Genetic Variants in Hormone-Related Genes and Risk of Breast Cancer

Sex hormones play a key role in the development of breast cancer. Certain polymorphic variants (SNPs and repeat polymorphisms) in hormone-related genes are associated with sex hormone levels. However, the relationship observed between these genetic variants and breast cancer risk has been inconsistent. We conducted a case-control study nested within two prospective cohorts to assess the relationship between specific genetic variants in hormone-related genes and breast cancer risk. In total, 1164 cases and 2111 individually-matched controls were included in the study. We did not observe an association between potential functional genetic polymorphisms in the estrogen pathway, SHBG rs6259, ESR1 rs2234693, CYP19 rs10046 and rs4775936, and UGT1A1 rs8175347, or the progesterone pathway, PGR rs1042838, with the risk of breast cancer. Our results suggest that these genetic variants do not have a strong effect on breast cancer risk.     

The ferrous-dioxygen intermediate in human cytochrome P450 3A4. Substrate dependence of formation and decay kinetics

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of approximately 30 ms at 5 degrees C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.     

Temporally extended gene expression of the ADP-Glc pyrophosphorylase large subunit (AgpL1) leads to increased enzyme activity in developing tomato fruit

Tomato plants (Solanum lycopersicum) harboring the allele for the AGPase large subunit (AgpL1) derived from the wild species Solanum habrochaites (AgpL1 ^H ) are characterized by higher AGPase activity and increased starch content in the immature fruit, as well as higher soluble solids in the mature fruit following the breakdown of the transient starch, as compared to fruits from plants harboring the cultivated tomato allele (AgpL1 ^E ). Comparisons of AGPase subunit gene expression and protein levels during fruit development indicate that the increase in AGPase activity correlates with a prolonged expression of the AgpL1 gene in the AgpL1 ^H high starch line, leading to an extended presence of the L1 protein. The S1 (small subunit) protein also remained for an extended period of fruit development in the AgpL1 ^H fruit, linked to the presence of the L1 protein. There were no discernible differences between the kinetic characteristics of the partially purified AGPase-L1^E and AGPase-L1^H enzymes. The results indicate that the increased activity of AGPase in the AgpL1 ^H tomatoes is due to the extended expression of the regulatory L1 and to the subsequent stability of the heterotetramer in the presence of the L1 protein, implying a role for the large subunit not only in the allosteric control of AGPase activity but also in the stability of the AGPase L1–S1 heterotetramer. The introgression line of S. lycopersicum containing the wild species AgpL1 ^H allele is a novel example of transgressive heterosis in which the hybrid multimeric enzyme shows higher activity due to a modulated temporal expression of one of the subunits.     

The one-electron autoxidation of human cytochrome P450 3A4

Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.     

Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4+ T Cells

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4⁺ T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4⁺ T cells, and latent infection in those cells may arise when infected CD4⁺ T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4⁺ T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo. Our study proposes a new mechanism for infection of resting CD4⁺ T cells in vivo and a new mechanism for latent infection in resting CD4⁺ T cells.     

Mechanism of pyocyanin- and 1-hydroxyphenazine-induced lung neutrophilia in sheep airways

Pyocyanin (Pyo)and 1-hydroxyphenazine (1-HP) are extracellular products ofPseudomonas aeruginosa. To testwhether these products were capable of producing an inflammatoryresponse in the airways, combinations of Pyo and 1-HP at concentrationsof 10⁴ and10⁵ M were instilled intosheep airways, and indexes of inflammation were assessed bybronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazinescaused a significant dose-dependent increase in the number of cells andneutrophils recovered by BAL. Control challenges produced no suchchanges. The lung neutrophilia was accompanied by an increasedconcentration of albumin in BAL. The increases in BAL neutrophils andalbumin could be blocked by treating the sheep with the 5-lipoxygenaseinhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophilswhen tested in vitro, but when alveolar macrophages (AM) were culturedin vitro in the presence of both Pyo and 1-HP (1 µM), thesupernatants caused neutrophil chemotaxis. Analysis of AM culturesupernatants incubated with the combination of pigments showedsignificant increases in leukotrieneB₄ and interleukin-8, and blockingthese mediators separately or together reduced AM supernatant-inducedneutrophil chemotaxis. We conclude that local instillation of Pyo and1-HP can initiate an inflammatory response in the airways of sheep invivo. This effect can be explained, in part, by the release ofchemotactic factors produced by AM.

     

Substrate Selection for Fundamental Studies of Electrocatalysts and Photoelectrodes: Inert Potential Windows in Acidic,Neutral, and Basic Electrolyte

The selection of an appropriate substrate is an important initial step for many studies of electrochemically active materials. In order to help researchers with the substrate selection process, we employ a consistent experimental methodology to evaluate the electrochemical reactivity and stability of seven potential substrate materials for electrocatalyst and photoelectrode evaluation. Using cyclic voltammetry with a progressively increased scan range, we characterize three transparent conducting oxides (indium tin oxide, fluorine-doped tin oxide, and aluminum-doped zinc oxide) and four opaque conductors (gold, stainless steel 304, glassy carbon, and highly oriented pyrolytic graphite) in three different electrolytes (sulfuric acid, sodium acetate, and sodium hydroxide). We determine the inert potential window for each substrate/electrolyte combination and make recommendations about which materials may be most suitable for application under different experimental conditions. Furthermore, the testing methodology provides a framework for other researchers to evaluate and report the baseline activity of other substrates of interest to the broader community.