Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 µM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 µM) induced a time-dependent increase in HIF-1a protein levels. Quercetin (30 µM) was also responsible for a rapid and long-lasting translocation of HIF-1a from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT–PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1a was reversed by addition of excess iron (100 µM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells

Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor

A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged amino-terminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR↓GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexahistidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized.A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged amino-terminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR↓GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexahistidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized

Comparison of explicit and mean-field models of cytoskeletal filaments with crosslinking motors

In cells, cytoskeletal filament networks are responsible for cell movement, growth, and division. Filaments in the cytoskeleton are driven and organized by crosslinking molecular motors. In reconstituted cytoskeletal systems, motor activity is responsible for far-from-equilibrium phenomena such as active stress, self-organized flow, and spontaneous nematic defect generation. How microscopic interactions between motors and filaments lead to larger-scale dynamics remains incompletely understood. To build from motor-filament interactions to predict bulk behavior of cytoskeletal systems, more computationally efficient techniques for modeling motor-filament interactions are needed. Here we derive a coarse-graining hierarchy of explicit and continuum models for crosslinking motors that bind to and walk on filament pairs. We compare the steady-state motor distribution and motor-induced filament motion for the different models and analyze their computational cost. All three models agree well in the limit of fast motor binding kinetics. Evolving a truncated moment expansion of motor density speeds the computation by $10^3$--$10^6$ compared to the explicit or continuous-density simulations, suggesting an approach for more efficient simulation of large networks. These tools facilitate further study of motor-filament networks on micrometer to millimeter length scales.Comment: 54 pages, 7 figures, 1 tabl

Evaluation of the use of sludge containing plutonium as a soil conditioner for food crops

An experiment was conducted to assess the potential hazard associated with the use of sludge containing plutonium as a soil conditioner for food crops. Conditions were chosen that would maximize exposure to the $sup 239$Pu in the sludge through resuspension and in plant content and thus approximated the maximum potential hazards due to the inhalation and ingestion pathways. The estimated 50-year radiation doses to the pulmonary region of the lung, bone, and liver based on the results of the inhalation experiment are 6 x 10$sup -4$ rem, 1.2 x 10$sup -3$ rem, and 0.55 x 10$sup -4$ rem, respectively. Similarly, the 50- year radiation doses attributable to ingestion of the sludge-grown vegetables were 2.2 x 10$sup -5$ rem to the bone and 1.5 x 10$sup -5$ rem to the liver. Thus, the inhalation pathway is the more critical of the two. The maximum permissible annual doses to the lungs, bone, and the liver for a member of the general public are 1.5, 3.0, and 1.5 rem, respectively. Thus, the maximum credible 50-year lung, bone, and liver dose commitments associated with the use of the $sup 239$Pu-contaminated sludge as a soil conditioner are approximately 4.0 x 10$sup -2$ percent of the annual maximum permissible dose. Under more realistic exposure circumstances, one might expect less drying of the sludge, less resuspension of dust and flying dirt before and during rototilling, and a much smaller sludge vegetable consumption rate. The conservative assumptions made in this analysis tend to assure that actual radiation doses would be even less than those calculated. (auth

Novel Human Rhinoviruses and Exacerbation of Asthma in Children1

To determine links between human rhinoviruses (HRV) and asthma, we used data from a case–control study, March 2003–February 2004, among children with asthma. Molecular characterization identified several likely new HRVs and showed that association with asthma exacerbations was largely driven by HRV-A and a phylogenetically distinct clade of 8 strains, genogroup C

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.</p

The use of a physiologically based pharmacokinetic model to evaluate deconvolution measurements of systemic absorption

BACKGROUND: An unknown input function can be determined by deconvolution using the systemic bolus input function (r) determined using an experimental input of duration ranging from a few seconds to many minutes. The quantitative relation between the duration of the input and the accuracy of r is unknown. Although a large number of deconvolution procedures have been described, these routines are not available in a convenient software package. METHODS: Four deconvolution methods are implemented in a new, user-friendly software program (PKQuest, ). Three of these methods are characterized by input parameters that are adjusted by the user to provide the "best" fit. A new approach is used to determine these parameters, based on the assumption that the input can be approximated by a gamma distribution. Deconvolution methodologies are evaluated using data generated from a physiologically based pharmacokinetic model (PBPK). RESULTS AND CONCLUSIONS: The 11-compartment PBPK model is accurately described by either a 2 or 3-exponential function, depending on whether or not there is significant tissue binding. For an accurate estimate of r the first venous sample should be at or before the end of the constant infusion and a long (10 minute) constant infusion is preferable to a bolus injection. For noisy data, a gamma distribution deconvolution provides the best result if the input has the form of a gamma distribution. For other input functions, good results are obtained using deconvolution methods based on modeling the input with either a B-spline or uniform dense set of time points

In vitro digestion and lactase treatment influence uptake of quercetin and quercetin glucoside by the Caco-2 cell monolayer

BACKGROUND: Quercetin and quercetin glycosides are widely consumed flavonoids found in many fruits and vegetables. These compounds have a wide range of potential health benefits, and understanding the bioavailability of flavonoids from foods is becoming increasingly important. METHODS: This study combined an in vitro digestion, a lactase treatment and the Caco-2 cell model to examine quercetin and quercetin glucoside uptake from shallot and apple homogenates. RESULTS: The in vitro digestion alone significantly decreased quercetin aglycone recovery from the shallot digestate (p < 0.05), but had no significant effect on quercetin-3-glucoside recovery (p > 0.05). Digestion increased the Caco-2 cell uptake of shallot quercetin-4'-glucoside by 2-fold when compared to the non-digested shallot. Despite the loss of quercetin from the digested shallot, the bioavailability of quercetin aglycone to the Caco-2 cells was the same in both the digested and non-digested shallot. Treatment with lactase increased quercetin recovery from the shallot digestate nearly 10-fold and decreased quercetin-4'-glucoside recovery by more than 100-fold (p < 0.05), but had no effect on quercetin recovery from apple digestates. Lactase treatment also increased shallot quercetin bioavailability to the Caco-2 cells approximately 14-fold, and decreased shallot quercetin-4'-glucoside bioavailability 23-fold (p < 0.05). These Caco-2 cells had lactase activity similar to that expressed by a lactose intolerant human. CONCLUSIONS: The increase in quercetin uptake following treatment with lactase suggests that dietary supplementation with lactase may increase quercetin bioavailability in lactose intolerant humans. Combining the digestion, the lactase treatment and the Caco-2 cell culture model may provide a reliable in vitro model for examining flavonoid glucoside bioavailability from foods