Regiospecific Reduction of 4,6-Dinitrobenzimidazoles: Synthesis,Characterization, and Biological Evaluation

The regiospecific reduction of 4,6-dinitrobenzimidazole derivatives leading to the corresponding 4-amino-6-nitrobenzimidazoles was studied. The identification of the formed product structures was accomplished by spectroscopic and X-ray diffraction data. The anticancer and antiparasitic activities of the synthesized compounds were examined, and promising activities against Toxoplasma gondii and Leishmania major parasites were discovered for certain 4,6-dinitrobenzimidazoles in addition to moderate anticancer activities of the 4-amino-6-nitrobenzimidazole derivatives against T. gondii cells. However, the tumor cell experiments revealed a promising sensitivity of p53-negative colon cancer cells to these compounds.     

An alliance between Ras GTPase-activating protein, filamin C, and Ras GTPase-activating protein SH3 domain-binding protein regulates myocyte growth

We have previously reported that Ras GTPase-activating protein (RasGAP) is involved in a pathway that regulates total cellular mRNA and protein synthesis in cardiac myocytes. A yeast two-hybrid screen resulted in identification of filamin C (FLN-C) as one of its targets. Knockdown of RasGAP or FLN-C, or severing their interaction, resulted in down-regulation of the RNA polymerase II kinase, cyclin-dependent kinase 7 (Cdk7). This appeared to be provoked by the release of cdk7 mRNA from RasGAP SH3 domain-binding protein, G3BP, and its subsequent degradation. In parallel, myocyte growth was also inhibited. On the other hand, overexpression of RasGAP induced a Cdk7- and FLN-C-dependent growth. Thus, we propose that the physical interaction between RasGAP and FLN-C facilitates an interaction between G3BP and cdk7 mRNA. This results in stabilization of cdk7 mRNA, an increase in its protein, which is required for cell growth.     

RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes

Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway.     

The autolytic phenotype of Bacillus thuringiensis

AIMS: To evaluate the autolytic phenotype of Bacillus thuringiensis. METHODS AND RESULTS: The autolytic rate of 87 strains belonging to different subsp. of B. thuringiensis was examined at pH 6, 6.5 and 8.5 in different buffers under starvation conditions. At pH 6 the extent of autolysis (average in the strain collection 38.3 +/- 21.1) was strain-dependent with wide variability, while at pH 6.5 and 8.5 (averages 72.0 +/- 9.0 and 63.1 +/- 8.2, respectively) it was much more uniform with only a few strains showing low autolytic rates. Forty-one per cent of the strains showed high resistance (>/=80%) to mutanolysin, a commercial muramidase from Streptomyces. The peptidoglycan hydrolase pattern was evaluated by renaturing SDS-PAGE using cells of B. thuringiensis subsp. tolworthi HD125 as indicator. The strain collection showed seven major lytic bands of about 90, 63, 46, 38, 32, 28 and 25 kDa, and in the stationary growth phase (72 h) there was a more intense 25 kDa band in the autolytic pattern. Using Micrococcus lysodeicticus and Listeria monocytogenes as the indicators lytic activity was retained, as seen by the bands of 63, 46, 38, 32 and 25 kDa. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases in the gel, but in the presence of KCl, MgCl(2), MnCl(2) and EDTA some activity was retained. At basic pH the lytic activity increased. CONCLUSIONS: The autolytic phenotype of B. thuringiensis was found to be strain-dependent, and different proteins exibited peptidoglycan hydrolase activity, particularly at alkaline pH. Several of these proteins retained lytic activity against other bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterisation of the autolytic phenotype of B. thuringiensis should expand the prospects of using this species in bacterial bio-control and field applications.     

Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival

The survival of a cell depends on its ability to meet its energy requirements. We hypothesized that the mitochondrial reserve respiratory capacity (RRC) of a cell is a critical component of its bioenergetics that can be utilized during an increase in energy demand, thereby, enhancing viability. Our goal was to identify the elements that regulate and contribute to the development of RRC and its involvement in cell survival. The results show that activation of metabolic sensors, including pyruvate dehydrogenase and AMP-dependent kinase, increases cardiac myocyte RRC via a Sirt3-dependent mechanism. Notably, we identified mitochondrial complex II (cII) as a target of these metabolic sensors and the main source of RRC. Moreover, we show that RRC, via cII, correlates with enhanced cell survival after hypoxia. Thus, for the first time, we show that metabolic sensors via Sirt3 maximize the cellular RRC through activating cII, which enhances cell survival after hypoxia.During normal/unstressed conditions, the cell runs on a fraction of its mitochondrial bioenergetics capacity, where the difference between the maximum respiratory capacity and basal respiratory capacity is referred to as the spare or reserve respiratory capacity (RRC). In the case when energy demand exceeds supply (e.g., an increase in workload or neuronal activity), the RRC has the potential to increase supply, thus, avoiding an ‘ATP crisis''. In accordance, RRC has been shown to correlate with enhanced cell survival¹ and, conversely, reduced RRC has been associated with neuronal cell death and disease.² RRC is a well-recognized phenomenon;^{3, 4, 5, 6, 7, 8, 9} however, its components or the factors that regulate it remain unknown, or, at best, minimally defined. Not surprisingly, one of the known factors that influence the extent of the RRC is substrate availability.⁷One potential source of RRC is a regulated increase of substrate entry into the TCA cycle that is synchronized with an increase in the electron transport chain (ETC) activity. Interestingly, mammalian complex II (cII) has the unique characteristic of being a common component that links the TCA cycle and the ETC and its role in cell survival and death is well established. For example, inactivating mutations in the subunit A (SDHA) are associated with Leigh''s syndrome, which is a progressive neurodegenerative disease associated with neuronal cell death.¹⁰ Likewise, at least one case report shows that a mutation in cII is associated with heart failure,¹¹ while in Drosophila a mutation in Sdhb causes an increase in ROS production and early mortality.¹² In contrast, inhibition of cII during ischemia/reperfusion attenuates ROS-induced damage.¹³ Indeed, while inhibiting cII has been shown to induce apoptosis,¹⁴ it is also recognized as an apoptosis sensor.¹⁵ One mechanism that has been described for cII-induced apoptosis involves its disassembly in the low pH environment of distressed cells that results in excessive production of ROS from the Sdha.^{16, 17} Thus, these results would suggest that a fully assembled cII is critical for cell health and survival, while the disassembled form participates in cell demise. In this report, we show that holo-cII is the source of the RRC, which increases the cells'' resistant to cell death.     

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells,Growth, Photosynthetic Capacity,and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.     

Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius

Coffee contamination by ochratoxigenic fungi affects both coffee quality as well as coffee price with harmful consequences on the economy of the coffee exporting countries for whom which is their main source of income. Fungal strains were isolated from coffee beans and identified as black Aspergilli. Ochratoxigenic moulds like Aspergillus carbonarius were screened and selected for detailed studies. Also lactic acid bacteria (LAB) were isolated from silage coffee pulp and their antifungal activity was tested on dual-culture agar plate. Ten of the isolated LAB demonstrated antifungal effect against A. carbonarius. API 50 CH and APIZYM were used to perform phenotypic identification. 16S rDNA sequencing was made to confirm the results.     

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.     

The Mu class glutathione transferase is abundant in striated muscle and is an isoform-specific regulator of ryanodine receptor calcium channels

Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.