Angiotensin II infusion induces marked diaphragmatic skeletal muscle atrophy

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD.     

Electrochemical screening of the indole/quinolone derivatives as potential protein kinase CK2 inhibitors

An electrochemical method based on the bioorganometallic Fc-ATP cosubstrate for kinase-catalyzed phosphorylation reactions was used for monitoring casein kinase 2 (CK2) phosphorylations in the absence and presence of five indole/quinolone-based potential inhibitors. Fc-phosphorylation of immobilized peptide RRRDDDSDDD on Au surfaces resulted in a current density at approximately 460 ± 10 mV. An electrochemical redox signal was significantly decreased in the presence of inhibitors. In addition, the electrochemical signal was concentration dependent with respect to the potential inhibitors 1 to 5, which proved to be viable CK2 drug targets with estimated IC₅₀ values in the nanomolar range.     

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.     

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.Influenza A viruses can infect a wide range of avian and mammalian species (49). Most avian strains of influenza virus infect wild waterfowl and domestic poultry but usually do not spread to humans. However, adaptation of pathogenic avian viruses to humans can occur either by mutation or reassortment, leading to potentially very serious pandemics, as was the case in 1918 when the “Spanish” flu caused 20 to 40 million deaths worldwide (33). Due to this ability to cross the species barrier, influenza A viruses are a permanent threat to human health. Since 2005 the spread of highly pathogenic H5N1 avian strains in Asia, Europe, and Africa has raised serious concern about the potential of this strain to cause an influenza pandemic (50). Since early 2009, an ongoing new, rapidly evolving pandemic threat has arisen from the emergence of a highly contagious, interhuman-transmissible “quadruple reassortant” swine H1N1 virus to which the world population is antigenically naïve (6).Influenza A viruses are enveloped viruses of the orthomyxovirus family whose genomes comprise eight negative-strand RNA segments (2). In contrast to many RNA viruses, the influenza virus genome is transcribed and replicated by the trimeric viral RNA polymerase (PA, PB1, and PB2) in the nuclei of the infected cells. Therefore, the polymerase subunits, which are produced in the cytoplasm, have to be imported into the nucleus and assembled into a functional trimer (2, 18). Many studies have demonstrated that the viral polymerase plays a major role in host specificity, probably due to the necessity for the polymerase subunits to adapt to host cell-interacting partners such as nuclear import factors (13, 16, 25, 37, 46). Due to the lack of in vivo data concerning the interactions between the polymerase subunits in the nucleus and the cytoplasm of the host cells, the mechanisms of polymerase assembly and nuclear import, as well as their spatial and temporal relationships, are still not completely understood. Putative nuclear localization signals (NLSs) have been identified on PB1 (31), PB2 (29), and PA (32), suggesting that each subunit could be imported separately. However, based on in vitro assembly observations and cellular localization studies (8, 9, 12), it has been proposed that PB1 and PA are imported into the nucleus as a subcomplex by import factor RanBP5 (a member of the importin β superfamily). PB2 is thought to enter the nucleus separately, probably via the canonical importin α/importin β pathway (46), and then associates with the PB1/PA heterodimer in the nucleus to form the functional trimeric polymerase. Nevertheless, alternative pathways have also been proposed. Naito et al. (30) suggested that the nuclear import of PB1 requires the formation of a PB2/PB1 heterodimer, stabilized by Hsp90, in the cytoplasm, while PA is transported in the nucleus separately. More recently, a pathway in which the PA/PB2 heterodimer would be formed in the cytoplasm and then imported into the nucleus has been proposed (17). It has also been recently shown that efficient assembly of the trimeric polymerase could be a major limiting factor in the viability of reassortant influenza viruses (26). Since gene reassortment is an evolutionary mechanism of influenza virus which can lead to new strains with pandemic potential, a precise understanding of the processes leading to the formation of an active viral polymerase in the nuclei of infected cells is of great importance.Recent publications have demonstrated that fluorescence cross-correlation spectroscopy (FCCS) is a method of choice to study protein-protein interactions in vivo (23, 27, 42). FCCS is the dual-color extension of fluorescence correlation spectroscopy (FCS), a technique based on the analysis of the temporal fluorescence fluctuations arising from single fluorescently labeled molecules diffusing in and out of the femtoliter-scale detection volume commonly obtained with a confocal microscope. From the autocorrelation of the fluctuating signal, it is possible to extract the local concentrations and mobilities of the molecules of interest (10, 28, 39). In the case of FCCS, signals from two spectrally separated dyes labeling two different molecules are recorded. If the two molecules interact with each other, they diffuse synchronously through the detection volume, resulting in correlated fluctuations in the fluorescence signals acquired in the two channels. The cross-correlation between the two signals is then a direct and quantitative readout of the interactions between the molecular species studied (22, 38, 40). To our knowledge, this study is the first application of FCCS to viral protein interactions and thus provides a general methodological framework to analyze the effects of different host range mutations and the interactions of viral proteins and host factors in the future.In this study, we applied FCCS to monitor the interactions between the subunits of influenza A virus RNA polymerase in live cells. Based both on the study of these interactions in the cytoplasm and nucleus and on the quantitative analysis of the intracellular localization of the subunits, we show that PB1 and PA form a heterodimer in the cytoplasm while PB2 remains a monomer in this compartment. Association of PB1/PA with PB2 to form the trimeric polymerase was detected only in the nucleus, arguing that the PB1/PA heterodimer is normally imported separately from PB2. Interestingly, when we impaired the nuclear import of PB2 by mutating its nuclear localization signal, this induced the aberrant presence of the trimeric polymerase in the cytoplasm and led to the retention of PB1 and PA outside the nucleus. Finally, by comparing the molecular brightnesses of the single polymerase subunits with that of the trimeric complex, we show that trimeric polymerase complexes can interact with each other in the nucleus to form higher-order oligomers.     

Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5

The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.     

Modulation of longevity by environmental sensing

Organisms from bacteria to humans have the capacity to gauge the quality of their respective environments. Recent advances in understanding how various types of environmental conditions are sensed and interpreted by cells and by organisms have established a critical role for these systems in the modulation of physiology, health, and aging.     

2-Alkyl(aryl)-quinazolin-4(3H)-thiones, 2-R-(quinazolin-4(3H)-ylthio)carboxylic acids and amides: synthesis,molecular docking,antimicrobial and anticancer properties

In this study, a series of novel 2-alkyl(aryl)-quinazolin-4(3H)-thiones, 2-R-(quinazolin-4(3H)-ylthio)carboxylic acids and amides were synthesized and evaluated for antimicrobial and anticancer activities. Their structure was confirmed by elemental analysis and spectral data (FT-IR, LC-MS, ¹H-NMR). Antimicrobial activity was tested in vitro against Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Candida albicans and NCI in vitro preliminary anticancer activity against nine different cancer types. The most active antibacterial and antifungal compounds were: 2.1, 2.2 and 2.4. The introduction of the carboxylic acid or amide residue into the fourth position of quinazolin-4(3H)-thione resulted in the absence of antimicrobial activity. Substance 3.8 inhibited renal cancer UO-31 line and 2.18 – leukemia CCRF-CEM. The results of in silico molecular docking for DHFR and CK2 kinase had no correlation with in vitro properties, proposing the presence of other biological activity pathways.     

Difference in stability of the N-domain underlies distinct intracellular properties of the E1064A and H1069Q mutants of copper-transporting ATPase ATP7B

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu(1064) and His(1069) is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.