RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

The Minimum Capture Proportion for Reliable Estimation in Capture–Recapture Models

Summary .   We derive estimates of the minimum capture proportion required to obtain a reliable estimate of the population size for several continuous and discrete-time capture–recapture models. The models considered are     , and     in the notation of Otis et al. , (1978, Wildlife Monograph 62 , 1–135). Numerical results with simulation studies are given, and two real examples for the model     are also considered. Potential applications of these results are suggested.     

Impedance analysis of renal vascular smooth muscle cells

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Z_cal) based on the model agreed well with the experimental measurement (Z_exp), and the percentage error defined as |(Z_cal – Z_exp)/Z_exp| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin     

Unusual structural characteristics and complete amino acid sequence of a protease inhibitor from Phaseolus acutifolius seeds.

Two isoforms of a protease inhibitor were isolated by ion-exchange chromatography of tepary bean (Phaseolus acutifolius G.) seed proteins. The main isoform was used to determine the amino acid sequence of the protein. It is an 80 amino acid residue protein with a molecular mass of 8765 Da, showing sequence homology with the Bowman-Birk family of protease inhibitors. Several regions with amino acid microheterogeneity were found, corroborating the possible presence of isoforms. Mass spectrometry analysis was carried out to confirm isoforms. The presence of dimer and trimer forms of the inhibitor was shown through electrophoresis and mass spectrometry. Another unusual characteristic for this inhibitor was its ability to bind metals. The presence of four sequential histidines at the N-terminal end of the protein could account for this binding. Mass spectrometry and atomic absorption spectroscopy support the presence of calcium in the native inhibitor.     

Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

In this study, we found that adding iodoacetamide to the homogenization buffer used in the preparation of mouse or rat liver plasma membranes resulted in an increase of insulin receptor autophosphorylation by 4-5-fold and receptor kinase activity by about 2-fold. Similar effects were obtained with iodoacetate and p-chloromercuriphenyl sulfonate. The effect of iodoacetamide was minimal when it was added to membranes prepared without the thiol reagent. The enhancing effect of iodoacetamide on insulin receptor autophosphorylation was the result of a more than 2-fold decrease in the Km and a more than 3-fold increase in Vmax for ATP. The presence of iodoacetamide in the preparation of plasma membranes also greatly increased the solubilization of the insulin receptor from the plasma membrane by Triton X-100. We propose that iodoacetamide acts to alkylate some unknown thiols released during tissue homogenization and that in its absence these thiols formed mixed disulfides with the insulin receptor, thus adversely affecting the process of receptor activation by insulin.     

A rapid and simple radioassay for inosinic acid: pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa.

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10^?5m allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) into the reaction mixture, the [8-¹⁴C]hypoxanthine (Hx) is converted only to [8-¹⁴C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase.Using [8-¹⁴C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 mm ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8-¹⁴C]IMP or [8-¹⁴C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value.Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.     

Alcohol Tax Policy and Related Mortality. An Age-Period-Cohort Analysis of a Rapidly Developed Chinese Population, 1981–2010

To delineate the temporal dynamics between alcohol tax policy changes and related health outcomes, this study examined the age, period and cohort effects on alcohol-related mortality in relation to changes in government alcohol policies. We used the age-period-cohort modeling to analyze retrospective mortality data over 30 years from 1981 to 2010 in a rapidly developed Chinese population, Hong Kong. Alcohol-related mortality from 1) chronic causes, 2) acute causes, 3) all (chronic+acute) causes and 4) causes 100% attributable to alcohol, as defined according to the Alcohol-Related Disease Impact (ARDI) criteria developed by the US Centers for Disease Control and Prevention, were examined. The findings illustrated the possible effects of alcohol policy changes on adult alcohol-related mortality. The age-standardized mortality trends were generally in decline, with fluctuations that coincided with the timing of the alcohol policy changes. The age-period-cohort analyses demonstrated possible temporal dynamics between alcohol policy changes and alcohol-related mortality through the period effects, and also generational impact of alcohol policy changes through the cohort effects. Based on the illustrated association between the dramatic increase of alcohol imports in the mid-1980s and the increased alcohol-related mortality risk of the generations coming of age of majority at that time, attention should be paid to generations coming of drinking age during the 2007–2008 duty reduction.