Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A^pro based on sequence comparison and homology modeling with human rhinovirus 2A^pro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A^pro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A^pro structure.     

Analysis of beta-hemolysis in human blood agars by Streptococcus pyogenes

The aim of the study was to assess the reliability of human blood agar media (HuBA) in identifying Streptococcus pyogenes by hemolysis analysis. We analyze several factors that might affect the accuracy of HuBA media for microbial analysis, including incubation time, blood group, Rh factor and presence of antistreptolysin-o.     

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.     

Composition, antimicrobial and antioxidant activity of the extracts of Eryngium palmatum Pančić and Vis. (Apiaceae)

The chemical composition, antimicrobial and antioxidant activity of Eryngium palmatum, an endemic plant species from the Balkan Peninsula, were investigated. The flavonoids apigenin (9.5±0.3 mg g^?1) and apigenin 7-O-glucoside (2.4±0.1 mg g^?1) were determined in a methanol extract of aerial parts using HPLC analysis. The methanol extract of roots contained catechin (5.0±0.1 mg g^?1), epicatechin (2.9±0.1 mg g^?1), chlorogenic acid (1.6±0.0 mg g^?1), gallic acid (0.9±0.0 mg g^?1) and rosmarinic acid (0.9±0.2 mg g^?1). GC-FID and GCMS analysis of a chloroform extract of aerial parts showed that the main volatile constituents were falcarinol, linoleic acid, hexadecanoic acid and methyl linoleate (comprising 32.6%; 24.4%; 19.9; 13.2% of the volatile fraction, respectively), while octanoic acid, tetradecanol and dodecanol dominated in the chloroform extract of the roots (34.9%; 25.8%; 22.2% of the volatile fraction, respectively). Investigation of antimicrobial activity by broth microdilution showed that the methanol and chloroform extracts of aerial parts and roots exerted a significant effect (MIC 3.5–15.6 μg mL^?1) against tested Gram-positive and Gram-negative bacteria. The methanol extracts of aerial parts or roots exerted moderate ferric reducing antioxidant power, DPPH radical scavenging activity and hydroxyl radical scavenging activity.     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.     

Mouse Protocadherin-1 Gene Expression Is Regulated by Cigarette Smoke Exposure In Vivo

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.     

Biochemical and biological properties of cortexillin III,a component of Dictyostelium DGAP1–cortexillin complexes

Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.     

Conformational Itinerary of Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase during Its Catalytic Cycle

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.     

Glutamatergic Activation of Hippocampal Phospholipase D: Postnatal Fading and Receptor Desensitization

Abstract: Phospholipase D (PLD) activity was determined in rat hippocampal slices between postnatal days 3 and 35. After birth, basal PLD activity was low and, within 2 weeks, increased to reach a plateau that was maintained up to the adult age. Likewise the response to glutamate developed postnatally to reach a maximum at day 8, but then faded rapidly and was almost absent at day 35. Activation of PLD by 4β-phorbol 12β,13α-dibutyrate (PDB) was independent of age, whereas the effect of aluminum fluoride (AlF₄⁻) increased to a plateau within the first week. At day 8, PLD stimulation by glutamate via metabotropic receptors involved protein kinase C activation, but was independent of Ca²⁺ influx; the time course of PLD activation by PDB or AlF₄⁻ was linear throughout the experiment, whereas the response to glutamate or 1-aminocyclopentane-1,3-dicarboxylic acid followed a biphasic pattern: the rapid "first phase activation" desensitized within a few minutes and disclosed a small, but maintained "second phase." Pretreatment experiments confirmed desensitization of PLD activation by glutamate, but not by AlF₄⁻ or PDB. The biphasic pattern of glutamatergic PLD activation changed during development, i.e., the first phase activation faded and the second phase remained. These results were fully confirmed by the time courses of the PLD-mediated efflux of choline evoked by glutamate. In conclusion, postnatal glutamatergic activation of hippocampal PLD is composed of a pronounced and desensitizing first phase activation and a small, but nondesensitizing second phase. The first, but not the second, phase activation fades rapidly during development. The hypothesis is discussed that the glutamatergic activation of PLD occurs along different pathways in neonate and adult tissue.