker gene T except for the basal portion. These results suggest that the conditional activation of RhoA signaling suppressed the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.     

Modification of cysteine residue in transthyretin and a synthetic peptide: analyses by electrospray ionization mass spectrometry

Cysteine and cystine in protein are modified to various derivatives in vitro and in vivo. By electrospray ionization mass spectrometry (ESI-MS), we previously found derivatives of serum transthyretin (TTR) in which cysteine residue at position 10 was changed to glycine residue and sulfocysteine residue. The change, cysteine to glycine, was unique and the origin of the sulfonic acid group was controversial. In the present paper, we show the molecular masses of various TTR derivatives including these two, and the modification process was studied using a synthetic peptide with the same sequence as cysteine containing part of TTR, i.e., SKCPLMVK. After incubation of the peptide at pH 8.3, various derivatives were generated, which showed changes of cysteine residue to glycine, dehydroalanine, S-thiocysteine, and S-sulfocysteine residues, which were confirmed by molecular mass and collision-induced dissociation spectra. Dehydroalanine may react with other amino acids and contribute to form cross-linking fibril, causing amyloidosis.     

Cell-matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca(2+) influx and PKC activation

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca(2+) influx or the activation of protein kinase C. Here we show that CD44-mediated cell-matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca(2+) influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca(2+) influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell-matrix interaction and cell migration.     

Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL-RANK interaction

It is well known that thyroid hormone excess causes bone loss. However, the precise mechanism of bone loss by thyroid hormone still remains unclear. When T(3) was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of indomethacin and IL-6 Ab. T(3) increased the expression of osteoprotegerin (OPG) messenger RNA (mRNA), but not of receptor activator of nuclear factor kappaB ligand (RANKL) in unfractionated bone cells, suggesting that the stimulatory effect of T(3) on osteoclast formation was not mediated by the RANKL/OPG system. We next examined the direct effect of T(3) on osteoclast precursors in the absence of osteoblasts, using hemopoietic blast cells derived from spleen cells. T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors. OPG did not inhibit T(3)-induced osteoclast formation from osteoclast precursor cells. The polymerase chain reaction (PCR) product corresponding in size to the mouse T(3) receptor alpha1 cDNA was detected in osteoclast precursors from mouse hemopoietic blast cells as well as mouse heart and mouse osteoblastic cell line MC3T3-E1 cells, suggesting that T(3) directly stimulated osteoclast-like cell formation from osteoclast precursors in the absence of osteoblasts. Further, T(3) increased the expression of c-Fos mRNA at 15 min and 24 h and Fra-1 mRNA at 2 and 6 h in osteoclast precursors. Consistent with the increased expression of c-Fos mRNA observed by RT-PCR, the activation of c-Fos occurred in osteoclast precursor cells stimulated by T(3), while the activation of neither NF-kappaB nor MAPKs was observed by immunoblot analysis. Antisense oligodeoxynucleotides (as-ODN) complementary to c-Fos mRNA at 1 microM significantly inhibited T(3)-induced osteoclast-like cell formation from osteoclast precursors in the absence of stromal cells while sense-ODN did not affect T(3)-induced osteoclast-like cell formation. These results indicate that T(3) directly stimulates osteoclast differentiation at least in part by up-regulation of c-fos protein in osteoclast precursor cells.     

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gbeta13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gbeta13F to the membrane, indicating an essential role of cortical Ggamma1-Gbeta13F signaling in asymmetric divisions. In Ggamma1 and Gbeta13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gbetagamma and other mutants indicates a predominant role of Partner of Inscuteable-Galphai in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.     

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.     

RNA interference directed against Poly(ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells

We established small interfering RNA (siRNA) directed against poly(ADP-ribose) polymerase 1 (PARP-1) that effectively reduces the expression of PARP-1 in two human cell lines. Established siRNA against PARP-1 significantly suppressed human immunodeficiency virus type 1 (HIV-1) replication, as well as the activation of the integrated HIV-1 long terminal repeat promoter. These results indicate that PARP-1 is required for efficient HIV-1 replication in human cells. We propose that PARP-1 may serve as a cellular target for RNA interference-mediated gene silencing to inhibit HIV-1 replication.