FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway, independently of mTOR/HIF-1α activation.

BACKGROUND AND PURPOSE: FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved. EXPERIMENTAL APPROACH: Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa. KEY RESULTS: Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients. CONCLUSIONS AND IMPLICATIONS: Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.

Simultaneous detection of two breast cancer-related miRNAs in tumor tissues using p19-based disposable amperometric magnetobiosensing platforms.

A novel magnetobiosensing approach for the rapid and simultaneous detection of two breast cancer-related miRs (miR-21 and miR-205) is reported. It involves the use of antimiR-21 and antimiR-205 specific probes, chitin-modified magnetic beads (Chitin-MBs), the p19 viral protein as capture bioreceptor and amperometric detection with the H2O2/hydroquinone (HQ) system at dual screen-printed carbon electrodes (SPdCEs). The use of SPdCEs allows the simultaneous independent amperometric readout for each target miR to be measured. The magnetosensor exhibited sensitive and selective detection with dynamic ranges from 2.0 to 10.0nM and detection limits of 0.6nM (6fmol) for both target miRs without any amplification step in less than 2h. The usefulness of the approach was evaluated by detecting the endogenous levels of both target miRs in total RNA (RNAt) extracted from metastatic breast cancer cell lines and human tissues.

Establishment and characterisation of a human carcinoma cell line with acquired resistance to Aplidin.

Aplidin (APL) is a new antitumoral drug from marine origin currently in phase II clinical trials against a wide multiplicity of cancers. As resistance may be, as with other drugs, an important obstacle to the APL therapeutic efficacy, we have established an acquired resistance cellular model by continuous exposure of HeLa cells to the drug. The stably resistant subline generated (HeLa-APL), possessing more than 1000-fold relative resistance to APL than parental cells, did not show crossresistance to a subset of clinically relevant antitumoral agents. In addition, resistance was not related to overexpression of P-glycoprotein or differences in overall drug accumulation. Comparing to parental cells, HeLa-APL cells did not present either significant differences in the growth rate or apparent alterations in the cell cycle distribution. Aplidin induced rapid and persistent phosphorylation of both JNK and p38 MAPKs, resulting in activation of the mitochondrial apoptotic pathway in parental cells, but, notably, in HeLa-APL-resistant cells MAPKs activation only occurred in a slight and transiently manner, failing to activate the above-mentioned apoptotic machinery. These results suggest that sustained activation of JNK and p38 is essential for triggering the apoptotic programme induced by APL and that HeLa-APL cells bypass this apoptotic response by preventing the specific mechanisms that prime and sustain the long-term activation of these signalling cascades. Although far from human tumour physiology in vivo, HeLa-APL cells represent a potentially useful tool in gaining insights into the mode of action of APL, in selecting non-crossresistant APL structural analogues, as well as in investigating and developing methods to prevent resistance to this drug.

Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris.

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.

SB-253514 and analogues; novel inhibitors of lipoprotein-associated phospholipase A2 produced by Pseudomonas fluorescens DSM 11579. I. Fermentation of producing strain, isolation and biological activity.

Two new classes of inhibitors of LpPLA2 have been identified in fermentations of Pseudomonas fluorescens. The two structurally isomeric series differ in the geometry of closure of the bicyclic carbamate and comprise a range of compounds varying only in length of their lipophilic sidechain. The most abundant species were extracted from the cells and purified by silica and C18 chromatography. Members of the more stable class were shown to be potent and selective competitive inhibitors of LpPLA2.

Synergistic neurite-outgrowth promoting activity of two related axonal proteins, Bravo/Nr-CAM and G4/Ng-CAM in chicken retinal explants.

In the developing chicken retina, optic fibres migrating to the tectum express on their surfaces several cell adhesion molecules, including Bravo/Nr-CAM and G4/Nr-CAM and G4/Ng-CAM. We have previously described differential distribution along the retinotectal projection and differential modulation by environmental cues for Bravo and G4 and here we further compare the characteristics of these immunoglobulin superfamily molecules. From day 6 of embryonic development (E6) to 20 (E20), Bravo and G4 were found to coexist in the retinal optic fibre layer. However, while G4 staining was confined to that layer, as development proceeded Bravo staining spread to plexiform layers and some radial structures of the retina. G4 displayed a dose-dependent neurite-outgrowth promoting activity for E6 retinal explants, while Bravo did not support neurite growth. Surprisingly, when the retinal explants were grown on mixtures of the two molecules, a much more vigorous growth of neurites was seen, revealing a synergistic effect. We propose that Bravo and G4, as well as other axonal surface molecules, affect axonal growth in different ways when they are present in combination than when they are alone.

Immobilization and single-step purification of fusion proteins using DEAE-cellulose.

We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain--beta-galactosidase chimera, which can be purified by this procedure and shows a high beta-galactosidase activity when immobilized in the column. A vector plasmid, pCUZ1, containing the lppp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-beta-D-galactoside procedure. A chimera between the choline-binding domain and the pneumococcal hemolysin was also constructed and purified using pCUZ1.

Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases.

The cloning in Escherichia coli of the 3' moieties of the lytA and cpl-1 genes is described, coding for the C-terminal regions of the lytic amidase of Streptococcus pneumoniae and the phage Cp-1 lysozyme, respectively. The truncated genes were overexpressed in E. coli and the purified polypeptides showed a great affinity for choline, although they were devoid of cell wall-degrading activity. Biochemical and circular dichroism analyses indicated that these are the domains responsible for the specific recognition of the choline-containing pneumococcal cell walls by the lytic enzymes. The data presented here suggested that these choline-binding domains can function independently of their catalytic domains.

Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages.

The nucleotide sequences of genes cpl7 and cpl9 of the Streptococcus pneumoniae bacteriophages Cp-7 and Cp-9, encoding the muramidases CPL-7 and CPL-9, respectively, have been determined. The N-terminal domains of CPL-7 and CPL-9 were virtually identical to that previously reported for the CPL-1 muramidase. The C-terminal domain of the CPL-7 muramidase, however, was different from those of the host amidase and the phage Cp-1 and Cp-9 lysozymes. Whereas all enzymes studied are characterized by repeated sequences at their C termini, the repeat-unit lengths are 20 amino acids (aa) in CPL-1, CPL-9 and in the host amidase, but 48 aa in CPL-7. Six repeated sequences represent the C-terminal domains of CPL-1, CPL-9 and the host amidase, and 2.8 perfect tandem repetitions that of CPL-7. The peculiar characteristics of the structure of CPL-7 muramidase correlate with its biochemical and biological properties. Whereas CPL-1, CPL-9 and the pneumococcal amidase strictly depend on the presence of choline-containing cell walls for activity, CPL-7 is able to degrade cell walls containing either choline or ethanolamine. These results support the previously postulated role for the C-terminal domain of these lytic enzymes in substrate recognition and provide further experimental evidence supporting the notion that the proteins have evolved by an exchange of modular units.

Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages.

A 2.9-kilobase Acc I fragment of the DNA of the pneumococcal bacteriophage Cp-1, containing the cpl gene, hybridizes with the lytA gene encoding the pneumococcal amidase. The nucleotide sequence of the cpl gene of Cp-1, encoding a muramidase (CPL), has been determined. The 3' regions of the cpl and lytA coding sequences show considerable nucleotide sequence homology and the carboxyl-terminal domains of the deduced amino acid sequences of these lysins are quite similar: 73 of the carboxyl-terminal 142 amino acid residues are identical, and of the 69 substitutions, 55 are conservative. Comparisons between CPL, the pneumococcal amidase, and the muramidase of the fungus Chalaropsis sp. (an enzyme that also degrades the pneumococcal cell wall) strongly suggest that the carboxyl-terminal domains of CPL and of the amidase might be responsible for the specific recognition of choline-containing cell walls, as well as for the noncompetitive inhibition of the catalytic activity of these enzymes by the pneumococcal lipoteichoic acid or by high concentrations of choline. In addition, the active center of these enzymes should be located in their amino-terminal domains. Our results suggest an evolutionary relationship between phage and host lysins.

Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae.

A pneumococcal recombinant plasmid, pRG2, containing the lytA gene that codes for the pneumococcal N-acetylmuramoyl-L-alanine amidase has been constructed using the pneumococcal plasmid pLS1 as a vector. pRG2 was introduced by genetic transformation into a mutant of Streptococcus pneumoniae (M31) that has a complete deletion of the lytA gene. The transformed strain (M51) grew at a normal growth rate as 'diplo' cells and underwent autolysis at the end of the exponential phase of growth, two properties that had been lost in the deleted mutant M31. M51 lysed very rapidly at the end of the exponential phase when the cells were grown in choline-containing medium probably because of the higher level of amidase activity present in this strain as compared to the lysis-prone strain M11. These findings show that the expression of the plasmid-linked gene was placed under the mechanism(s) of control of the cell during the exponential phase. Our results demonstrate that the physiological role of the pneumococcal amidase was to catalyze the separation of the daughter cells at the end of the cell division to produce diplo cells; in addition we have also confirmed the basic role of this autolysin in the bacteriolytic nature of beta-lactam antibiotics.

3'-end modifications of the Streptococcus pneumoniae lytA gene: role of the carboxy terminus of the pneumococcal autolysin in the process of enzymatic activation (conversion).

Plasmids containing modifications at the 3' end of the lytA gene encoding the pneumococcal amidase were constructed by DNA recombinant techniques. Several deleted and fused amidases were obtained. These modified amidases were capable of degrading cell walls containing choline residues in their teichoic acid components without need of conversion (i.e., change of the inactive E form of amidase to the active C form). The reintroduction of as few as the terminal 11 amino acid (aa) residues present in the wild-type (wt) amidase into the sequence of the most extensively deleted form of the autolysin obtained in this work (E-520) partially restored the need of conversion. Our results demonstrate the importance of the C terminus for the catalytic activation of the wt amidase.

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae.

A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.

Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene.

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the beta-lactam antibiotics.

Molecular characterization of an autolysin-defective mutant of Streptococcus pneumoniae.

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.