Actin Content Research Articles

Cryoprotective effect of low-sweetness additives on protein denaturation of threadfin bream surimi (Nemipterus spp.) during frozen storage

The cryoprotective effects of lactitol, maltodextrin, palatinit, polydextrose, trehalose on the protein denaturation of threadfin bream surimi during six months of frozen storage were investigated. The characteristics analyzed were Ca2+-ATPase activity, protein solubility, sulfhydryl (SH) content, electrophoresis, surface morphology, and thermal stability. The effectiveness of all cryoprotectants generally decreased as the storage time increased. Polydextrose was able to maintain Ca2+-ATPase activity, protein solubility, and SH content of 33.5%, 71.3%, 71.8%, respectively. Sucrose was able to maintain Ca2+-ATPase activity, protein solubility, and SH content of 32.4%, 68.1%, 75.6%, respectively. Electrophoresis showed that the myosin heavy chain (MHC) and actin band intensity almost disappeared by the end of storage. Analysis by scanning electron microscopy (SEM) demonstrated that the number of pores formed increased after storage. Thermal stability analysis showed a decrease in myosin but no significant change in actin content. This study suggests that polydextrose as a cryoprotectant is able to maintain protein from denaturation as the same as sucrose.

Notch1 intracellular domain increases cytoplasmic EZH2 levels during early megakaryopoiesis

Notch pathway is a well-known factor in the development of lymphoid lineage. However, its role in the myeloid lineage has remained ambiguous. We looked into the effect of Notch1 on the megakaryocytic lineage commitment and found an increase in megakaryocyte-specific lineage markers upon transfection with Notch1 intracellular domain (NICD). This effect was mediated by Akt whereby constitutive activation of Akt increased the megakaryocyte markers, whereas inhibition of Akt signalling reduced these marker levels. Along with the change in differentiation status, NICD-induced initiation of early megakaryopoiesis was accompanied by an increased cytoplasmic enhancer of zeste homolog-2 (EZH2) expression. This process was found to be Akt-dependent, and inhibition or overexpression of Akt lead to concurrent changes in EZH2 levels. To elucidate the function of EZH2 in the cytoplasm, novel cytoplasmic interactors of EZH2 were identified by co-immunoprecipitation followed by matrix-assisted laser desorption ionization MS/MS-based protein identification, and thus, PDIA1 and LIM domain kinase-1 (LIMK1) were identified. Interaction of EZH2 with LIMK1 changed the activity of cofilin (a downstream target of LIMK1) towards actin filaments, thereby leading to lower filamentous actin content within these cells. Thus, Notch1 not only induces early megakaryopoiesis but also prepares these cells for subsequent morphological changes.

Alpha-1 Giardin is an Annexin with Highly Unusual Calcium-Regulated Mechanisms

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host.

Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [(35) S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4(+) CCR4(+) T cells were determined. The basal [(35) S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pK(a) = 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [(35) S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells.

Cerebral Myogenic Reactivity and Blood Flow in Type 2 Diabetic Rats: Role of Peroxynitrite in Hypoxia-Mediated Loss of Myogenic Tone

Dysregulation of cerebral vascular function and, ultimately, cerebral blood flow (CBF) may contribute to complications such as stroke and cognitive decline in diabetes. We hypothesized that 1) diabetes-mediated neurovascular and myogenic dysfunction impairs CBF and 2) under hypoxic conditions, cerebral vessels from diabetic rats lose myogenic properties because of peroxynitrite (ONOO(-))-mediated nitration of vascular smooth muscle (VSM) actin. Functional hyperemia, the ability of blood vessels to dilate upon neuronal stimulation, and myogenic tone of isolated middle cerebral arteries (MCAs) were assessed as indices of neurovascular and myogenic function, respectively, in 10- to 12-week control and type 2 diabetic Goto-Kakizaki rats. In addition, myogenic behavior of MCAs, nitrotyrosine (NY) levels, and VSM actin content were measured under normoxic and hypoxic [oxygen glucose deprivation (OGD)] conditions with and without the ONOO(-) decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl) prophyrinato iron (III), chloride (FeTPPs). The percentage of myogenic tone was higher in diabetes, and forced dilation occurred at higher pressures. Functional hyperemia was impaired. Consistent with these findings, baseline CBF was lower in diabetes. OGD reduced the percentage of myogenic tone in both groups, and FeTPPs restored it only in diabetes. OGD increased VSM NY in both groups, and although FeTPPs restored basal levels, it did not correct the reduced filamentous/globular (F/G) actin ratio. Acute alterations in VSM ONOO(-) levels may contribute to hypoxic myogenic dysfunction, but this cannot be solely explained by the decreased F/G actin ratio due to actin nitration, and mechanisms may differ between control and diabetic animals. Our findings also demonstrate that diabetes alters the ability of cerebral vessels to regulate CBF under basal and hypoxic conditions.

Pyruvate protects the HT22 neuronal cells treated with rtPA against hypoxia‐reoxygenation damage

Recombinant tissue plasminogen activator (rtPA) is the only FDA approved treatment for ischemic stroke, but delayed rtPA treatment may be neurotoxic. Pyruvate has proven neuroprotective in animal models of stroke. We examined pyruvate + rtPA cotreatment of neuronal cells subjected to an in vitro stroke model.MethodsHT22 cells were exposed to 2 or 6 h hypoxia (0.5% O2) + glucose deprivation (OGD), followed by 24 h reoxygenation. Pyruvate (8 mM) and/or rtPA (10 μg/ml) were applied upon reoxygenation. Cell viability was assessed with Calcein AM. DCF‐DA and mitosox staining detected reactive oxygen species (ROS). Matrix metalloproteinase (MMP) activities were measured by gelzymography; MMP, tissue inhibitor of metalloproteinase 2, HIF‐1α, EPO and actin contents were determined by immunoblot.ResultsOGD lowered cell viability by 50% vs. normoxic control (P <0.05). Although rtPA treatment did not increase post‐OGD cell death, it intensified ROS production and activated MMP. Pyruvate‐induced cytoprotection was not compromised by rtPA cotreatment, and pyruvate dampened rtPA‐induced ROS and MMP. Delayed rtPA treatment intensified cell death 2 h after OGD, but pyruvate salvaged 50% of these cells.ConclusionPyruvate attenuates rtPA‐induced MMP activation and ROS formation, thereby protecting reoxygenated HT22 cells. Pyruvate could be a useful cotreatment with rtPA for ischemic stroke. NIH support: R01NS054687, R01NS054651, R01NS076975, T32AG020494

T cell derived IL-6 and IL-13 drive fibroblast fibrosis: implications for systemic sclerosis

Systemic Sclerosis (SS) is an autoimmune disease of unknown aetiology that is characterised by inflammation, vasculopathy and excessive extracellular matrix deposition. The extracellular matrix deposition is primarily in the skin...

Inducible Nitric Oxide Synthase Inhibition Attenuates Physical Stress-Induced Lung Hyper-Responsiveness and Oxidative Stress in Animals with Lung Inflammation

Mechanisms involved in stress-induced asthmatic alterations have been poorly characterised. We assessed whether inducible nitric oxide synthase (iNOS) inhibition modulates the stress-amplified lung parenchyma responsiveness, oxidative stress and extracellular matrix remodelling that was previously increased by chronic lung inflammation. Guinea pigs were subjected to 7 exposures to ovalbumin (1–5 mg/ml) or saline (OVA and SAL groups) over 4 weeks. To induce behavioural stress, animals were subjected to a forced swimming protocol (5 times/week, over 2 weeks; SAL-Stress and OVA-Stress groups) 24 h after the 4th inhalation. 1400W (iNOS-specific inhibitor) was administered intraperitoneally in the last 4 days of the protocol (SAL-1400W, OVA-1400W, SAL-Stress+1400W and OVA-Stress+1400W groups). Seventy-two hours after the last inhalation, animals were anaesthetised and exsanguinated, and adrenal glands were removed. Lung tissue resistance and elastance were evaluated by oscillatory mechanics and submitted for histopathological evaluation. Stressed animals had higher adrenal weights compared to non-stressed groups, which were reduced by 1400W treatment. Behavioural stress in sensitised animals amplified the resistance and elastance responses after antigen challenge, numbers of eosinophils and iNOS+ cells, actin content and 8-iso-PGF2α density in the distal lung compared to the OVA group. 1400W treatment in ovalbumin-exposed and stressed animals reduced lung mechanics, iNOS+ cell numbers and 8-iso-PGF2α density compared to sensitised and stressed animals that received vehicle treatment. We concluded that stress amplifies the distal lung constriction, eosinophilic inflammation, iNOS expression, actin content and oxidative stress previously induced by chronic lung inflammation. iNOS-derived NO contributes to stress-augmented lung tissue functional alterations in this animal model and is at least partially due to activation of the oxidative stress pathway.

N-Cofilin Can Compensate for the Loss of ADF in Excitatory Synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.

Differential Sensitivity to Angiotensin II and Norepinephrine in Human Uterine Arteries

During pregnancy, uteroplacental responses to norepinephrine (NE) exceed systemic responses. In contrast, uteroplacental responses to angiotensin II (ANG II) are less than systemic. The explanation for these differences in uteroplacental sensitivity remain unclear but may reflect type 2 ANG II receptor (AT(2)R) predominance in uterine artery (UA) vascular smooth muscle (VSM). The objective of the study was to examine VSM sensitivity to KCl, NE, and ANG II in UA from nonpregnant (NP) and pregnant (P) women and determine VSM ANG II receptor subtype expression. Responses to KCl, NE, and ANG II were examined in endothelium-denuded UA rings from NP (n = 28) and P (n = 13; 34-40 wk gestation) women, and ANG II receptor subtype, α(1)-receptor and contractile proteins were measured. KCl and NE dose dependently contracted UA (P < 0.001), P exceeding NP 2-fold or greater; but α(1)-receptor expression was unchanged. ANG II did not elicit dose effects in NP or P UA; however, P responses exceeded NP approximately 2-fold (P < 0.001) and were approximately 2.5-fold less than NE (P < 0.001). AT(2)R and AT(1)R expression were similar (P > 0.1) in VSM from NP and term P women. AT(1)R blockade abolished ANG II contractions (P < 0.001); AT(2)R blockade did not enhance ANG II sensitivity in UA with or without endothelium. Actin contents increased approximately 2-fold in term UA. Sensitivity to α-stimulation exceeds ANG II in NP and P UA, explaining the differential uteroplacental sensitivity in pregnancy. Because AT(2)R predominate in UA VSM throughout reproduction, this contributes to the inherent refractoriness to ANG II in the uterine vasculature. The increase in UA contractile proteins at term P suggests remodeling, explaining the enhanced contractility seen.

Spice up the hypertension diet - curcumin and piperine prevent remodeling of aorta in experimental L-NAME induced hypertension

BackgroundIncrease of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension.MethodsWistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated.ResultsThe increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself.ConclusionsAdministration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.

Activity of platelet hemostasis in newborn calves

Healthy newborn Simmental and Black Pied calves with a normal number of platelets in bloodstream don’t have reliable dynamics of the aggregation function of blood platelets with adenosine diphosphate, collagen, thrombin, ristocetin, epinephrine, as well as combinations of ADP + epinephrine, ADP + collagen, and collagen + epinephrine inducers. The level of discocytes in blood on days 1–2 was 78.5, not reliably changing until the end of the neonatal stage. In this case, the number of disco-echinocytes, spherocytes, spheroechinocytes, and bipolar forms of platelets in the bloodstream remained stably low. Maintenance during colostrum feeding of a low intensity of metabolism of endogenous arachidonic acid in platelets and of a low content of ATP, ADP, actin, and myosin in them can be considered important mechanisms providing stability of platelet aggregation activity in calves in the neonatal stage.

Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters

Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.

The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell–matrix adhesion

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell–matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells.

Encapsulation of Active Cytoskeletal Protein Networks in Cell-Sized Liposomes

We demonstrate that cytoskeletal actin-myosin networks can be encapsulated with high efficiency in giant liposomes by hydration of lipids in an agarose hydrogel. The liposomes have cell-sized diameters of 10-20 μm and a uniform actin content. We show by measurements of membrane fluorescence intensity and bending rigidity that the majority of liposomes are unilamellar. We further demonstrate that the actin network can be specifically anchored to the membrane by biotin-streptavidin linkages. These protein-filled liposomes are useful model systems for quantitative studies of the physical mechanisms by which the cytoskeleton actively controls cell shape and mechanics. In a broader context, this new preparation method should be widely applicable to encapsulation of proteins and polymers, for instance, to create polymer-reinforced liposomes for drug delivery.

Hypoxic exercise training causes erythrocyte senescence and rheological dysfunction by depressed Gardos channel activity

Despite enhancing cardiopulmonary and muscular fitness, the effect of hypoxic exercise training (HE) on hemorheological regulation remains unclear. This study investigates how HE modulates erythrocyte rheological properties and further explores the underlying mechanisms in the hemorheological alterations. Twenty-four sedentary males were randomly divided into hypoxic (HE; n = 12) and normoxic (NE; n = 12) exercise training groups. The subjects were trained on 60% of maximum work rate under 15% (HE) or 21% (NE) O(2) condition for 30 min daily, 5 days weekly for 5 wk. The results demonstrated that HE 1) downregulated CD47 and CD147 expressions on erythrocytes, 2) decreased actin and spectrin contents in erythrocytes, 3) reduced erythrocyte deformability under shear flow, and 4) diminished erythrocyte volume changed by hypotonic stress. Treatment of erythrocytes with H(2)O(2) that mimicked in vivo prooxidative status resulted in the cell shrinkage, rigidity, and phosphatidylserine exposure, whereas HE enhanced the eryptotic responses to H(2)O(2). However, HE decreased the degrees of clotrimazole to blunt ionomycin-induced shrinkage, rigidity, and cytoskeleton breakdown of erythrocytes, referred to as Gardos effects. Reduced erythrocyte deformability by H(2)O(2) was inversely related to the erythrocyte Gardos effect on the rheological function. Conversely, NE intervention did not significantly change resting and exercise erythrocyte rheological properties. Therefore, we conclude that HE rather than NE reduces erythrocyte deformability and volume regulation, accompanied by an increase in the eryptotic response to oxidative stress. Simultaneously, this intervention depresses Gardos channel-modulated erythrocyte rheological functions. Results of this study provide further insight into erythrocyte senescence induced by HE.

Novel Application of Western Blotting for Human Skeletal Muscle Fiber Type Specific Protein Content Assessment

Little is known about protein profiles in slow-twitch (MHC I) and fast-twitch (MHC IIa and MHC IIx) muscle fiber types from human skeletal muscle. PURPOSE:To develop a method of assessing fiber type specific protein content with low amounts of protein via Western blotting following single fiber SDS-PAGE MHC identification. METHODS:Two hundred and sixty four individual muscle fibers from a muscle biopsy of the vastus lateralis were isolated. Approximately ¼ of each single fiber was clipped and fiber-typed based on MHC composition using SDS-PAGE combined with silver staining. The remaining ¾ was stored in individually labeled wells of an RNALater filled 96-well plate. Following MHC identification, the preserved portions were combined into pools of 20 like fibers. Homogenized pools of known fiber types were loaded onto Western blots and probed for GAPDH, Actin, and total p38 to establish reliable protein detection using this methodology. RESULTS:At low amounts of protein, good reliability of this method was shown as MHC I and MHC IIa pools loaded in quintuplicate displayed 11% and 4% variance in GAPDH expression, respectively. When the amount of sample loaded on the blot doubled, GAPDH expression doubled in both MHC I and MHC IIa samples, implying consistent transferring and equal protein loading. Actin content did not differ between fiber types and was therefore found to be an appropriate loading control. GAPDH content was 67% (MHC IIa) and 84% (MHC IIx) more abundant when compared to MHC I pools. Similarly, total p38 expression was 40% greater in MHC IIa fibers when compared to MHC I fibers. CONCLUSIONS:These data establish a novel application of Western blotting for fiber type specific protein quantification in human skeletal muscle. These initial results show content of specific proteins are 40-84% higher in fast-twitch muscle fibers as compared to slow-twitch muscle fibers (MHC IIx>MHC IIa>MHC I). Application of these methods can enhance our knowledge on fiber type specific differences in relation to skeletal muscle health profiles among physically active and clinically based populations.

Goniodomin A, an antifungal polyether macrolide, increases the filamentous actin content of 1321N1 human astrocytoma cells.

Goniodomin A, a polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax, caused morphological change in 1321N1 human astrocytoma cells. Observation by scanning electron microscopy revealed that the shape of the cell became refractile from flat in response to goniodomin A. This morphological change was accompanied by the outgrowth of a needle-like structure from the cell surface. Goniodomin A increased the intracellular content of filamentous actin in a concentration- and time-dependent manner. It also caused the change in filamentous actin distribution in the cells. The elongation of filamentous actin was observed in goniodomin A-treated cells. These results suggest that goniodomin A induces morphological change by increasing the content of filamentous actin in non-muscle cells.

Rho Kinase‐mediated Regulation of Actin Cytoskeleton Dynamics Participates in NFATc3 Nuclear Transport

Chronic hypoxia activates the Nuclear Factor of Activated T-cells isoform c3 (NFATc3) via endothelin-1 (ET-1) and NFATc3 is required for chronic hypoxia-induced pulmonary hypertension. ET-1 elevates Ca2+activating calcineurin which dephosphorylates NFATc3 allowing its nuclear import. RhoA/Rho kinase (ROK) has also been implicated in NFAT activation but the mechanism is unknown. We hypothesized that ET-1 via ROK increases actin cytoskeleton polymerization to mediate NFATc3 nuclear transport. We found that ROK blockade (fasudil) and destabilization of actin cytoskeleton (cytochalasin B) inhibited ET-1-induced increases in NFAT transcriptional activity in isolated pulmonary arteries from NFAT-luciferase reporter mice. The same treatments as well as stabilization of actin cytoskeleton (jasplakinolide) also inhibited ET-1-induced NFATc3-GFP nuclear import in pulmonary arterial smooth muscle cells. None of the drugs affected the increase in cytosolic Ca2+ caused by ET-1 as detected by fura 2. ET-1 also increased filamentous actin content and its co-localization with NFATc3. Both responses were inhibited by fasudil and cytochalasin B. Our results suggest NFATc3 nuclear transport depends on Ca2+ and ROK-mediated actin cytoskeleton polymerization. Funding source: R01 HL088151.