Dynamical systems analysis of arterial blood pressure signals in relation to heart rate fluctuations in chick embryos

We attempted a new approach based on a modern dynamical system theory to reconstruct the arterial blood pressure signals in relation to heart rate fluctuations of developing chick embryos. The dynamical systems approach in general is to model a phenomenon that is presented by a single time series record and approximate the dynamical property (e.g. heart rate fluctuations) of a system based only on information contained in a single-variable (arterial blood pressure) of the system. The time-series data of the arterial blood pressure was reconstructed in 3-dimensional space to draw characteristic orbits. Since the reconstructed orbits of the blood pressure should retain information contained in the pressure signals, we attempted to derive instantaneous heart rate (IHR) from the reconstructed orbits. The derived IHR presenting HR fluctuations coincided well with the IHR obtained conventionally from the peak-to-peak time intervals of the maximum blood pressure. Movements of the reconstructed orbits of the arterial blood pressure in 3-dimensional space reflected HR fluctuations (i.e. transient decelerations and accelerations).     

Mechanisms for resistance to anticancer agents and the reversal of the resistance

MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps. Lung resistance related protein (LRP) is also supposed to be involved in MDR. The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated. cMOAT is homologous to MRP1 and supposed to be involved in drug resistance. Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin. The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT. Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol. Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype. The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells. ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells. The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody. Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme. The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells. These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.     

Strand asymmetry of +1 frameshift mutagenesis at a homopolymeric run by DNA polymerase III holoenzyme of Escherichia coli

We have recently shown that single-base frameshifts were predominant among mutations induced within the rpsL target sequence upon oriC plasmid DNA replication in vitro. We found that the occurrence of +1 frameshifts at a run of 6 residues of dA/dT could be increased proportionally by increasing the concentration of dATP present in the in vitro replication. Using single-stranded circular DNA containing either the coding sequence of the rpsL gene or its complementary sequence, the +1 frameshift mutagenesis by DNA polymerase III holoenzyme of Escherichia coli was extensively examined. A(6) --> A(7) frameshifts occurred 30 to 90 times more frequently during DNA synthesis with the noncoding sequence (dT tract) template than with the coding sequence (dA tract). Excess dATP enhanced the occurrence of +1 frameshifts during DNA synthesis with the dT tract template, but no other dNTPs showed such an effect. In the presence of 0.1 mM dATP, the A(6) --> A(7) mutagenesis with the dT tract template was not inhibited by 1.5 mM dCTP, which is complementary to the residue immediately upstream of the dT tract. These results strongly suggested that the A(6) --> A(7) frameshift mutagenesis possesses an asymmetric strand nature and that slippage errors leading to the +1 frameshift are made during chain elongation within the tract rather than by misincorporation of nucleotides opposite residues next to the tract.     

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.     

Establishment and characterization of a new human glioblastoma cells line, NYGM

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.     

Simultaneous monitoring of acetylcholine and catecholamine release in the in vivo rat adrenal medulla

To simultaneously monitor acetylcholine release from pre-ganglionic adrenal sympathetic nerve endings and catecholamine release from post-ganglionic adrenal chromaffin cells in the in vivo state, we applied microdialysis technique to anesthetized rats. Dialysis probe was implanted in the left adrenal medulla and perfused with Ringer's solution containing neostigmine (a cholinesterase inhibitor). After transection of splanchnic nerves, we electrically stimulated splanchnic nerves or locally administered acetylcholine through dialysis probes for 2 min and investigated dialysate acetylcholine, choline, norepinephrine and epinephrine responses. Acetylcholine was not detected in dialysate before nerve stimulation, but substantial acetylcholine was detected by nerve stimulation. In contrast, choline was detected in dialysate before stimulation, and dialysate choline concentration did not change with repetitive nerve stimulation. The estimated interstitial acetylcholine levels and dialysate catecholamine responses were almost identical between exogenous acetylcholine (10 microM) and nerve stimulation (2 Hz). Dialysate acetylcholine, norepinephrine and epinephrine responses were correlated with the frequencies of electrical nerve stimulation, and dialysate norepinephrine and epinephrine responses were quantitatively correlated with dialysate acetylcholine responses. Neither hexamethonium (a nicotinic receptor antagonist) nor atropine (a muscarinic receptor antagonist) affected the dialysate acetylcholine response to nerve stimulation. Microdialysis technique made it possible to simultaneously assess activities of pre-ganglionic adrenal sympathetic nerves and post-ganglionic adrenal chromaffin cells in the in vivo state and provided quantitative information about input-output relationship in the adrenal medulla.     

FORTE: a profile-profile comparison tool for protein fold recognition

We present FORTE, a profile-profile comparison tool for protein fold recognition. Users can submit a protein sequence to explore the possibilities of structural similarity existing in known structures. Results are reported via email in the form of pairwise alignments.     

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.