Effect of peroxisome proliferators on the methylation and protein level of the c-myc protooncogene in B6C3F1 mice liver

Peroxisome proliferators in general are nongenotoxic mouse liver carcinogens for which DNA hypomethylation and altered gene expression are proposed mechanisms. Therefore, the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D), dibutyl phthalate (DBP), gemfibrozil, and Wy-14,643 were evaluated for the ability to alter the methylation and expression of the c-myc protooncogene. Male B6C3F1 mice were administered for 6 days in their diet Wy-14,643 (5-500 ppm), 2,4-D (1,680 ppm), DBP (20,000 ppm), or gemfibrozil (8,000 ppm). All four peroxisome proliferators caused hypomethylation of the c-myc gene in the liver. Wy-14,643 appeared to be the most efficacious with a threshold between 10 and 50 ppm. The level of the c-myc protein was increased by Wy-14,643, but not the other peroxisome proliferators. When female B6C3F1 mice received a two-thirds partially hepatectomy and 16 h later were administered 50 mg/kg Wy-14,643 by gavage, hypomethylation of the gene occurred 24 h later. Hypomethylation was not found in mice that received Wy-14,643 following a sham operation. Hypomethylation of the c-myc gene within 24 h of administering Wy-14,643 after a partial hepatectomy but not after a sham operation supports the hypothesis that the peroxisome proliferators prevent methylation of hemimethylated sites formed by DNA replication.     

癫痫发作敏感大鼠前深梨状皮层T区GABA免疫反应活性变化

γ-氨基丁酸(γ-aminobutyric acid,GABA)是脑内最重要的抑制性神经递质,在阻断兴奋扩布及传导中起重要作用,其参与抗癫痫作用已被证实.有研究表明:前深梨状皮层T区是颞叶癫痫的始动部位.我们从前的工作已证明:惊厥剂量的红藻氨酸(Kainic acid,A)诱发SD大鼠出现急性癫痫发作后的5～7 d,动物出现癫痫发作敏感性长期增强,而蝎毒(scorpion venom,V)可通过增强海马结构内GABA的表达,对抗其癫痫发作敏感性的形成.本研究采用免疫组化技术,并探讨其与癫痫发作敏感长期增强的可能关系,检测癫痫发作敏感大鼠和经SV处理后癫痫发作敏感性明显降低的大鼠前深梨状皮层T区的GABA免疫反应活性变化.     

The toxicity test and hypothetical model ofBacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.     

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Interactions between heme d and heme b595 in quinol oxidase bd from Escherichia coli: a photoselection study using femtosecond spectroscopy

Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O(2)-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b(595) on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b(595) allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b(595) transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b(595), causing induction/loss of an absorption band centered at 435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b(595) relative to the static spectrum. No evidence for transient binding of CO to heme b(595) after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of < 15 ps is found to be diminished more than 3-fold when heme b(595) is oxidized rather than reduced. In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.