The gene distribution in the genomes of pea, tomato and date palm

The vast majority of genes of maize, rice, barley and wheat are contained in long gene-rich regions (collectively called the 'gene space') separated by long gene-empty regions. The gene space covers a narrow, 0.8-1.6%, GC range, possibly because of the presence of abundant transposons. Here we report that the gene space is not an exclusive property of Gramineae, because it also exists in the large genome of pea (5000 Mb). Moreover, the gene space is not just dependent upon genome size, since a gene space is found in rice (415 Mb), but not in Arabidopsis (120 Mb), nor in two other plants investigated in the present work, date palm (250 Mb) and tomato (1000 Mb).     

Unsteady and three-dimensional simulation of blood flow in the human aortic arch

A three-dimensional and pulsatile blood flow in a human aortic arch and its three major branches has been studied numerically for a peak Reynolds number of 2500 and a frequency (or Womersley) parameter of 10. The simulation geometry was derived from the three-dimensional reconstruction of a series of two-dimensional slices obtained in vivo using CAT scan imaging on a human aorta. The numerical simulations were obtained using a projection method, and a finite-volume formulation of the Navier-Stokes equations was used on a system of overset grids. Our results demonstrate that the primary flow velocity is skewed towards the inner aortic wall in the ascending aorta, but this skewness shifts to the outer wall in the descending thoracic aorta. Within the arch branches, the flow velocities were skewed to the distal walls with flow reversal along the proximal walls. Extensive secondary flow motion was observed in the aorta, and the structure of these secondary flows was influenced considerably by the presence of the branches. Within the aorta, wall shear stresses were highly dynamic, but were generally high along the outer wall in the vicinity of the branches and low along the inner wall, particularly in the descending thoracic aorta. Within the branches, the shear stresses were considerably higher along the distal walls than along the proximal walls. Wall pressure was low along the inner aortic wall and high around the branches and along the outer wall in the ascending thoracic aorta. Comparison of our numerical results with the localization of early atherosclerotic lesions broadly suggests preferential development of these lesions in regions of extrema (either maxima or minima) in wall shear stress and pressure.     

Computational study of the effect of geometric and flow parameters on the steady flow field at the rabbit aorto-celiac bifurcation

Arterial hemodynamic forces may play a role in the localization of early atherosclerotic lesions. We have been developing numerical techniques based on overset or "Chimera" type formulations to solve the Navier-Stokes equations in complex geometries simulating arterial bifurcations. This paper presents three-dimensional steady flow computations in a model of the rabbit aorto-celiac bifurcation. The computational methods were validated by comparing the numerical results to previously-obtained flow visualization data. Once validated, the numerical algorithms were used to investigate the sensitivity of the computed flow field and resulting wall shear stress distribution to various geometric and hemodynamic parameters. The results demonstrated that a decrease in the extent of aortic taper downstream of the celiac artery induced looping fluid motion along the lateral walls of the aorta and shifted the peak wall shear stress from downstream of the celiac artery to upstream. Increasing the flow Reynolds number led to a sharp increase in spatial gradients of wall shear stress. The flow field was highly sensitive to the flow division ratio, i.e., the fraction of total flow rate that enters the celiac artery, with larger values of this ratio leading to the occurrence of flow separation along the dorsal wall of the aorta. Finally, skewness of the inlet velocity profile had a profound impact on the wall shear stress distribution near the celiac artery. While not physiological due to the assumption of steady flow, these results provide valuable insight into the fluid physics at geometries simulating arterial bifurcations.     

Cdk2 Activity Is Dispensable for the Onset of DNA Replication during the First Mitotic Cycles of the Sea Urchin Early Embryo

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and inXenopusextracts. In the present report we analyzein vivothe involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. UnfertilizedSphaerechinus granulariseggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studyingin vivothe onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained inXenopusegg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with thisin vitrosystem in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21^Cip1does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensablein vivofor the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast toXenopusegg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.     

Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

Mathematical modeling of intracellular calcium in presence of receptor: a homeostatic model for endothelial cell

Biomechanics and Modeling in Mechanobiology - Calcium is a ubiquitous molecule and second messenger that regulates many cellular functions ranging from exocytosis to cell proliferation at different...     

Measurement of menadione in urine by HPLC

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C₃₀ column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H₂O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R²) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Trophoblast migration under flow is regulated by endothelial cells

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.