Effects of dietary protein contents and habitual endurance exercise on supplemental leucine oxidation in mice

The effects of dietary protein contents and regular exercise on the oxidation of supplemented leucine were examined. In the short-term study, male BALB/cCrSlc mice were fed diets containing 0, 10, 20, 35, and 60% protein: energy ratios for 1 week. In the long-term study, exercised and sedentary mice were fed diets containing 20, 35, and 60% protein ratios for 9 weeks. After the feeding periods, the mice were a bolus administered oral supplements of l-[1-¹³C] leucine. Expired gas was analyzed, and oxidized leucine was expressed as a relative ¹³CO₂/¹²CO₂ ratio. In the short-term study, the peak ¹³CO₂/¹²CO₂ ratio significantly increased with diet protein concentrations. Moreover, the long-term study also showed that the peak ¹³CO₂/¹²CO₂ ratio was significantly increased by high protein diets in both exercised and sedentary mice. Our results indicate that supplemental leucine oxidation is associated with consumption of a high-protein diet, irrespective of exercise status.

Abbreviations: AUC: area under the curve; EX: exercise; RQ: respiratory quotient; SED: sedentary; VO₂/W: oxygen uptake per body weight     

Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.     

Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.     

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.     

Effects of Chronic Administration of Interferon α A/D on Serotonergic Receptors in Rat Brain

The effects of chronic administration of interferon (IFN; recombinant human IFN -A/D) on serotonergic binding sites in rat brain were investigated. IFN was injected daily for 2 weeks at a dose of 100000 I.U./kg, (i.p.) in male Wistar rats. IFN did not alter either [³H]ketanserin binding to 5-HT_2A receptors or [³H]paroxetine binding to 5-HT transporters. Scatchard analysis of [³H]8-hydroxy-dipropylaminotetraline (8-OH-DPAT) binding to 5-HT_1A receptors demonstrated the presence of high- and low-affinity binding sites in both treatment and control groups. IFN significantly increased both Kd and Bmax measures of [³H]8-OH-DPAT binding at low-affinity binding sites, but not at the high-affinity sites. These results suggest that IFN affects the low-affinity 5-HT_1A receptors sites and may be involved in the development of IFN-induced psychiatric disturbances.