2,3,7,8-tetrachlorodibenzo-p-dioxin mechanism of action to reduce lipoprotein lipase activity in the 3T3-L1 preadipocyte cell line

Lipoprotein lipase (LPL) is important in the process of triglyceride storage in adipose tissue. Depression of LPL activity in adipose tissue is associated with 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD)-induced wasting syndrome and may have a role in the associated serum hyperlipidemia produced by TCDD. The 3T3-L1 cell line was used as an in vitro model, independent of hormonal, nutritional, or other interfering factors associated with in vivo studies, in order to systematically examine the mechanism of action of TCDD. TCDD produced a statistically significant (P < 0.05) time- and dose-dependent decrease in LPL activity. Results of experiments with Ah-receptor blockers and structure activity studies with different polychlorinated biphenyl (PCB) and dioxin congeners were consistent with reduction of LPL activity being mediated by the Ah receptor. Culturing of 3T3-L1 cells without glucose or with cytochalasin B, a blocker of facilitative glucose transporters (GLUT), was effective in reducing LPL activity (P < 0.05). TCDD did not further reduce LPL activity in cytochalasin B pretreated 3T3-L1 cells or in 3T3-L1 cells cultured in glucose-free media. Dexamethasone pretreatment, which is known to increase GLUT expression in 3T3-L1 cells, prevented the reduction of LPL activity by TCDD. Protein tyrosine kinase activities, assayed using γ-³²P-ATP and RR-SRC, a src specific peptide substrate, were significantly increased (P < 0.05) over control levels by both TCDD and glucose deprivation. Furthermore, results of experiments treating 3T3-L1 cells with either insulin, EGF, 8-Br-cAMP, TPA, or genistein, alone or in combination with TCDD, were generally consistent with the hypothesis that lowered intracellular glucose and altered cellular kinase activities may be involved in reduction of LPL activities by TCDD. Further work is needed to confirm and better understand the role protein phosphorylation plays in TCDD-mediated alteration of glucose disposition and LPL activity. In summary, TCDD reduced LPL activity in 3T3-L1 cells as seen in vivo. Manipulation of glucose transport through a number of experimental approaches produced changes in 3T3-L1 LPL activity consistent with results of previous investigators showing glucose to be a positive regulator of LPL activity and consistent with our hypothesis that TCDD-mediated reduction of glucose transport is an important factor in the down regulation of LPL activity by TCDD. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 29–39, 1998     

Hemagglutinating activity of the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

Abstract The hemagglutinating activity of the heat-labile enterotoxin (LT_p) isolated from porcine enterotoxigenic Escherichia coli was studied by hemagglutination inhibition. The hemagglutinating activity of LT_p was enhanced 64–512-fold with pronase- and neuraminidase-treated human erythrocytes although both intact human and sheep erythrocytes were not agglutinated by LT_p at the highest concentration used. No enhancement was found in hemagglutination of neuraminidase-treated sheep erythrocytes by LT_p. Hemagglutination of pronase-treated human type A erythrocytes induced by LT_p was inhibited by melibiose and galactose among mono-, di-, and polysaccharides used as inhibitors. Galactose was a slightly better inhibitor than melibiose. These findings suggest that LT_p is a bacterial lectin specific for galactose.     

DNA replication in vivo of linear DNA killer plasmids pGKL1 and pGKL2 in Saccharomyces cerevisiae

Abstract Electron microscopic observation demonstrated that linear DNA plasmids, pGKL1 and pGKL2, were replicated by a strand displacement mechanism similar to adenovirus and Bacillus subtilis ø 29 phage. Moreover, their DNA replication was prevented by α-factor, a mating hormone which prevents the replication of chromosomal DNA and 2 μm plasmid in Saccharomyces cerevisiae mating type a cells. This result suggests that the replication of pGKL plasmids is controlled by the same genes that control the initiation or maintenance of chromosomal DNA and 2 μm plasmid replications.     

Isolation and characterization of enterotoxigenic Escherichia coli mutants that produce abnormal heat-labile enterotoxins

Abstract Bacteroides fragilis populations were separated according to the size of surface structure. Subculture of the separated populations produced cultures enriched for 3 different structures; a large capsule, a small capsule and an electron-dense layer (EDL). The ability of these subpopulations to haemagglutinate (HA) erythrocytes from a number of species was examined. Populations which produced either a large or s small capsule did not have HA activity, whereas those with an extracellular EDL did. By mixing populations with EDL and those with either the large or small capsule, the degree of HA could be altered. HA was dependent on the proportion of EDL-bearing bacteria present. Fimbriae were not observed on electron microscopy.     

The linker of calmodulin lacking Glu84 is elongated in solution,although it is bent in the crystal

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 Å and 62.5 Å, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 Å and 55.0 Å, and smaller than those expected from the crystal structure of wild type, 22.8 Å and 67.5 Å. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether. © 1996 Wiley-Liss, Inc.     

K+ Excretion: The Other Purpose for Puddling Behavior in Japanese Papilio Butterflies

To elucidate the purpose of butterfly puddling, we measured the amounts of Na⁺, K⁺, Ca²⁺, and Mg²⁺ that were absorbed or excreted during puddling by male Japanese Papilio butterflies through a urine test. All of the butterflies that sipped water with a Na⁺ concentration of 13 mM absorbed Na⁺ and excreted K⁺, although certain butterflies that sipped solutions with high concentrations of Na⁺ excreted Na⁺. According to the Na⁺ concentrations observed in naturally occurring water sources, water with a Na⁺ concentration of up to 10 mM appears to be optimal for the health of male Japanese Papilio butterflies. The molar ratio of K⁺ to Na⁺ observed in leaves was 43.94 and that observed in flower nectars was 10.93. The Na⁺ amount in 100 g of host plant leaves ranged from 2.11 to 16.40 mg, and the amount in 100 g of flower nectar ranged from 1.24 to 108.21 mg. Differences in host plants did not explain the differences in the frequency of puddling observed for different Japanese Papilio species. The amounts of Na⁺, K⁺, Ca²⁺, and Mg²⁺ in the meconium of both male and female butterflies were also measured, and both males and females excreted more K⁺ than the other three ions. Thus, the fluid that was excreted by butterflies at emergence also had a role in the excretion of the excessive K⁺ in their bodies. The quantities of Na⁺ and K⁺ observed in butterfly eggs were approximately 0.50 μg and 4.15 μg, respectively; thus, female butterflies required more K⁺ than male butterflies. Therefore, female butterflies did not puddle to excrete K⁺. In conclusion, the purpose of puddling for male Papilio butterflies is not only to absorb Na⁺ to correct deficiencies but also to excrete excessive K⁺.     

The tail structure of bacteriophage T4 and its mechanism of contraction

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.     

Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.     

Taxol derivatives are selective inhibitors of DNA polymerase alpha

During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase alpha (pol.alpha) and beta (pol.beta). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.alpha and beta. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.alpha activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.beta inhibitors. With both pol.alpha and beta, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.