Integrins regulate Rac targeting by internalization of membrane domains

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.     

Localized Rac activation dynamics visualized in living cells

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.     

Supplementation of test meals with fat-free phytosterol products can reduce cholesterol micellarization during simulated digestion and cholesterol accumulation by Caco-2 cells

Phytosterols have been shown to reduce cholesterol absorption in humans. Supplementing phytosterols in fat-free formulations, however, has yielded controversial results. In the present study, we investigated the effect of supplementing test meals with different fat-free phytosterol products on cholesterol incorporation into mixed micelles during simulated digestion and accumulation of micellar cholesterol by Caco-2 cells: control orange juice (OJ), orange juice supplemented with either multivitamin/multimineral tablets (MVT), multivitamin/multimineral tablets containing phytosterols (MVT+P), and phytosterol powder (PP). These combinations were added to Ensure-based test meals and spiked with cholesterol of natural isotopic composition or 13C2-cholesterol to differentiate external from endogenous cholesterol. After simulated gastric/small intestinal digestion, micelle fractions were analyzed for cholesterol enzymatically (n = 6-20/product) and by high-performance liquid chromatography-tandem mass spectrometry (n = 12/product) and added to Caco-2 cells to determine the accumulation of 13C2-cholesterol (n = 10-24/product). As compared to OJ, PP and MVT+P significantly decreased cholesterol micellarization (determined enzymatically) by 70 +/- 39 (mean +/- SD) and 70 +/- 39%, respectively (P < 0.001, Bonferroni). The stable isotope experiments revealed that both PP and MVT+P reduced cholesterol micellarization [by 25 +/- 12 (P = 0.055) and 21 +/- 8% (P = 0.020), respectively, Fisher's protected LSD test] and Caco-2 cell accumulation (by 28 +/- 8 and 10 +/- 8%, respectively; P < 0.010, Bonferroni). OJ+P did not inhibit micellarization or accumulation of cholesterol by Caco-2 cells. This study shows that fat-free phytosterol-containing products can significantly inhibit cholesterol micellarization and Caco-2 cell bioaccessibility, albeit to different extents depending on individual formulations. This is most likely explained by inhibition of cholesterol micellarization.     

The effect of body condition on ovarian activity of free ranging tropical jennies (Equus asinus)

Serial ultrasonography was performed on seven jennies to study the effect of seasonally fluctuating body condition on ovarian activity during the dry, short rainy and long rainy seasons. Kendall Tau correlations and differences of least square mean values were compared. A distinct seasonal pattern was observed both in body condition and ovarian activity. The mean (+/-SD) Body Condition Score (BCS) was lowest during the dry season 2.6 +/- 0.45 when compared with 3.5 +/- 0.52 and 3.9 +/- 0.58 during the short and long rainy seasons, respectively. The mean (+/-SD) number of total follicles was also proportionally low 7.3 +/- 1.6 during the dry season compared with 9.6 +/- 2.4 and 11.3 +/- 3.3 found during the short and the long rainy seasons, respectively. The BCS was positively correlated to the total number of follicles and the diameter of the largest follicle during all seasons. Periods of increasing BCS and transition between seasons with both increasing and decreasing body conditions were strongly correlated to the number and size of ovarian follicles. Seasonal fluctuation in the body condition was found to be the result of variation in feed resource and ovarian activity was also closely following this seasonal pattern. The improvement in body condition has generally, a positive impact on emergence of small growing follicles there by having influence on the total count of ovarian follicles.     

Simultaneously mitigating near-term climate change and improving human health and food security

Tropospheric ozone and black carbon (BC) contribute to both degraded air quality and global warming. We considered ~400 emission control measures to reduce these pollutants by using current technology and experience. We identified 14 measures targeting methane and BC emissions that reduce projected global mean warming ~0.5°C by 2050. This strategy avoids 0.7 to 4.7 million annual premature deaths from outdoor air pollution and increases annual crop yields by 30 to 135 million metric tons due to ozone reductions in 2030 and beyond. Benefits of methane emissions reductions are valued at $700 to $5000 per metric ton, which is well above typical marginal abatement costs (less than $250). The selected controls target different sources and influence climate on shorter time scales than those of carbon dioxide-reduction measures. Implementing both substantially reduces the risks of crossing the 2°C threshold.     

Two dogs with iatrogenic discospondylitis caused by meticillin-resistant Staphylococcus aureus

Two dogs developed discospondylitis caused by meticillin-resistant Staphylococcus aureus following thoracolumbar hemilaminectomy. Diagnoses were established by magnetic resonance imaging and radiography, respectively, in conjunction with culturing of microbial swabs. Treatment with β-lactam antibiotics was first initiated. As soon as culturing results, confirming meticillin-resistant Staphylococcus aureus infection, and antibiograms became available, antimicrobial therapy was changed to gentamicin and trimethoprim/sulphadiazine. One dog, however, deteriorated further and was euthanased. The other dog improved on appropriate therapy. The first attempt to discontinue drug therapy four months after surgery led to a relapse. Antimicrobial therapy with chloramphenicol was then initiated and maintained for an additional four months. This dog is free of any relapses for 2·5 years. The veterinary surgeon should be aware of the possible involvement of meticillin-resistant Staphylococcus aureus in postsurgical discospondylitis when choosing an antibiotic for initial antimicrobial therapy while culturing results are still pending.     

Discriminating between organic matter in soil from grass and forest by near‐infrared spectroscopy

The absorbance of near‐infrared (NIR) radiation by plants depends on the overtones of vibration, bending and stretching of the chemical bonds within their organic components. The wide variety of these bonds gives rise to characteristic NIR spectra, or ‘fingerprints’, of plant materials. Do these fingerprints remain in the soil when the plants decompose and allow discrimination between soils that have supported different types of vegetation? We have compared the NIR spectra of grassland soil with those of soil under forest in the Vosges mountains in France. Near‐infrared reflectances, R, from 370 samples of soil were recorded as A = log₁₀(1/R) at wavelengths between 1100 and 2500 nm and averaged over 10‐nm intervals to give spectra each with 140 values. A canonical variate analysis (CVA) of the raw spectra discriminated well, but not perfectly, between grassland and forest soil, with Mahalanobis distance, D, of 4.87. Standardization along the spectra to remove effects of varied mineral composition achieved complete separation between the two sources with D = 9.81. Canonical variate analysis of first and second derivatives of the spectra distinguished the two groups even better, with D = 12.27 and D = 16.65, respectively. The results show much promise for inferring past vegetative cover from NIR spectra deriving from the organic matter in the soil. The next step will be to extend these studies to see how well we can distinguish other types of vegetation from such spectra.     

Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids

A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P < 0.05) by addition of TG (2.5% v/w) to the meal and was dependent on fatty acyl chain length in structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These data suggest that the amount of TG in a typical meal does not limit the bioaccessibility of carotenoids.