Conformations of adducts and kinetics of binding to DNA of the optically pure enantiomers of anti-benzo(a)pyrene diol epoxide

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (-) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (-) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.     

Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting "fibrillar" structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

Zip6-attenuation promotes epithelial-to-mesenchymal transition in ductal breast tumor (T47D) cells

Breast cancer is associated with zinc (Zn) hyper-accumulation in breast tissue which is postulated to be potentiated by the over-expression of Zn importing proteins. Zip6 (LIV-1) over-expression has been documented in estrogen receptor-positive (ER+) breast tumors. Anti-estrogens, such as fulvestrant, are typically prescribed for ER+ breast cancer and thus may play a role in modulating cellular Zn hyper-accumulation. Herein, we investigated the physiological relevance of Zip6 over-expression and the consequences of Zip6-attenuation in breast tumor cells as a mechanism in the development of anti-estrogen resistance. We documented that over-expression of Zip6 was associated with significantly higher cellular Zn levels in tumor cells compared with normal breast cells. Fulvestrant significantly reduced Zn accumulation in tumor cells, without robust effects on Zip6 protein abundance. Zip6-attenuation significantly reduced cellular Zn pools, which was associated with increased mitochondrial membrane potential (ΔΨm) and decreased apoptotic stimuli (cytoplasmic cytochrome C release, caspase- 3 and - 9 activities). Importantly, decreased apoptosis significantly increased tumor colony formation in soft agar and was associated with reduced E-cadherin expression. Our data suggest that anti-estrogen treatment regulates Zn level and importantly verify that Zip6 over-expression is not an underlying mechanism initiating breast cancer, but in fact may play a “tumor-constraining” role.     

Purification of mixed-function amine oxidase from rat liver microsomes

To clarify the metabolism of carcinogenic aminoazo dyes in target tissues, mixed function amine oxidase (MFAO) was purified from rat liver. The MFAO was solubilized from microsomes with Triton X in the presence of 20 glycerol and 1 mM EDTA and purified successively with DEAE Sepharose CL-6B, 2',5'-ADP Sepharose 4B and Hydroxyapatite column chromatography. The purified enzyme yielded a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight was about 59,000. When dimethylaniline (DMA) was used as a substrate, the specific activity of the enzyme fortified with NADPH was about 430 nmol DMA N-oxide formed/mg protein/min with a yield of about 15%. N-Demethylation of dimethylaminoazobenzene (DAB) with the enzyme proceeded only when iron was added to the reaction system.     

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of α-tocopherol and plastoquinone-9 precursors. The biosynthesis of α-tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in α-tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of α-tocopherol in spinach chloroplasts.     

Irreversible inactivation of beta-adrenergic receptors of C6 glioma cells. Synthesis and study of a thiol derivative of propranolol

The beta-adrenergic receptor of C6 glioma cells contains a disulfide bridge which can be reduced by dithiothreitol (DTT). On intact cells, N-ethylmaleimide (NEM) (5 mM) does not change the affinity of [3H] H2-alprenolol ([3H] DHA) but reduces the total number of beta-adrenergic cell receptors by 21 +/- 3 per cent ; (N = 3). After receptor reduction by DTT, NEM irreversibly blocks the accessibility of the beta-adrenergic receptors to [3H]DHA. On isolated membranes, incubation in the presence of either NEM (5 mM) or isoproterenol (5.10(-7) M) does not significantly modify the total number of beta-adrenergic receptors accessible to [3H]DHA. Incubation of membranes with both NEM and isoproterenol reduces the number of binding sites by 33 +/- 2 per cent ; (N = 3). A thiol derivative of propranolol was synthetized. Its affinity is 10 times lower than that of propranolol. This sulfur derivative reduces the total number of beta-adrenergic receptors by 22 +/- 3 per cent (N = 3) when incubated with the native receptor and by 55 +/- 4 per cent (N = 4) when incubated with the reduced receptor. DTT does not significantly reverse the blockade induced by propranolol-SH. A model is proposed for explaining these results.