Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.     

Diversity of pituitary cells in primary cell culture. An immunocytochemical study

A bioassay system for rapid detection of carcinogenic agents has been developed using male Fischer 344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system, called the medium-term liver bioassay, is fundamentally based on the 2-stage hypothesis of tumor production, employing initiation by diethylnitrosamine (200 mg/kg, i.p.) in the first stage and test chemical administration during the second, in combination with two-thirds partial hepatectomy. It requires only 8 wk for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 291 chemicals/substances have already been analyzed in our laboratory. Among 63 chemicals that were proved to be carcinogenic in the liver of rat and/or mouse, 57 (90%) gave positive results irrespective of their mutagenicity. Negative compounds include peroxisome proliferators and tamoxifen. Even nonhepatocarcinogens were positive at a rate of 24%. Eighty-six percent (12/14) of mouse liver carcinogens were also positive. On the other hand, only 2 out of 45 noncarcinogens showed very weak positivity. Thus, the efficacy of the system for hepatocarcinogens has been well established. This bioassay is increasingly regarded as an appropriate alternative test for carcinogenicity risk assessment and is practically used for a rapid evaluation of hepatocarcinogenicity of chemicals.     

Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.     

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system

The hypothesis of Geisler (Brain Res. 212 (1981) 198-201), in which the different spontaneous-rate classes of primary auditory neurons were accounted for by the different sizes of uniquantal EPSPs relative to the gap between resting membrane and threshold potentials, was represented with an expanded model which included relative refractory effects. The spike rates generated by the expanded model, when plotted vs. estimated sound level, are qualitatively similar to those of experimentally obtained rate-level curves. The hypothesis is also consistent with recent ultrastructural data which suggest that average quantal-release rates for any particular primary auditory neuron are inversely related to its spontaneous rate. The model's recovery processes following spike generation (hazard functions) are also similar to those observed experimentally.     

Major internal nuclear matrix proteins are common to different human cell types

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.     

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.     

Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity

The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 2 x 10(-5) to 1 x 10(-3).     

Inhibiting and deactivating effects of water on the selective catalytic reduction of nitric oxide with ammonia over MnOx/Al2O3

The effect of water on the selective catalytic reduction (SCR) of nitric oxide with ammonia over alumina supported with 2–15 wt.-% manganese oxide was investigated in the temperature range 385–600 K, with the emphasis on the low side of this temperature window. Studies on the effect of 1–5 vol.-% water vapour on the SCR reaction rate and selectivity were combined with TPD experiments to reveal the influence of water on the adsorption of the single SCR reactants. It turned out that the activity decrease due to water addition can be divided into a reversible inhibition and an irreversible deactivation. Inhibition is caused by molecular adsorption of water. TPD studies showed that water can adsorb competitively with both ammonia and nitric oxide. Additional kinetic experiments revealed that adsorbed ammonia is present in excess on the catalyst surface, even in the presence of water. Reduced nitric oxide adsorption is responsible for the observed reversible decrease in the reaction rate; the fractional reaction order changes from 0.79 in the absence of water to 1.07 in its presence. Deactivation is probably due to the dissociative adsorption of water, resulting in the formation of additional surface hydroxyls. As the amount of surface hydroxyls formed is limited to a saturation level, the deactivating effect on the catalyst is limited too. The additional hydroxyls condense and desorb in the temperature range 525–775 K, resulting in a lower degree of deactivation at higher temperature. A high temperature treatment at 775 K results in a complete regeneration. The amount of surface hydroxyls formed per unit surface area decreases at increasing MnO_x-loading. The selectivity to the production of nitrogen is enhanced significantly by the presence of gas phase water.     

Nitric oxide synthase-immunoreactive neurons in human and porcine respiratory tract

The presence of nitric oxide synthase (NO-synthase), the enzyme responsible for the production of nitric oxide (NO) from L-arginine, is shown immunocytochemically in the intrinsic neurons of the human and porcine respiratory tract. NO-synthase immunoreactivity is demonstrated in a subpopulation of neurons of the microganglia present in the wall of the extra- and intrapulmonary bronchi as well as in the hilar region of the lung in relation to blood vessels. The immunostaining was also found in some nerve fibers of the respiratory nervous system. Human and porcine lung gave similar results. The possible involvement of NO in the nonadrenergic noncholinergic (NANC) nervous regulation of the lung is discussed.