The use of c-src knockout mice for the identification of the main toxic signaling pathway of TCDD to induce wasting syndrome

The effect of single intraperitoneal injection of 115 microg/kg of TCDD (i.e., approximately 1/2 of LD50) to male C57BL/6 mice on the liver mRNA expression changes of several growth factor related genes was assessed at 3 h, 24 h, 10 days, and 30 days posttreatment. The results revealed that the most consistently elevated mRNAs during the entire test period were those of c-Src, TGFalpha, and PDGFa. In contrast, those observed to be consistently suppressed were mRNAs for EGF receptor (EGFR), Ki-Ras, SAPKK, Sp-1, C/EBPbeta, and NFkB. Elevation of mRNAs for TGFbeta and STAT3 was observed only on day 10 and day 30. To assess the role of c-Src in the above action of TCDD, we conducted a parallel study with congenic C57BL/6 male c-src -/- mice. The results showed that in scr -/- mice the effect of TCDD was less in the case of mRNA expression of PDGF(AA), STAT3, C/EPBbeta, NMT-1, and AP-2gamma in addition to c-src as compared to scr +/+ mice. Those affected least by the absence of c-Src were SAPKK, and surprisingly, EGF receptor mRNAs, both of which were consistently downregulated in both strains. In most of the other cases, the extent of TCDD-induced changes were generally less pronounced in src -/- mice as compared to +/+ mice. These observations support the notion that c-Src is an important mediator of the effects of TCDD on TGFalpha, PDGF(AA), and C/EBPalpha, beta.     

Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric alpha-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast alpha-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A.     

Inhibition of DNA polymerases and DNA topoisomerase II by triterpenes produced by plant callus

We found that some triterpene compounds could not only selectively inhibit the activities of mammalian DNA polymerase alpha (pol alpha) and beta (pol beta), but could also potently inhibit DNA topoisomerase II (topo II) [Biochem. J. 350 (2000) 757]. Here, we report that natural triterpenes produced by callus from an ancient Chinese medicinal plant were also inhibitors of the enzymes, and some were more selective than others. The natural triterpenes with a carboxyl group equally inhibited the activities of pol alpha, pol beta, and topo II, while the olide-type triterpenes with a ketone group suppressed the activities of pol beta and topo II, but not pol alpha. The other triterpenes from the callus hardly influenced these enzyme activities. As also described previously [J. Biochem. 130 (2001) 657], pol beta and topo II have a three-dimensionally similar triterpene-binding region, which is a pocket in which specific compounds can insert. The newly found triterpene inhibitors might structure-dependently insert into the pocket, and the pocket structure of each enzyme might, three-dimensionally but slightly, differ among them. The triterpene frames could be used for screening new inhibitors of the enzymes, and computer-simulated drug design using the frame and pocket structure may in theory be a possible approach to develop new inhibitors.     

P15 and P3, the tail completion proteins of bacteriophage T4, both form hexameric rings

Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.     

Molecular action mode of Hippospongic acid A,an inhibitor of gastrulation of starfish embryos

Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.     

Isolation of chromosaponin I-specific antibody by affinity chromatography

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.     

Neuroactive and other free amino acids in seed and young plants of Panax ginseng

The seeds and one to three years old plants of Asian ginseng (Panax ginseng C.A. Meyer) were analyzed for their free amino acid contents. The neuro-excitatory beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid), suggested to be the cause of the crippling neurolathyrism, was the major component in the seed extract (70% of the total free amino acids detected) and showed the highest concentration (0.43% by wt) compared to that in the different parts of young plants. beta-ODAP concentration was higher in the shoots as compared to roots and declined in older plants. The amount of beta-ODAP in the roots may be considered as an indirect measure of age and quality. Another neuro-active non-protein amino acid, GABA (gamma-aminobutyric acid), increased dramatically after germination and reached highest concentration in different parts of 3 year-old plants. Glutamine and arginine were the two major free proteinogenic amino acids in the ginseng plants and together they constituted over 50% of all the free amino acids detected in the root.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.