Academic literature on the topic 'Actin remodeling proteins'

Actin participates in several intracellular trafficking pathways. We now find that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment mixing stage of homotypic vacuole fusion. The Cdc42p GTPase is known to be required for vacuole fusion. We now show that proteins of the Cdc42p-regulated actin remodeling cascade (Cdc42p → Cla4p → Las17p/Vrp1p → Arp2/3 complex → actin) are enriched on isolated vacuoles. Vacuole fusion is dramatically altered by perturbation of the vacuole-bound actin, either by mutation of the ACT1 gene, addition of specific actin ligands such as latrunculin B or jasplakinolide, antibody to the actin regulatory proteins Las17p (yeast Wiskott-Aldrich syndrome protein) or Arp2/3, or deletion of actin regulatory genes. On docked vacuoles, actin is enriched at the “vertex ring” membrane microdomain where fusion occurs and is required for the terminal steps leading to membrane fusion. This role for actin may extend to other trafficking systems.

ABSTRACT Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase–dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.

Abstract The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation.

Members of the actin family have well-characterized cytoskeletal functions,but actin and actin-related proteins (ARPs) have also been implicated in nuclear activities. Previous analyses of the actin family have identified four conserved subfamilies, but many actin-related proteins (ARPs) do not fall into these groups. A new systematic phylogenetic analysis reveals that at least eight ARP subfamilies are conserved from humans to yeast, indicating that these ARPs are part of the core set of eukaryotic proteins. Members of at least three subfamilies appear to be involved in chromatin remodeling,suggesting that ARPs play ancient, fundamental roles in this nuclear process.

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.

Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

Darmepithelzellen entwickeln eine spezielle apikale Oberfläche zur Aufnahme von Nährstoffen aus dem Darminhalt. Diese Oberfläche besteht aus F-Aktin Protrusionen und werden als Mikrovilli (MV) bezeichnet. MV regulieren die kommensalen Bakterien und schützen die inneren Gewebe gegen den Angriff pathogener Mikroorganismen. Dennoch kann das Enteropathogen Salmonella enterica (Salmonella) die MV auslöschen und zerstört durch sein Typ-3-Sekretionssystem und dessen sekretierte Virulenzsproteine die Epithelschicht. Diese Virulenzproteine werden in das Zytoplasma der Wirtzellen injiziert und führen während des Eindringens von Salmonella zur F-Aktin Umlagerung. Durch Untersuchungen des Einflusses einiger T3SS-1 Effektorproteine auf die Zerstörung der MV konnte nachgewiesen werden, dass allein die Translokation von SopE die MV-Auslöschung verursachte und ausreichend für die Wiederherstellung der Invasion war. Echtzeitlebend-zellmikroskopie zeigte, dass MV ausgelöscht werden während Membranausstülpungen (Ruffles) gebildet werden. Diese Ruffle-Bildung vereinfachte ein paralleles Eindringen nicht-invadierender Stämme von Salmonella. Es konnte beobachtet werden, dass die Ausschaltung von Villin und Myosin 1a durch shRNA in C2BBe1 Zellen die Invasionsrate von Salmonella ermäßigte. Darüber hinaus wurde Ezrin zu den intrazellulären Bakterien aber nicht zur apikalen Seite rekrutiert. Außerdem verhinderte die durch das SopE verursachte Umlagerung des F-Aktins, welche die MV-Auflösung zur Folge hatte, die Makropinozytose der infizierten Zellen. Es lässt sich daraus schließen, dass die Zerstörung der MV für eine effiziente Invasion von Salmonella nötig ist. Die F-Aktin Umlagerung begünstigt zudem das Eindringen von nicht-invadierenden Bakterien. Des Weiteren benötigt Salmonella MV-Proteine zur F-Aktin Polymerisierung und Invasion in polarisierten Epithelzellen, was die Makropinozytose der Zellen beeinträchtigt. Möglicherweise tragen diese Phänotypen zur Infektion in vivo bei und verursachen das klinische Bild des Durchfalls.

Le cancer colorectal représente la troisième forme de cancer la plus fréquente au Canada. Malgré les récents développements, aucun traitement curatif n’est disponible pour les patients diagnostiqués à un stade avancé de la maladie. Plusieurs évidences supportent un rôle important des prostaglandines dans cette maladie. En effet, une surexpression des récepteurs de type EP et FP est remarquée. Ces derniers ainsi que les molécules de signalisations intracellulaires qu’ils activent représentent donc de nouvelles cibles pour traiter ce cancer. Nous et d’autres groupes avons démontré que les protéines G monomériques sont des petits interrupteurs moléculaires importants dans la signalisation intracellulaire engagée par les récepteurs dans des cellules saines mais qu’un dérèglement de celles-ci est associé au cancer. L’objectif principal de ce projet de recherche vise donc à identifier les voies de signalisations par lesquelles le récepteur FP contribue aux capacités invasives des cellules tumorales d’origine colorectale. Notre hypothèse est que la protéine ARF6, une des 6 isoformes des ARFs, et la protéine RhoA, agissent pour coordonner l’activation des voies de signalisations associées à la migration et l’invasion cellulaire. Nos résultats ont indiqué que la stimulation des cellules HEK293 exprimant de façon stable le récepteur FP (HA-FP) ainsi que les cellules SW480, une lignée invasive de cancer colorectal, par le PGF2α augmentait les niveaux d’activation d’ARF6 mais également de la protéine RhoA. De plus, d’autres médiateurs et intermédiaires associés à la réorganisation du cytosquelette comme la cofiline et la chaine légère de la myosine (MLC) ont été hautement phosphorylés suite à la stimulation par la prostaglandine. Ces observations sont associées avec une augmentation des fibres de stress dans les cellules. Nous avons tenté de déterminer si l’inhibition de ces protéines G affectait la capacité du PGF2α à activer ces intermédiaires de signalisation ainsi que certains effets biologiques. Ainsi, nos expériences contribueront à identifier de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer colorectal.
Colorectal cancer is the third most common form of cancer in Canada. Despite recent developments, no curative treatment is available for patients diagnosed at an advanced stage of the disease. Several evidences support an important role of prostaglandins in this disease. Indeed, overexpression of EP-like and FP receptors is noticed. These and intracellular signaling molecules that are activated following receptor stimulation, represent new targets for treating cancer. We and others have demonstrated that monomeric G proteins are important molecular switches coordinating intracellular signaling cascades initiated by receptors in normal cells but that disruption thereof is associated with cancer. The main objective of this research project aims to identify the signaling pathways by which the FP receptor contributes to the invasive capacity of tumor cells of colorectal origin. Our hypothesis is that the proteins ARF6, one of the 6 ARF isoforms, and RhoA protein, act to coordinate the activation of signaling pathways associated with cell migration and invasion. Our results show that stimulation of HEK293 cells that stably express the FP receptor (HA-FP) and SW480 cells, a lineage of invasive colorectal cancer that endogenously express FP receptor, by PGF2α, increased the level of activation of ARF6, but also that of RhoA. In addition, other mediators and intermediates associated with cytoskeleton reorganization such as cofilin and the myosin light chain (MLC) were found highly phosphorylated following stimulation by prostaglandin PGF2α. These observations are associated with an increase of stress fibers in cells. Finally, we have examinated whether inhibition of ARF6 and RhoA, affected the ability of PGF2α to activate these effectors and some biological effects. Thus, our results will contribute to identify new potential therapeutic targets for the treatment of colorectal cancer.

Les récepteurs couplés aux protéines-G (RCPGs) constituent la première étape d’une série de cascades signalétiques menant à la régulation d’une multitude de processus physiologiques. Dans le modèle classique connu, la liaison du ligand induit un changement de conformation du récepteur qui mène à sa forme active. Une fois activés, les RCPGs vont réguler l’activité d’une protéine membranaire cible qui peut être tant une enzyme qu’un canal ionique. L’interaction entre le récepteur et la cible nécessite l’intermédiaire d’une protéine hétérotrimérique appelée « protéine G », qui est activée pour favoriser l’échange du GDP (guanosine diphosphate) pour un GTP (guanosine triphosphate) et assurer la transduction du signal du récepteur à l’effecteur. Les mécanismes moléculaires menant à l’activation des effecteurs spécifiques via l’activation des RCPGs par les protéines G hétérotrimériques sont encore plutôt méconnus. Dans notre étude nous nous sommes intéressés aux récepteurs FP et PAF, à leurs ligands naturels, la PGF2α et le Carbamyl-PAF respectivement, et à des ligands à action antagoniste sur ces récepteurs. Des ligands considérés comme agonistes, sont des molécules qui interagissent avec le récepteur et induisent les mêmes effets que le ligand naturel. Les antagonistes, par contre, sont des molécules qui interagissent avec le récepteur et bloquent l’action du ligand naturel en prévenant le changement conformationnel du complexe, et ils peuvent avoir une action compétitive ou non-compétitive. Nous avons étudié aussi des ligands orthostériques et allostériques du récepteur FP des prostaglandines et du récepteur PAF. Un ligand orthostérique peut se comporter comme agoniste ou antagoniste en se fixant au site de liaison du ligand (agoniste) naturel. Un ligand allostérique est un agoniste ou antagoniste se fixant à un site autre que celui du ligand naturel entraînant un changement de conformation ayant pour conséquence soit une augmentation (effecteur positif), soit une diminution (effecteur négatif) de l'activité du ligand naturel.
G protein coupled receptors (GPCRs) are involved in the first step of most signalling pathways that regulate a variety of physiological events. The classical view of GPCR activation suggests that ligand binding to the inactive receptor will trigger a conformational change leading to an active conformation of the receptor. The GPCRs activated regulate the activity of a target membrane protein which can then activate other signalling proteins such as enzymes and ionic channels. The interaction between the receptor and the target requires an intermediary, in this case an heterotrimeric protein named « G protein », which is activated in order to facilitate the exchange of GDP (guanosine diphospate) for a GTP (guanosine triphosphate) and allow the transduction of the signal from the receptor to the effector. The molecular mechanisms leading to the activation of signalling effectors via the activation of GPCRs by its heterotrimeric G protein have not yet been well characterized. We focused our study on two GPCRs, the FP and PAF receptors, their natural ligands, PGF2α and Carbamyl-PAF respectively, and their antagonist ligands. Agonists are ligands that bind to the target receptor and trigger the same effects as the natural ligand of the GPCR. In contrast with agonists, antagonist ligands are molecules that prevent the effects of the natural ligand by keeping the GPCR from changing to its active conformation and can be competitive or non-competitive. We have also studied orthosteric and allosteric ligands of the FP and PAF receptors. An orthosteric ligand binds the same site as the natural ligand of the receptor and can act as an agonist or an antagonist. In the contrary, an allosteric ligand will rather have a different binding site then the natural ligand (agonist) and can positively or negatively modulate the effects of the natural ligand.

Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.

The atria serve a combination of reservoir, sensor, and neuroendocrine roles that help the heart to adapt to variations in blood volume, heart rate, and ventricular filling. When stressed by high-rate activity or increased haemodynamic load (due to hypertension, valve disease, or heart failure), the atria respond with increased oxidant production (oxidative stress) that promotes transcriptional changes that reversibly remodel electromechanical activity, with shortened action potential duration and effective refractory period, slowed and heterogeneous conduction, and impaired contractility. When the stresses persistent, the atria undergo persistent structural changes including chamber dilatation and increased interstitial fibrosis. The combination of electrical and structural remodelling leads to increased risk and persistence of atrial fibrillation and stroke. Accumulation of dysfunctional proteins that are normally recycled by the proteasome may contribute to the susceptibility to development of atrial fibrillation. Changes in ion channel expression are most often associated with the development of persistent atrial fibrillation. While many atrial fibrillation therapies have focused on targeting of atrial ion channels, efforts to target atrial proteostasis may have promise as a therapeutic atrial fibrillation treatment or prevention strategy.

RNA or ribonucleic acid constitutes of nucleotides, which are ribose sugars coupled to nitrogenous bases and phosphate groups. Nitrogenous bases include adenine, guanine, cytosine and uracil. Messenger RNA, ribosomal RNA and Transfer RNA are three main types of RNA that are involved in protein synthesis. Apart from its primary role in synthesis of protein, RNA comes in variety of forms like snRNA, miRNA, siRNA, antisense RNA, LncRNA etc., that are involved in DNA replication, post-transcriptional modification, and gene regulation etc. LncRNAs regulate gene expression by various ways including at, transcriptional, post-transcriptional, translational, post-translational and epigenetic levels by interacting principally with mRNA, DNA, protein, and miRNA. Among other biological functions, they are involved in chromatin remodelling, transcriptional interference, transcriptional activation, mRNA translation and RNA processing. In this chapter we shall be discussing the origin of lncRNAs, their biogenesis, their mechanism of action and their role in many biological and pathological processes like epigenetics, genome imprinting, several cancers and autoimmune diseases.

Cyclic stretching of endothelial cells (ECs), such as occurs in arteries during the cardiac cycle, induces ECs and their actin stress fibers to orient perpendicular to the direction of maximum stretch. This perpendicular alignment response is strengthened by increasing the magnitudes of stretch and cell contractility (1). The actin cytoskeleton is a dynamic structure that regulates cell shape changes and mechanical properties. It has been shown that actin stress fibers are ‘prestretched’ under normal, non-perturbed, conditions (2), consistent with the ideas of ‘prestress’ that have motivated tensegrity cell models (3). It has also been shown that ‘tractional forces’ generated by cells at focal adhesions tend to increase proportionately with increasing focal adhesion area, thus suggesting that cells tend to maintain constant the stress borne by a focal adhesion (4). By implication, this suggests that cells try to maintain constant the stress in actin stress fibers. Thus, it seems that cells reorganize or turnover cytoskeletal proteins and adhesion complexes so as to maintain constant a preferred mechanical state. Mizutani et al. (5) referred to this as cellular tensional homeostasis, although they did not suggest a model or theory to account for this dynamic process.

Cell spreading is governed by two cooperative cellular processes: the association of binding proteins to form focal adhesions, and the active remodeling of the actin cytoskeleton as the cell spreads [1]. The interaction between these two processes is poorly understood, and previous computational models have only examined each process in isolation. Previous studies have established that cells possess the ability to sense and react to their physical environment, for example cells seeded on substrates of varying stiffness exhibit a different cytoskeletal response [2,3].

Tropomodulin is an actin-capping protein in cardiac muscle, and is associated with both sarcomeric and cytoskeletal actin filaments. Homozygous knockout of erythrocyte tropomodulin (E-Tmod) is embryonically lethal, but heterozygous knockout (+/-) mice survive. Heterozygous E-Tmod knockout resulted in smaller right ventricle (RV) cavities and free walls compared to wild type. To investigate the effect of heterozygous tropomodulin knockout on mouse cardiac function and remodeling, mice (n=6 to 9) were subjected to 5 weeks of hypoxia to increase loading conditions on the RV via pulmonary hypertension. The effect of loading was determined by measuring the volume of RV anatomical features, and surface strain during the cardiac cycle. Although there was no significant change due to loading on RV cavity or free wall volume for wild type, geometrical measurements suggest that tissue had been redistributed. Under equal loading conditions, knockout mice exhibited a significant increase in volume for both RV features. RV epicardial function showed an increase in surface area strain at peak systole for hypoxic knockout mice. Thus it appears that heterozygous knockout of E-Tmod affects RV volume under normal and adverse loading conditions, as well as RV function.

The ability of cells to make or break specific attachments to extracellular matrix (ECM) and other cells is important in cell migration, proliferation and wound repair. Specific attachment proteins believed to be involved in mediating these interactions comprise functional domains joined by protease sensitive segments. Proteases can conceivably modulate cellular interactions by releasing functional domains from parent molecules. Tissue plasminogen activator (t-pA) is known to participate in various pathophysiological processes. That t-pA may also act directly on structural proteins has not been investigated. We have studied the direct proteolytic action of melanoma t-pA on fibronectin (FN), vitronectin (VN) and laminin (LN). These were incubated with t-pA for 0 to 48 h in 50 mM Tris HCi, pH 7.4. The cleavage products were separated on polyacrylamide slab gels and blotted onto nitrocellulose strips. FN and VN fragments with cell attachment properties were identified by incubating the strips with human gingiva fibroblasts and staining with Amido black. Monoclonal antibodies to FN were used to identify heparin, cell and gelatin binding fragments. VN was converted to a major 55 Kd product as a function of time. Lower molecular weight species migrating at 45 Kd, 30 Kd and 15 Kd positions were also identified. Most of these fragments possessed cell attachment properties. LN became susceptible to t-pA digestion after dénaturation with H2O2. The catalytic activity of t-pA could be inhibited in the presence of nitrophenyl-p-guinidino benzoate (a synthetic inhibitor of plasminogen activator), whereas O-phenanthroline (a metalloprotease inhibitor), α 2-antiplasmin and trasylol had no effect. A monoclonal IgG preparation (HI 72 A1, kindly provided by Dr. David J. Loskutoff) that specifically inhibits t-pA also inhibited the protelyotic action of t-pA on FN. These data suggest that direct proteolytic action of t-pA on adhesive proteins may modulate cellular behaviour in various normal and pathological conditions which involve dynamic interactions between cells and ECM and where plasminogen activator levels are elevated either transiently or permanently, for example during tissue remodelling, wound-related repair and thrombolytic therapy.

In the cardiovascular system, the flow of blood within the complex vascular network creates hemodynamic shear forces experienced by cells. Situated between the circulating blood and the bulk vascular tissue, the endothelium is a cell monolayer acting as a barrier that protects the underlying arterial tissue. Shear forces have been seen to interact with and regulate endothelial cells through mechanotransduction induced cytoskeletal changes within the cell [1]. Shear forces can be beneficial in cases of laminar flow, which are thought to be atheroprotective by aligning and organizing endothelial cells [2]. However, disturbances to a smooth flow field, caused by vessel bifurcations or obstructions like an implanted stent or a bulging atherosclerotic lesion, can cause recirculation zones to form downstream. In these flow regions, detrimental monolayer remodeling occurs which breaks down the endothelial barrier function [3]. Biochemically, it has been seen that shear forces drive signaling cascades in the Rho/Rac pathways that cascade into morphological changes in the cytoskeleton [4].