Postnatal development of transmural gradients in expression of ion channels and Ca²⁺-handling proteins in the ventricle

Transmural gradients in myocyte action potential duration (APD) and Ca(2+)-handling proteins are argued to be important for both the normal functioning of the ventricle and arrhythmogenesis. In rabbit, the transmural gradient in APD (left ventricular wedge preparation) is minimal in the neonate. During postnatal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in the expression of ion channels and also Ca(2+)-handling proteins in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2-7 days of age) and adult male (~6 months of age) New Zealand White rabbits using quantitative PCR and also, when possible, in situ hybridisation and immunohistochemistry. In the adult, there were significant and substantial transmural gradients in Ca(v)1.2, KChIP2, ERG, K(v)LQT1, K(ir)2.1, NCX1, SERCA2a and RyR2 at the mRNA and, in some cases, protein level-in every case the mRNA or protein was more abundant in the epicardium than the endocardium. Of the eight transmural gradients seen in the adult, only three were observed in the neonate and, in two of these cases, the gradients were smaller than those in the adult. However, in the neonate there were also transmural gradients not observed in the adult: in HCN4, Na(v)1.5, minK, K(ir)3.1 and Cx40 mRNAs - in every case the mRNA was more abundant in the endocardium than the epicardium. If the postnatal changes in ion channel mRNAs are used to predict changes in ionic conductances, mathematical modelling predicts the changes in APD observed experimentally. It is concluded that many of the well known transmural gradients in the ventricle develop postnatally.     

Role of the β1 integrin molecule in T-cell activation and migration

β1 integrins play crucial roles in a variety of cell processes such as adhesion, migration, proliferation, and differentiation of lymphocytes. To understand the molecular mechanisms of these various biological effects, it is particularly important to analyze cell signaling through the β1 integrins. Our previous study showed that PLC-γ, pp125FAK (focal adhesion kinase), pp105, paxillin, p59fyn, p56lck, and ERK1/2 are phosphorylated in their tyrosine residues upon engagement of β1 integrins. We identified pp105 as Cas (Crk-associated substrate)-related protein and successfully cloned its cDNA. pp105 is a Cas homologue predominantly expressed in the cells of lymphoid lineage, which led us to designate it Cas-L. Like p130Cas, Cas-L contains a single SH3 domain and multiple SH2-binding sites (YXXP motif), which are suggested to bind SH2 domains of Crk, Nck, and SHPTP2. Subsequent studies revealed that pp125FAK binds Cas-L on its SH3 domain and phosphorylates its tyrosine residues upon β1 integrin stimulation. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. We have shown that Cas-L is involved in the T-cell receptor (TCR)/CD3 signaling pathway as well as the β1 integrin signaling pathway. Cas-L is transiently phosphorylated following CD3 crosslinking and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant (Cas-LΔSH3), which lacks the binding site for FAK, is still tyrosine-phosphorylated upon CD3 crosslinking but not upon β1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Finally, we have identified a crucial role of Cas-L in β1 integrin-mediated T-cell co-stimulation. We have found that this co-stimulatory pathway is impaired in the Jurkat T-cell line, and that the expression level of Cas-L is reduced in the Jurkat cells compared to peripheral T-cells. The transfection of Cas-L cDNA into Jurkat cells restored the β1 integrin-mediated co-stimulation, while the transfection of Cas-LΔSH3 mutant failed to do so, which contrasts with the case of CD3-mediated signaling. These results indicate that Cas-L plays a key role, through the association and phosphorylation by FAK, in β1 integrin-mediated T-cell co-stimulation. Moreover, tyrosine phosphorylation of Cas-L is critical for T-cell receptor and β1 integrin-induced T-lymphocyte migration. Taken together, Cas-L might be the bi-modal docking protein which assembles the signals through β1 integrins and TCR/CD3, and which participates in a variety of T-cell functions. Received: August 24, 1999 / Accepted: August 31, 1999     

Monitoring the roles of prothrombin activation fragment 1 and 2 (F1 + 2) in patients with atrial fibrillation receiving rivaroxaban and apixaban

Journal of Thrombosis and Thrombolysis - Factor Xa (FXa) inhibitors are recommended for use in fixed doses without laboratory monitoring. However, prior studies reported the importance of...     

Differential roles of integrins α2β1 and αIIbβ3 in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin α₂β₁ and possibly to other receptors. In this study, we have compared the role of integrins α₂β₁ and α_IIbβ₃ in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A₂ (TxA₂) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and α_IIbβ₃ activation. This adhesion was substantially mediated by α₂β₁. The α_IIbβ₃ antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that α_IIbβ₃ outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of α₂β₁ and α_IIbβ₃ in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA₂ greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to α₂β₁.     

Immunohistologic markers of immune activation and changes of glycosylation of serum proteins in primary Sjögren's syndrome

OBIECTIVE: To assess the possible correlations between the immune activation of certain surface antigens at the lip salivary gland (LSG) level, and changes in glycosylation of serum proteins in primary Sj?gren's syndrome (SS). METHODS: LSG biopsy samples were obtained from 22 SS patients (mean age 56.3 years; mean disease duration 70.8 months) and prepared for immunohistochemical analysis using murine monoclonal antibodies for interleukin-2 receptor (IL-2R) (CD25) and for the class II major histocompatibility antigen HLA-DR. The glycosylation of serum proteins was evaluated in all patients by an enzyme-linked lectin assay (ELLA) using concanavalin A (Con A). RESULTS: In LSG specimens the presence of IL-2R was observed at the infiltrating level, mainly periductally, in 13 (59%) cases and on the epithelial cells of 14 (64%) patients. In 13 out of 22 SS patients (59%) a marked positivity both of the infiltrates and of the epithelium was found for anti-HLA-DR monoclonal antibody. The degree of expression of different antigens on LSG samples was correlated with their histologic class according to Tarpley evaluation. The positivity for IL-2R and HLA-DR molecules on glandular tissues was correlated. A significant increase in the total Con A reactivity of serum proteins was found in those patients expressing IL-2R and HLA-DR antigens on LSG specimens. CONCLUSIONS: The co-expression of IL-2R and HLA-DR antigens on both the epithelium and infiltrates of LSG is consistent with a participation of these cells in the immune process of SS. Moreover, changes in the glycosylation of serum proteins seem to be related to the presence of these immunoactivation markers of the disease at the LSG level, suggesting that the control of protein glycosylation could be mediated by the same mechanisms involved in the tissue damage of SS.     

What are the roles of metalloproteinases in cartilage and bone damage?

A role for metalloproteinases in the pathological destruction in diseases such as rheumatoid arthritis and osteoarthritis, and the irreversible nature of the ensuing cartilage and bone damage, have been the focus of much investigation for several decades. This has led to the development of broad spectrum metalloproteinase inhibitors as potential therapeutics. More recently it has been appreciated that several families of zinc dependent proteinases play significant and varied roles in the biology of the resident cells in these tissues, orchestrating development, remodelling, and subsequent pathological processes. They also play key roles in the activity of inflammatory cells. The task of elucidating the precise role of individual metalloproteinases is therefore a burgeoning necessity for the final design of metalloproteinase inhibitors if they are to be employed as therapeutic agents.     

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli (E(?) coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine fibrobllast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E(?)coli strains BL21(DE3) and M15[pREP4] respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain,E(?)coli Origami(DE3). The 4 recombinant E(?)coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E(?)coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv] was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from inclusion body in M15[pQE-HBscFv] was 30-35 mg/L and the affinity constant was 1.98×107 mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E(?)coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.     

The relationship between activation of TLR4 and partial hepatic ischemia/reperfusion injury in mice

BACKGROUND: Toil-like receptors (TLRs) are a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens. In this study, we elucidated the relationship between activation of TLR4 and liver injury in partial hepatic ischemia/reperfusion (I/R) injury in mice. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury, and the changes of TLR4 gene expression in ischemic liver lobes were detected with real-time polymerase chain reaction (RT-PCR). The levels of plasma ALT and endotoxin in the portal vein were measured. TLR4-deficient mice (C3H/Hej) and wild type mice (C3H/Heouj) were used in a model of I/R injury; liver function impairment and the level of serum TNF-α were observed. RESULTS: After one hour ischemia, the expression of TLR4 mRNA increased at the 1st, 3rd hour of reperfusion, indicating the value of △Ct(1st hour: 1.21±0.87vs. 5.85±1.07, t=13.72, P<0.01; 3rd hour: 0.85±0.92vs. 6.11±1.24, t=16.33, P<0.01). No endotoxemia developed in every group of mice. At the 3rd hour of reperfusion, the level of serum TNF-α was significantly higher than that of sham group (Hej: 152±43 pg/ml vs. 18±10pg/ml, t=5.26, P<0.01; Heouj: 249±52pg/ml vs. 25±13 pg/ml, t=7.24, P<0.01). At the 1st, 3rd hour reperfusion, the level of plasmid ALT in Hej mice was lower than that in Heouj mice (1st hour 662±106 U/Lvs. 1216±174 U/L, t=4.21, P<0.01; 3rd hour 1145±132U/L vs. 29584±187 U/L, t=13.72, P<0.01). The level of serum TNF-αwas lower than that in Heouj mice (1524.43 U/L vs. 2494±52 U/L, t=3.94, P<0.01) at the 3rd hour reperfusion. CONCLUSION: TLR4 activation causes partial hepatic I/R injury through release of TNF-α.     

The molecular and cellular mechanisms of itch and the involvement of TRP channels in the peripheral sensory nervous system and skin

Itch is an unpleasant cutaneous sensation that can arise following insect bites, exposure to plant ingredients, and some diseases. Itch can also have idiopathic causes. Itch sensations are thought to protect against external insults and toxic substances. Although itch is not directly lethal, chronic and long lasting itch in certain diseases can worsen quality of life. Therefore, the mechanisms responsible for chronic itch require careful investigation. There is a significant amount of basic research concerning itch, and the effect of various itch mediators on primary sensory neurons have been studied. Interestingly, many mediators of itch involve signaling related to transient receptor potential (TRP) channels. TRP channels, especially thermosensitive TRP channels, are expressed by primary sensory neurons and skin keratinocytes, which receive multimodal stimuli, including those that cause itch sensations. Here we review the molecular and cellular mechanisms of itch and the involvement of TRP channels in mediating itch sensations.     

A novel player in cellular hypertrophy: Giβγ/PI3K-dependent activation of the RacGEF TIAM-1 is required for α₁-adrenoceptor induced hypertrophy in neonatal rat cardiomyocytes

Activation of α(1)-adrenoceptors (α(1)-AR) by high catecholamine levels, e.g. in heart failure, is thought to be a driving force of cardiac hypertrophy. In this context several downstream mediators and cascades have been identified to potentially play a role in cardiomyocyte hypertrophy. One of these proteins is the monomeric G protein Rac1. However, until now it is unclear how this essential G protein is activated by α(1)-AR agonists and what are the downstream targets inducing cellular growth. By using protein-based as well as pharmacological inhibitors and the shRNA technique, we demonstrate that in neonatal rat cardiomyocytes (NRCM) Rac1 is activated via a cascade involving the α(1A)-AR subtype, G(i)βγ, the phosphoinositide-3'-kinase and the guanine nucleotide exchange factor Tiam1. We further demonstrate that this signaling induces an increase in protein synthesis, cell size and atrial natriuretic peptide expression. We identified the p21-activated kinase 2 (PAK2) as a downstream effector of Rac1 and were able to link this cascade to the activation of the pro-hypertrophic kinases ERK1/2 and p90RSK. Our data thus reveal a prominent role of the α(1A)-AR/G(i)βγ/Tiam1-mediated activation of Rac1 and its effector PAK2 in the induction of hypertrophy in NRCM.     

The association between the decreased expression levels of FOXJ1 and the activation of NF-κB pathway in interstitial lung disease of MRL/Lpr mice

Background: Pulmonary manifestations of systemic lupus erythematosus (SLE) are appearing in 4-5% of patients involving lung in almost half of the cases during the disease course. Objective: We compared the autoimmune pulmonary inflammation in the lung tissue of mice to determine the association between decreased expression levels of Forkhead Box J1 (FOXJ1) and the activation of the NF-κB pathway in autoimmune pulmonary inflammation of MRL/Lpr mice. Methods: The female BALB/c mice (n=6) and MRL/Lpr mice (n=30) were divided into 5 groups including a control group (BALB/c), and five MRL/Lpr mice groups (8W, 12W, 16W, 24W, and 32W). The infiltration of the inflammatory cells was determined in lung tissue by performing the histological analysis. The western blotting was used to examine the expression levels of the age-related FOXJ1, and p50 and p65 proteins in the lungs of MRL/Lpr mice. The expression levels of MMP2 and MMP9 were determined via immunohistochemistry and immunofluorescence. Results: There were severe infiltrates of lung cells with high levels of tracheal damage, perivascular injury and interstitial inflammatory cell infiltration when the MRL/Lpr mice from 16w to 32w comparing to the 8w old healthy MRL/Lpr mice in the control group (p <0.05). Moreover, the reduced expression levels of FOXJ1 were associated with the activation of the NF-κB pathway in interstitial lung disease of MRL/Lpr mice via the modulation of p50 and p65. In addition, the expression levels of MMP2 and MMP9 pro-inflammation factors increased in the lungs of the MRL/Lpr mice from 16w to 32w. Conclusions: The expression level of FOXJ1 might be an indicator of the degree of lung disease in lupus-prone mice.     

Controversial issues regarding the roles of IL-10 and IFN-γ in active/inactive chronic hepatitis B

According to the important roles played by cytokines in induction of appropriate immune responses against hepatitis B virus(HBV),Dimitropoulou et al have examined the important cytokines in their patients.They showed that the serum levels of interleukin 10(IL-10)and interferon-γ(IFN-γ)were decreased in patients with HBeAg-negative chronic active hepatitis B compared with the inactive hepatitis B virus carriers(Dimitropoulou et al 2013).The controversy can be considered regarding the decreased serum levels of IFN-γin the HBeAg-negative chronic active hepatitis B patients.They concluded that subsequent to decreased expression of IFN-γ,the process of HBV proliferation led to liver diseases.Previous studies stated that HBV is not directly cytopathic for the infected hepatocytes and immune responses are the main reason for destruction of hepatocytes(Chisari et al,2010).Scientists believe that immune responses against HBV are stronger in active forms of chronic HBV infected patients than inactive forms(Zhang et al,2012).Therefore,the findings from Dimitropoulou et al may deserve further attention and discussion.Additionally,downregulation of IL-10 inchronically active hepatitis B infected patients has also confirmed our claim.IL-10 is an anti-inflammatory cytokine and its expression is increased in inactive forms in order to downregulate immune responses(Arababadi et al,2012).Thus,based on the results from Dimitropoulou et al,it can be concluded that increased immune responses in chronically active hepatitis B infected patients are related to declined expression of IL-10 and interestingly IFN-γis not involved in induction of immune responses in these patients.     

N-acetylcysteine inhibits activation of toll-like receptor 2 and 4 gene expression in the liver and lung after partial hepatic ischemia-reperfusion injury in mice

BACKGROUND:Toll-like receptor 2 and 4(TLR2/4)may play important roles in ischemia-reperfusion(I/R)injury, and N-acetylcysteine(NAC)can prevent the generation of reactive oxygen species(ROS)induced by I/R injury.This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS:BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group(SH),a hepatic ischemia/ reperfusion group(I/R)or a NAC pretreated,hepatic I/R group(I/R-NAC).The levels of TNF-αin the portal vein and plasma alanine aminotransferase(ALT)were measured at 1 and 3 hours after reperfusion.The lung wet-to-dry ratio was measured,and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS:Compared with the I/R group,the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05).The levels of portal vein TNF-αand plasma ALT increased continuously in the I/R group at 1 and 3 hours of reperfusion compared with the SH group;however,they declined significantly in the group pretreated with NAC (P<0.05).The extent of lung edema was relieved in the I/ R-NAC group compared with the I/R group(P<0.05).CONCLUSIONS:TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury.NAC inhibited the activation of TLR2/4 and the induction of TNF-α resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.