Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.     

Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)

Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.     

Rosiglitazone treatment improves cardiac efficiency in hearts from diabetic mice

Isolated perfused hearts from type 2 diabetic (db/db) mice show impaired ventricular function, as well as altered cardiac metabolism. Assessment of the relationship between myocardial oxygen consumption (MVO(2)) and ventricular pressure-volume area (PVA) has also demonstrated reduced cardiac efficiency in db/db hearts. We hypothesized that lowering the plasma fatty acid supply and subsequent normalization of altered cardiac metabolism by chronic treatment with a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist will improve cardiac efficiency in db/db hearts. Rosiglitazone (23 mg/kg body weight/day) was administered as a food admixture to db/db mice for five weeks. Ventricular function and PVA were assessed using a miniaturized (1.4 Fr) pressure-volume catheter; MVO(2) was measured using a fibre-optic oxygen sensor. Chronic rosiglitazone treatment of db/db mice normalized plasma glucose and lipid concentrations, restored rates of cardiac glucose and fatty acid oxidation, and improved cardiac efficiency. The improved cardiac efficiency was due to a significant decrease in unloaded MVO(2), while contractile efficiency was unchanged. Rosiglitazone treatment also improved functional recovery after low-flow ischemia. In conclusion, the present study demonstrates that in vivo PPARgamma-treatment restores cardiac efficiency and improves ventricular function in perfused hearts from type 2 diabetic mice.     

Ecosystem respiration depends strongly on photosynthesis in a temperate heath

We measured net ecosystem CO₂ flux (F _n) and ecosystem respiration (R _E), and estimated gross ecosystem photosynthesis (P _g) by difference, for two years in a temperate heath ecosystem using a chamber method. The exchange rates of carbon were high and of similar magnitude as for productive forest ecosystems with a net ecosystem carbon gain during the second year of 293 ± 11 g C m⁻² year⁻¹ showing that the carbon sink strength of heather-dominated ecosystems may be considerable when C. vulgaris is in the building phase of its life cycle. The estimated gross ecosystem photosynthesis and ecosystem respiration from October to March was 22% and 30% of annual flux, respectively, suggesting that both cold-season carbon gain and loss were important in the annual carbon cycle of the ecosystem. Model fit of R _E of a classic, first-order exponential equation related to temperature (second year; R ² = 0.65) was improved when the P _g rate was incorporated into the model (second year; R ² = 0.79), suggesting that daytime R _E increased with increasing photosynthesis. Furthermore, the temperature sensitivity of R _E decreased from apparent Q ₁₀ values of 3.3 to 3.9 by the classic equation to a more realistic Q ₁₀ of 2.5 by the modified model. The model introduces R _photo, which describes the part of respiration being tightly coupled to the photosynthetic rate. It makes up 5% of the assimilated carbon dioxide flux at 0°C and 35% at 20°C implying a high sensitivity of respiration to photosynthesis during summer. The simple model provides an easily applied, non-intrusive tool for investigating seasonal trends in the relationship between ecosystem carbon sequestration and respiration.     

Deposition of iron and beta-amyloid plaques is associated with cortical cellular damage in rabbits fed with long-term cholesterol-enriched diets

Hypercholesterolemia is a potential trigger of Alzheimer's disease, and is thought to increase brain levels of beta-amyloid (Abeta) and iron. However, animal models to address the mechanisms by which Abeta and iron accumulation may cause neuronal damage are poorly defined. To address this question, we fed adult rabbits a 1% cholesterol-enriched diet for 7 months. This diet was associated with increased regional deposition of both iron and Abeta peptide in the brain. Iron preferentially accumulated around Abeta plaques in the adjacent cortex, but was not found in the hippocampus. Co-localization of iron and Abeta was accompanied by apoptosis, DNA damage, blood-brain barrier (BBB) disruption, as well as dysregulation in the level of the iron-regulatory proteins, ferritin and heme-oxygenase-1. We further demonstrate that the cholesterol diet-induced apoptosis is mediated by the activation of the endoplasmic reticulum stress pathway, involving the down-regulation of the endoplasmic reticulum chaperones, calreticulin, grp78 and grp94, and the activation of the growth and arrest DNA damage protein, gadd153. Our results suggest that BBB damage and disturbances in iron metabolism may render the cortex more vulnerable than the hippocampus to the cholesterol-induced cellular stress.     

Phospholipid transfer protein activity is associated with inflammatory markers in patients with cardiovascular disease

Plasma phospholipid lipid transfer protein (PLTP) has several known key functions in lipoprotein metabolism. Recent studies suggest that it also may play a role in the inflammatory response. Inflammatory cell activity contributes to the development of atherosclerosis. To seek further evidence for the association of PLTP with inflammation, we studied the relationship between PLTP activity and five inflammatory markers [C-reactive protein (CRP), serum amyloid A (SAA), interleukin 6 (IL-6), white blood cells (WBC), and fibrinogen] in 93 patients with low HDL and cardiovascular disease (CVD). Plasma PLTP activity had the strongest correlation with CRP (r=0.332, P<0.001) followed by SAA (r=0.239, P=0.021). PLTP, CRP, and SAA were significantly associated with body mass index (BMI), insulin or glucose, apolipoprotein (apo) B, and/or apo E level (r=0.264-0.393, P<0.01). PLTP, SAA, and IL-6 also were associated with the concentration of HDL particles without apo A-II [Lp(A-I)](r=0.373-0.472, P<0.005, n=56), but not particles with apo A-II. Smoking was associated with increased PLTP activity, CRP, and WBC, and hypertension with increased PLTP activity. In linear models, CRP remained significantly associated with PLTP after adjustment of CVD risk factors and insulin resistance. Also, much of the variability of plasma PLTP activity was explained by CRP, BMI, Lp(A-I), smoking, glucose, and blood pressure. These findings show for the first time that plasma PLTP activity is associated positively with CRP in CVD, a state of chronic inflammation.     

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.     

Uniconazole-induced thermotolerance in soybean seedling root tissue

Soybean [Glycine max(L.) Merr. cv. A2] seeds were germinated in 0 or 1 mg 1¹ (3.4 uM) uniconazole, after which seedling roots were excised and exposed to 22 or 48°C for 90 min. Prior to the temperature treatments there were few ultrastructural differences between uniconazole-treated seedling roots and the controls. Following exposure to 48°C, electron micrographs revealed near complete loss of normal ultrastructure in control epidermal root cells, whereas cellular integrity was maintained in treated roots, indicating that uniconazole conferred tolerance to high temperature. Total electrolyte, sugar and K⁺ leakage were all greater from control roots than treated roots during exposure to 48°C. Proline content in the roots was unaffected by uniconazole at 22°C but was 25–30% greater in treated tissue than in controls following exposure to 48°C. Malondialdehyde content was unaffected by uniconazole at 22°C but was nearly 20% less in treated tissue than in controls following high temperature exposure. This indicates that uniconazole decreased high-temperature-induced lipid peroxidation. Uniconazole elevated several antiox-idant systems in the roots, including water-soluble sulfhydryl concentration and catalase, peroxidase and superoxide dismutase activities. These findings are consistent with the hypothesis that uniconazole-induced stress tolerance is due, at least in part, to enhanced antioxidant activity which reduces stress-related oxidative damage to cell membranes.