Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.     

Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection.

A total of 276 sequential serum samples from 34 men with antibodies to the human immunodeficiency virus (HIV) followed up for two to seven years were analysed for HIV antigen and antibodies to the viral core and envelope proteins. Results were correlated with clinical outcome and CD4 T lymphocyte count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients with antigenaemia compared with one out of 18 patients without antigenaemia. Low counts of CD4 cells (less than 0.5 X 10(9)/l) were found in 14 of the 16 patients with antigenaemia and five of the 18 without antigenaemia. Nine patients seroconverted to HIV during the study; two of these developed antigenaemia 14 and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test is simple to do and interpret, it may therefore be useful for selecting patients for antiviral treatment.     

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.     

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.     

Fine‐scale variation within urban landscapes affects marking patterns and gastrointestinal parasite diversity in red foxes

1. Urban areas are often considered to be a hostile environment for wildlife as they are highly fragmented and frequently disturbed. However, these same habitats can contain abundant resources, while lacking many common competitors and predators. The urban environment can have a direct impact on the species living there but can also have indirect effects on their parasites and pathogens. To date, relatively few studies have measured how fine‐scale spatial heterogeneity within urban landscapes can affect parasite transmission and persistence.
2. Here, we surveyed 237 greenspaces across the urban environment of Edinburgh (UK) to investigate how fine‐scale variation in socio‐economic and ecological variables can affect red fox (Vulpes vulpes) marking behavior, gastrointestinal (GI) parasite prevalence, and parasite community diversity.
3. We found that the presence and abundance of red fox fecal markings were nonuniformly distributed across greenspaces and instead were dependent on the ecological characteristics of a site. Specifically, common foraging areas were left largely unmarked, which indicates that suitable resting and denning sites may be limiting factor in urban environments. In addition, the amount of greenspace around each site was positively correlated with overall GI parasite prevalence, species richness, and diversity, highlighting the importance of greenspace (a commonly used measure of landscape connectivity) in determining the composition of the parasite community in urban areas.
4. Our results suggest that fine‐scale variation within urban environments can be important for understanding the ecology of infectious diseases in urban wildlife and could have wider implication for the management of urban carnivores.

     

Nongenomic effects of thyroid hormones on the immune system cells: New targets, old players

It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvβ3 has been identified as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors. Through the integrin αvβ3 receptor the hormone can activate both the ERK1/2 and phosphatidylinositol 3-kinase pathways, with downstream effects including intracellular protein trafficking, angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New results demonstrate the capability of T(3) or T(4) to induce in the short time range important responses related to the immune function, such as reactive oxygen species production and cell migration in THP-1 monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of rapid nongenomic responses interacting with the classical nuclear response.     

Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.     

Removal of toluene in waste gases using a biological trickling filter

The removal of toluene from waste gas was studied in a trickling biofilter. A high level of water recirculation (4.7 m h^–1) was maintained in order to keep the liquid phase concentration constant and to achieve a high degree of wetting. For loads in the range from 6 to 150 g m^–3 h^–1 the maximum volumetric removal rate (elimination capacity) was 35±10 g m^–3 h^–1, corresponding to a zero order removal rate of 0.11±0.03 g m^–2 h^–1 per unit of nominal surface area. The surface removal was zero order above the liquid phase concentrations of approximately 1.0 g m^–3, corresponding to inlet gas concentrations above 0.7–0.8 g m^–3. Below this concentration the surface removal was roughly of first order. The magnitude of the first order surface removal rate constant, k_1A , was estimated to be 0.08–0.27 m h^–1 (k_1A a=24–86 h^–1). Near-equilibrium conditions existed in the gas effluent, so mass transfer from gas to liquid was obviously relatively fast compared to the biological degradation. An analytical model based on a constant liquid phase concentration through the trickling filter column predicts the effluent gas concentration and the liquid phase concentration for a first and a zero order surface removal. The experimental results were in reasonable agreement with a very simple model valid for conditions with an overall removal governed by the biological degradation and independent of the gas/liquid mass transfer. The overall liquid mass transfer coefficient, K_La, was found to be a factor 6 higher in the system with biofilm compared to the system without. The difference may be explained by: 1. Difference in the wetting of the packing material, 2. Mass transfer occurring directly from the gas phase to the biofilm, and 3. Enlarged contact area between the gas phase and the biofilm due to a rough biofilm surface.