Rac inhibits thrombin-induced Rho activation: evidence of a Pak-dependent GTPase crosstalk

The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility, contractility, and growth. In this regard, the prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a still poorly understood mechanism. Indeed, we found that constitutively active forms of Rac inhibit stress fiber formation and Rho stimulation by thrombin. Surprisingly, a mutant of Rac that is unable to activate Pak1 failed to inhibit thrombin signaling to Rho. To explore the underlying mechanism, we investigated whether Pak1 could regulate guanine nucleotide exchange factors (GEFs) for Rho. We found that Pak1 associates with P115-RhoGEF but not with PDZ-RhoGEF or LARG, and knock down experiments revealed that P115-RhoGEF plays a major role in signaling from thrombin receptors to Rho in HEK293T cells. Pak1 binds the DH-PH domain of P115-RhoGEF, thus suggesting a mechanism by which Rac stimulation of Pak1 may disrupt receptor-dependent Rho signaling. In agreement, expression of a dominant-negative Pak-Inhibitory Domain potentiated the activation of Rho by thrombin, and prevented the inhibition of Rho by Rac. These findings indicate that Rac interferes with receptor-dependent Rho stimulation through Pak1, thus providing a mechanism for cross-talk between these two small-GTPases

Activation of ERK and p38 MAP Kinases in Human Fibroblasts during Collagen Matrix Contraction

Studies were carried out to characterize changes in MAP kinase activation during contraction of collagen matrices by fibroblasts under isometric tension. We found that both ERK and p38 MAP kinases were activated during contraction, as determined by immunoblotting and in vitro kinase assays. ERK activation was biphasic, with peaks at 10 min and 2 h; whereas p38 activation was monophasic, with a single peak at 10 min. Activation of ERK, but not p38, appeared to depend at least in part on the Gi class of heterotrimeric G proteins. The results show that ERK and p38 cooperate in contraction-stimulated activation of c-fos transcription

Increased c-fos mRNA Expression By Human Fibroblasts Contracting Stressed Collagen Matrices

We studied early changes in gene expression during fibroblast contraction of stressed collagen matrices. The level of c- fos mRNA increased dramatically and peaked 50 to 60 min after matrix contraction was initiated. This response did not require serum and could not be accounted for simply by disruption of the actin cytoskeleton. Increased c- fos mRNA levels required Ca 2+ influx but not the cyclic AMP or extracellular signal-regulated kinase (ERK 1/2) signaling pathways, both of which are activated when fibroblasts contract stressed collagen matrices. The levels of two other immediate-early genes, fosb and c- jun , also increased transiently after fibroblast contraction, whereas the levels of fra-1 , fra-2 , c- myc , and the transcription factor NF-κB remained the same, indicating that fibroblast contraction caused changes in a selective group of genes. The increase in c- fos mRNA during contraction of stressed collagen matrices may reflect a unique role for c- fos in mechanoregulated events at the end of wound repair

Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells

The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a Gi-independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores partially blocked increases in [Ca2+]i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma

Thermodynamic Study of the Interaction of Bovine Serum Albumin and Amino Acids with Cellulose Nanocrystals

The interaction of bovine serum albumin (BSA) with sulfated, carboxylated, and pyridinium-grafted cellulose nanocrystals (CNCs) was studied as a function of the degree of substitution by determining the adsorption isotherm and by directly measuring the thermodynamics of interaction. The adsorption of BSA onto positively charged pyridinium-grafted cellulose nanocrystals followed Langmuirian adsorption with the maximum amount of adsorbed protein increasing linearly with increasing degree of substitution. The binding mechanism between the positively charged pyridinum-grafted cellulose nanocrystals and BSA was found to be endothermic and based on charge neutralization. A positive entropy of adsorption associated with an increase of the degree of disorder upon addition of BSA compensated for the unfavorable endothermic enthalpy and enabled formation of pyridinium-g-CNC-BSA complexes. The endothermic enthalpy of adsorption was further found to decrease as a function of increasing degree of substitution. Negatively charged cellulose nanocrystals bearing sulfate and/or carboxylic functionalities were found to not interact significantly with the BSA protein. To investigate in more detail the role of single amino acids in the adsorption of proteins onto cellulose nanocrystals, we also studied the interaction of different types of amino acids with CNCs, i.e., charged (lysine, aspartic acid), aromatic (tryptophan, tyrosine), and polar (serine) amino acids. We found that none of the single amino acids bound with CNCs irrespective of surface charge and that therefore the binding of proteins with CNCs appears to require larger amino acid sequences that induce a greater entropic contribution to stabilize binding. Single amino acids are thus not adsorbed onto cellulose nanocrystals.status: publishe

Effect of Source on the Properties and Behavior of Cellulose Nanocrystal Suspensions

Sulfuric acid hydrolysis of native cellulose fibers results in colloidally stable suspensions of cellulose nanocrystals (CNCs). We have investigated the effect of the cellulose source on the suspension properties of CNCs extracted from cotton and wood sources using a comparable preparation strategy. The structural properties were revealed to be similar within the given standard deviation and prevalent polydispersity, whereas other properties such as liquid crystalline phase behavior, viscosity, diffusion coefficients, and surface tension were found to differ significantly. This study shows that ostensibly similar suspensions may exhibit rather differing behaviors and attempts to interpret this phenomenon. This finding shows that full characterization and a detailed description of the preparation of the nanocrystals used in publications are extremely important and should be reported in detail in all instances

Effect of Source on the Properties and Behavior of Cellulose Nanocrystal Suspensions

© 2018 American Chemical Society. Sulfuric acid hydrolysis of native cellulose fibers results in colloidally stable suspensions of cellulose nanocrystals (CNCs). We have investigated the effect of the cellulose source on the suspension properties of CNCs extracted from cotton and wood sources using a comparable preparation strategy. The structural properties were revealed to be similar within the given standard deviation and prevalent polydispersity, whereas other properties such as liquid crystalline phase behavior, viscosity, diffusion coefficients, and surface tension were found to differ significantly. This study shows that ostensibly similar suspensions may exhibit rather differing behaviors and attempts to interpret this phenomenon. This finding shows that full characterization and a detailed description of the preparation of the nanocrystals used in publications are extremely important and should be reported in detail in all instances.status: publishe

Effect of Source on the Properties and Behavior of Cellulose Nanocrystal Suspensions

Sulfuric acid hydrolysis of native cellulose fibers results in colloidally stable suspensions of cellulose nanocrystals (CNCs). We have investigated the effect of the cellulose source on the suspension properties of CNCs extracted from cotton and wood sources using a comparable preparation strategy. The structural properties were revealed to be similar within the given standard deviation and prevalent polydispersity, whereas other properties such as liquid crystalline phase behavior, viscosity, diffusion coefficients, and surface tension were found to differ significantly. This study shows that ostensibly similar suspensions may exhibit rather differing behaviors and attempts to interpret this phenomenon. This finding shows that full characterization and a detailed description of the preparation of the nanocrystals used in publications are extremely important and should be reported in detail in all instances