Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.     

Prevalent role of Akt and ERK activation in cardioprotective effect of Ca2+ channel- and beta-adrenergic receptor blockers

We studied cardioprotective as well as Akt and extracellular signal-activated kinase (ERK) activating effect of a Ca(2+) antagonist and a beta-adrenergic receptor blocker during ischemia-reperfusion, and compared these properties of the substances with that of a poly(ADP-ribose) polymerase (PARP) inhibitor used as a positive control throughout the experiments. Langendorff-perfused isolated rat hearts were subjected to 25 min global ischemia followed by 45 min reperfusion, and recovery of energy metabolism as well as functional cardiac parameters were monitored. Although to varying extents, all substances improved recovery of creatine phosphate, ATP, intracellular pH, and reutilization of inorganic phosphate. These favorable changes were accompanied by improved recovery of heart function parameters and reduced infarct size. In addition and again to varying extents, all studied substances decreased oxidative damage (lipid peroxidation and protein oxidation), and activated Akt, glycogen synthase kinase (GSK)-3beta, and ERK1/2. Correlation between cardioprotective and kinase activating effectivity of the compounds proved to be statistically significant. Physiological significance of these kinase activations was established by demonstrating that inhibition of Akt by LY294002 and ERK1/2 by PD98059 compromised the cardioprotective effect of all the substances studied. In conclusion, we demonstrated for the first time that activation of phosphatidylinositol-3-kinase (PI-3K)-Akt and ERK2 pathways significantly contributed to cardioprotective effects of a Ca(2+) antagonist and a beta-adrenergic receptor blocker. Furthermore, we found a strong correlation between cardioprotective and kinase-activating potencies of the substances studied (Verapamil, Metoprolol and two PARP inhibitors), which indicated the potentiality of these kinases as drug-targets in the therapy of ischemic heart disease.     

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162

Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5–100 μM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 μmol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p < 0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.     

Effects of fibre type and diffusion distance on mouse skeletal muscle glycogen content in vitro

In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin‐stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia‐induced‐factor‐1‐alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations. J. Cell. Biochem. 107: 1189–1197, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis and optical resolution of 1‐[(3‐carboxy‐1,1′‐biphenyl)‐2‐yl]‐1H‐pyrrole‐2‐carboxylic acid

Site selective mono‐ and dimetalation methods have been developed for the functionalization of 1‐[(1,1′‐biphenyl)‐2‐yl]‐1H‐pyrrole. Optical resolution of the prepared 1‐[(3‐carboxy‐1,1′‐biphenyl)‐2‐yl]pyrrole‐2‐carboxylic acid provided new atropisomeric 1‐arylpyrrole derivatives. The absolute configuration of the pure dicarboxylic acid enantiomers was determined by single crystal X‐ray diffraction and CD spectroscopy. Chirality 2009. © 2009 Wiley‐Liss, Inc.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

Keystone species and food webs

Different species are of different importance in maintaining ecosystem functions in natural communities. Quantitative approaches are needed to identify unusually important or influential, ‘keystone’ species particularly for conservation purposes. Since the importance of some species may largely be the consequence of their rich interaction structure, one possible quantitative approach to identify the most influential species is to study their position in the network of interspecific interactions. In this paper, I discuss the role of network analysis (and centrality indices in particular) in this process and present a new and simple approach to characterizing the interaction structures of each species in a complex network. Understanding the linkage between structure and dynamics is a condition to test the results of topological studies, I briefly overview our current knowledge on this issue. The study of key nodes in networks has become an increasingly general interest in several disciplines: I will discuss some parallels. Finally, I will argue that conservation biology needs to devote more attention to identify and conserve keystone species and relatively less attention to rarity.     

Investigation of iron pools in cucumber roots by Mössbauer spectroscopy: direct evidence for the Strategy I iron uptake mechanism

Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without ⁵⁷Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM ⁵⁷Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe^III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe^II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM ⁵⁷Fe^III-citrate for 30 min contained significant amount of Fe^II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe^II–Fe^III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants.     

Peptide conjugates of therapeutically used antitubercular isoniazid—design,synthesis and antimycobacterial effect

Tuberculosis (TB) is a bacterial infectious disease caused by Mycobacterium tuberculosis, a slow‐growing, powerful human pathogen which can survive in the host macrophages. In the chemotherapy of such intracellular pathogens it is necessary to achieve relatively high level of the drug in blood to attain therapeutically effective concentration in infected cells, which presumably has several serious side effects on healthy tissues. The elimination of M. tuberculosis from infected phagocytes could be more efficient with target cell‐directed delivery of antituberculars. A particularly promising approach is to conjugate a drug moiety to a peptide based carrier. The conjugates are chemically constructed to target release by hydrolysis (enzymatic and/or chemical) to liberate the active compound. Here we report the synthesis, characterisation and antimycobacterial evaluation of isoniazid (INH) peptide conjugates. As carrier moiety T‐cell epitope of immundominant 16‐kDa protein of M. tuberculosis and tuftsin‐derived peptides were used. To conjugate INH two synthetic methods were developed, where INH was coupled directly to the peptides or through a heterobifunctional reagent. We found that all of the INH conjugates were effective against M. tuberculosis and the minimal inhibitory concentration (MIC) values were comparable to the free INH moiety. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca²⁺ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α‐helical conformation in 50% CD₃OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca²⁺ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.