The dermomyotome ventrolateral lip is essential for the hypaxial myotome formation

Background The myotome is the primitive skeletal muscle that forms within the embryonic metameric body wall. It can be subdivided into an epaxial and hypaxial domain. It has been shown that the formation of the epaxial myotome requires the dorsomedial lip of the dermomyotome (DML). Although the ventrolateral lip (VLL) of the dermomyotome is believed to be required for the formation of the hypaxial myotome, experimentally evidence for this statement still needs to be provided. Provision of such data would enable the resolution of a debate regarding the formation of the hypaxial dermomyotome. Two mechanisms have been proposed for this tissue. The first proposes that the intermediate dermomyotome undergoes cellular expansion thereby pushing the ventral lateral lip in a lateral direction (translocation). In contrast, the alternative view holds that the ventral lateral lip grows laterally. Results Using time lapse confocal microscopy, we observed that the GFP-labelled ventrolateral lip (VLL) of the dermomyotome grows rather than translocates in a lateral direction. The necessity of the VLL for lateral extension of the myotome was addressed by ablation studies. We found that the hypaxial myotome did not form after VLL ablation. In contrast, the removal of an intermediate portion of the dermomyotome had very little effect of the hypaxial myotome. These results demonstrate that the VLL is required for the formation of the hypaxial myotome. Conclusion Our study demonstrates that the dermomyotome ventrolateral lip is essential for the hypaxial myotome formation and supports the lip extension model. Therefore, despite being under independent signalling controls, both the dorsomedial and ventrolateral lip fulfil the same function, i.e. they extend into adjacent regions permitting the growth of the myotome.

Rare variants in LRRK1 and Parkinson's disease

Approximately 20 % of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset PD to identify 15 potentially causal variants. Segregation analysis and frequency assessment in 862 PD cases and 1,014 ethnically matched controls highlighted variants in EEF1D and LRRK1 as the best candidates. Mutation screening of the coding regions of these genes in 862 cases and 1,014 controls revealed several novel non-synonymous variants in both genes in cases and controls. An in silico multi-model bioinformatics analysis was used to prioritize identified variants in LRRK1 for functional follow- up. However, protein expression, subcellular localization, and cell viability were not affected by the identified variants. Although it has yet to be proven conclusively that variants in LRRK1 are indeed causative of PD, our data strengthen a possible role for LRRK1 in addition to LRRK2 in the genetic underpinnings of PD but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.

Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi

BACKGROUND: Integrin-linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes. METHODS: The impact of ILK inhibition on integrin αII b β3 activation and degranulation was assessed with the ILK-specific inhibitor QLT0267, and a conditional ILK-deficient mouse model was used to assess the impact of ILK deficiency on in vivo platelet aggregation and thrombus formation. RESULTS: Inhibition of ILK reduced the rate of both fibrinogen binding and α-granule secretion, but was accompanied by only a moderate reduction in the maximum extent of platelet activation or aggregation in vitro. The reduction in the rate of fibrinogen binding occurred prior to degranulation or translocation of αII b β3 to the platelet surface. The change in the rate of platelet activation in the absence of functional ILK led to a reduction in platelet aggregation in vivo, but did not change the size of thrombi formed following laser injury of the cremaster arteriole wall in ILK-deficient mice. It did, however, result in a marked decrease in the stability of thrombi formed in ILK-deficient mice. CONCLUSION: Taken together, the findings of this study indicate that, although ILK is not essential for platelet activation, it plays a critical role in facilitating rapid platelet activation, which is essential for stable thrombus formation.

Diallyl disulfide-induced apoptosis in a breast-cancer cell line (MCF-7) may be caused by inhibition of histone de-acetylation

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

Hsc70 is a novel interactor of NF-kappaB p65 in living hippocampal neurons

Signaling via NF-κB in neurons depends on complex formation with interactors such as dynein/dynactin motor complex and can be triggered by synaptic activation. However, so far a detailed interaction map for the neuronal NF-κB is missing. In this study we used mass spectrometry to identify novel interactors of NF-κB p65 within the brain. Hsc70 was identified as a novel neuronal interactor of NF-κB p65. In HEK293 cells, a direct physical interaction was shown by co-immunoprecipitation and verified via in situ proximity ligation in healthy rat neurons. Pharmacological blockade of Hsc70 by deoxyspergualin (DSG) strongly decreased nuclear translocation of NF-κB p65 and transcriptional activity shown by reporter gene assays in neurons after stimulation with glutamate. In addition, knock down of Hsc70 via siRNA significantly reduced neuronal NF-κB activity. Taken together these data provide evidence for Hsc70 as a novel neuronal interactor of NF-κB p65.

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

Regulation of phospholipases C and D in rat ventricular myocytes: stimulation by endothelin-1, bradykinin and phenylephrine.

The physiological activator of protein kinase C (PKC), diacylglycerol, is formed by hydrolysis of phosphoinositides (PI) by phospholipase C (PLC) or phosphatidylcholine by phospholipase D (PLD). We have measured activation of these phospholipases by endothelin-1 (ET-1), bradykinin (BK), or phenylephrine (PE) in ventricular myocytes cultured from neonatal rat. The stimulation of PI hydrolysis after 10 min by 0.1 microM ET-1 (about 12-fold) was much greater than for BK or PE (each about four-fold), and did not correlate with translocation of nPKC delta or nPKC epsilon (Clerk A. Bogoyevitch MA. Andersson MB. Sugden PH, 1994. J Biol Chem 269: 32848-32857: Clerk A, Gillespie-Brown J, Fuller SJ, Sugden PH, 1996. Biochem J 317: 109-118). However, ET-1 and BK stimulated a similar rapid increase in [3H]InsP, formation (< 30 s), which was much greater than that seen with PE. This early phase correlated with PKC translocation. Acute or chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment with Ro-31-8220 showed that the stimulation of PI hydrolysis by PE, but not ET-1 or BK, was inhibited by activation of PKC. Furthermore, ET-1 and BK heterologously desensitized the stimulation of PI hydrolysis by PE, ET-1 or BK homologously uncoupled their own receptors from [3H]InsP3 formation, but there was no evidence of heterologous desensitization with these two agonists. Anomalously, chronic exposure to TPA increased the stimulation of PI hydrolysis by BK, but this probably resulted from an increase in BK receptor density. PLD was also rapidly activated by TPA. ET-1, BK or PE. Experiments with Ro-31-8220 showed that the stimulation of PLD by ET-1 and BK was mediated through activation of PKC. We discuss the characteristics of the activation of PI hydrolysis and PLD by ET-1, BK, and PE with respect to the translocation of PKC.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits.

A conscious rabbit model was used to study the effect of ischemic preconditioning (PC) on stress-activated kinases [c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK)] in an environment free of surgical trauma and attending external stress. Ischemic PC (6 cycles of 4-min ischemia/4-min reperfusion) induced significant activation of protein kinase C (PKC)-epsilon in the particulate fraction, which was associated with activation of p46 JNK in the nuclear fraction and p54 JNK in the cytosolic fraction; all of these changes were completely abolised by the PKC inhibitor chelerythrine. Selective enhancement of PKC-epsilon activity in adult rabbit cardiac myocytes resulted in enhanced activity of p46/p54 JNKs, providing direct in vitro evidence that PKC-epsilon is coupled to both kinases. Studies in rabbits showed that the activation of p46 JNK occurred during ischemia, whereas that of p54 JNK occurred after reperfusion. A single 4-min period of ischemia induced a robust activation of the p38 MAPK cascade, which, however, was attenuated after 5 min of reperfusion and disappeared after six cycles of 4-min ischemia/reperfusion. Overexpression of PKC-epsilon in cardiac myocytes failed to increase the p38 MAPK activity. These results demonstrate that ischemic PC activates p46 and p54 JNKs via a PKC-epsilon-dependent signaling pathway and that there are important differences between p46 and p54 JNKs with respect to the subcellular compartment (cytosolic vs. nuclear) and the mechanism (ischemia vs. reperfusion) of their activation after ischemic PC.

Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses

The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype.