Comparison of the Uptake and Metabolism of Retinol Delivered to Primary Mouse Keratinocytes Either Free or Bound to Rat Serum Retinol-binding Protein

Serum retinol-binding protein (RBP) is believed to be responsible for the transport of retinol from its storage site in the liver to vitamin A requiring target cells such as keratinocytes. We have used primary mouse keratinocytes as a model system to compare the uptake and metabolism of [3H] retinol delivered to them either free in solution or bound to RBP. RBP was purified from rat serum, loaded with [3H]retinol, and the [3H]retinol-RBP complex purified by affinity chromatography on human transthyretin-Sepharose. Keratinocytes incubated with either free [3H]retinol or [3H]retinal-RBP complex accumulated [3H]retinol in a time and temperature dependent manner, However, cells incubated with free [3H]retinol acquired 15- to 20-fold more ligand than if the retinol was delivered via RBP. The uptake of free [3H]retinol or [3H]retinol from RBP was not inhibited by excess unlabeled free retinol. The uptake of [3H]retinol from RBP was inhibited by high concentrations of holo-RBP, with half maximal inhibition occurring at 3μM holo-RBP. However, no specific binding of 125I-labeled RBP to monolayers of keratinocytes or membranes prepared from them was found indicating the absence of a high affinity RBP receptor on keratinocytes. Surprisingly, 50% of the [3H]retinol delivered to the keratinocytes during a 30-min uptake period was released from them within 30-min irrespective of whether or not it was initially delivered to them as free [3H]retinol or bound to RBP. The remaining 50% was lost at a much slower rate, but only 20% remained 24-h after delivery. Studies on retinol metabolism demonstrated that 7%12% of the total cell-associated [3H]retinol delivered during a 90-min uptake period was esterified (mostly as retinyl palmitate) whether or not it was given free in solution or bound to RBP

Retinoic Acid Resistance at Late Stages of Human Papillomavirus Type 16-Mediated Transformation of Human Keratinocytes Arises Despite Intact Retinoid Signaling and Is Due to a Loss of Sensitivity to Transforming Growth Factor-β

AbstractIn our in vitro model of human cell carcinogenesis, normal human foreskin keratinocytes (HKc) transfected with human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through several phenotypically defined and reproducible “steps” that include immortalization, growth factor independence (HKc/GFI), differentiation resistance (HKc/DR), and ultimately malignant conversion. While HKc/HPV16 are very sensitive to growth inhibition by all-trans-retinoic acid (RA) at early passages, they lose their sensitivity to RA during progression in culture. However, gel mobility shift assays using the retinoid response elements DR1 and DR5 showed no changes in binding activity of nuclear extracts obtained from HKc/HPV16 at different stages of in vitro progression. Similarly, Western blot analyses for retinoic acid receptor γ-1 and the retinoid X receptors failed to reveal any decreases in the levels of these retinoid receptors throughout progression. In addition, luciferase activity driven by the SV40 promoter with a DR5 enhancer element was activated following RA treatment of HKc/DR that were resistant to growth inhibition by RA. Since RA induces transforming growth factor-β2 (TGF-β2) in normal HKc and HKc/HPV16, we investigated whether this response changed during progression. Again, RA induced TGF-β2 mRNA in early and late passage HKc/HPV16, HKc/GFI, and HKc/DR approximately to the same extent, confirming that the RA signaling pathways remained intact during in vitro progression despite the fact that the cells become resistant to growth inhibition by RA. We then investigated the sensitivity of HKc/HPV16 to growth inhibition by TGF-β. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-β1 and TGF-β2, the cells became increasingly resistant to both TGF-β isotypes during in vitro progression. In addition, while both RA and TGF-β produced a decrease in the levels of mRNA for the HPV16 oncogenes E6 and E7 in early passage HKc/HPV16, this effect was also lost at later stages of progression. Finally, blocking anti-TGF-β antibodies partially prevented RA inhibition of growth and E6/E7 expression in early passage HKc/HPV16. Taken together, these data strongly suggest that inhibition of growth and HPV16 early gene expression in HKc/HPV16 by RA is mediated by TGF-β and that a loss of RA sensitivity is linked to TGF-β resistance rather than alterations in RA signaling

LC–MS-based absolute metabolite quantification:Application to metabolic flux measurement in trypanosomes

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself

Analytical studies of Hawking radiation and quasinormal modes in rotating linear dilatonic black hole

The rotating linear dilatonic black hole is an asymptotically non-flat solution to Einstein-Maxwell-Dilaton-Axion gravity theory due to the existence of non-trivial matter fields. We have analytically studied the wave equation of scalar field in this background and shown that the radial wave equation can be solved in terms of hypergeometric function. By determining the ingoing and the outgoing fluxes at the asymptotic infinity, we have found the analytical expressions for reflection coefficient and greybody factor for certain scalar modes. In the high frequency regime, we obtain the Hawking temperature by comparing the blackbody spectrum with the radiation spectrum resulting from reflection coefficient. It is shown that the Hawking temperature, which depends only on the linear dilatonic background parameter, does not agree with the temperature calculated from surface gravity. At last, the quasinormal modes of scalar field perturbation are presented, which shows that the rotating linear dilationic black hole is unstable for certain modes apart from the superradiant modes.Comment: 7 pages, 2 figures Comments are welcom

Lack of Knowledge of HIV Status a Major Barrier to HIV Prevention, Care and Treatment Efforts in Kenya: Results from a Nationally Representative Study

BACKGROUND: We analyzed HIV testing rates, prevalence of undiagnosed HIV, and predictors of testing in the Kenya AIDS Indicator Survey (KAIS) 2007. METHODS: KAIS was a nationally representative sero-survey that included demographic and behavioral indicators and testing for HIV, HSV-2, syphilis, and CD4 cell counts in the population aged 15-64 years. We used gender-specific multivariable regression models to identify factors independently associated with HIV testing in sexually active persons. RESULTS: Of 19,840 eligible persons, 80% consented to interviews and blood specimen collection. National HIV prevalence was 7.1% (95% CI 6.5-7.7). Among ever sexually active persons, 27.4% (95% CI 25.6-29.2) of men and 44.2% (95% CI 42.5-46.0) of women reported previous HIV testing. Among HIV-infected persons, 83.6% (95% CI 76.2-91.0) were unaware of their HIV infection. Among sexually active women aged 15-49 years, 48.7% (95% CI 46.8-50.6) had their last HIV test during antenatal care (ANC). In multivariable analyses, the adjusted odds ratio (AOR) for ever HIV testing in women ≥35 versus 15-19 years was 0.2 (95% CI: 0.1-0.3; p<0.0001). Other independent associations with ever HIV testing included urban residence (AOR 1.6, 95% CI: 1.2-2.0; p = 0.0005, women only), highest wealth index versus the four lower quintiles combined (AOR 1.8, 95% CI: 1.3-2.5; p = 0.0006, men only), and an increasing testing trend with higher levels of education. Missed opportunities for testing were identified during general or pregnancy-specific contacts with health facilities; 89% of adults said they would participate in home-based HIV testing. CONCLUSIONS: The vast majority of HIV-infected persons in Kenya are unaware of their HIV status, posing a major barrier to HIV prevention, care and treatment efforts. New approaches to HIV testing provision and education, including home-based testing, may increase coverage. Targeted interventions should involve sexually active men, sexually active women without access to ANC, and rural and disadvantaged populations