Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan

It was observed that levansucrase from Bacillus natto became unstable and was easily inactivated when the salts were removed from the enzyme solution, while the enzyme was stable for long time in a buffered saline. After modification with periodate oxidized yeast glucomannan, the enzyme increased thermal stability up to 45°C, in which it conserved more than 90% of its activity after 15 min treatement. The optimum temperature was also shifted from 40°C in the case of original enzyme to 50°C for the modified enzyme after 10 min reaction time. The half-life time increased significantly from 9 min to 55 min at 50°C, however it increased from 30 min and 22 min respectively at 40°C and 45°C to more than 1 h at the same temperature. The content of carbohydrates of modified enzyme was 25% that increases the molecular weight from 57 KDa to 80 KDa. The products from sucrose by the modified enzyme were the same as the case using original enzyme. Namely, the products confirmed were levan and 3 kestoses (6-, 1-, and neo-kestose).     

New insights into the ear region anatomy and cranial blood supply of advanced stem Strepsirhini: Evidence from three primate petrosals from the Eocene of Chambi,Tunisia

We report the discovery of three isolated primate petrosal fragments from the fossiliferous locality of Chambi (Tunisia), a primate-bearing locality dating from the late early to the early middle Eocene. These fossils display a suite of anatomical characteristics otherwise found only in strepsirhines, and as such might be attributed either to Djebelemur or/and cf. Algeripithecus, the two diminutive stem strepsirhine primates recorded from this locality. Although damaged, the petrosals provide substantial information regarding the ear anatomy of these advanced stem strepsirhines (or pre-tooth-combed primates), notably the patterns of the pathway of the arterial blood supply. Using μCT-scanning techniques and digital segmentation of the structures, we show that the transpromontorial and stapedial branches of the internal carotid artery (ICA) were present (presence of bony tubes), but seemingly too small to supply enough blood to the cranium alone. This suggests that the ICA was not the main cranial blood supply in stem strepsirhines, but that the pharyngeal or vertebral artery primitively ensured a great part of this role instead, an arterial pattern that is reminiscent of modern cheirogaleid, lepilemurid lemuriforms and lorisiforms. This could explain parallel loss of the ICA functionality among these families. Specific measurements made on the cochlea indicate that the small strepsirhine primate(s) from Chambi was (were) highly sensitive to high frequencies and poorly sensitive to low frequencies. Finally, variance from orthogonality of the plane of the semicircular canals (SCs) calculated on one petrosal (CBI-1-569) suggests that Djebelemur or cf. Algeripithecus likely moved (at least its head) in a way similar to that of modern mouse lemurs.     

Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate     

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6′β-hydroxy-lovastatin, 3′α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

     

Isolation and Characterization of Moderately Halophilic Bacteria from Tunisian Solar Saltern

Bacterial screenings from solar saltern in Sfax (Tunisia) lead to the isolation of 40 moderately halophilic bacteria which were able to grow optimally in media with 5–15% of salt. These isolates were phylogenetically characterized using 16S rRNA gene sequencing. Two groups were identified including 36 strains of Gamma-Proteobacteria (90%) and 4 strains of Firmicutes (10%). The Gamma-Proteobacteria group consisted of several subgroups of the Halomonadaceae (52.5%), the Vibrionaceae (15%), the Alteromonadaceae (10%), the Idiomarinaceae (7.5%), and the Alcanivoracaceae (5%). Moreover, three novel species: 183ZD08, 191ZA02, and 191ZA09 were found, show <97% sequence similarity of the 16S rRNA sequences while compared to previously published cultivated species. Most of these strains (70%) were able to produce hydrolases: amylases, proteases, phosphatases, and DNAases. Over the isolates, 60% produced phosphatases, 15.0% proteases, 12.5% amylases and DNAases equally. This study showed that the solar saltern of Sfax is an optimal environment for halophilic bacterial growth, where diverse viable bacterial communities are available and may have many industrial applications.     

In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

In Vitro Synergistic Activities of the Hybrid Antimicrobial Peptide MelitAP-27 in Combination with Conventional Antibiotics Against Planktonic and Biofilm Forming Bacteria

The extensive use of antibiotics for the treatment of human infections during the last few decades has led to a dramatic increase in the emergence of multidrug-resistant bacteria (MDRB) among various bacterial strains. Global research is currently focused on finding novel alternative agents with different mechanisms of action rather than the use of conventional antibiotics to counteract the threat of bacterial and biofilm infections. Antimicrobial peptides represent promising alternative agents for conventional antibiotics as these molecules display a broad spectrum of activity against several microorganisms. Recently, we have designed a novel hybrid antimicrobial peptide named MelitAP-27. This peptide has been found to display potent broad spectrum and selective in vitro antimicrobial activities against a wide range of Gram-positive and Gram-negative bacteria. In the present study, the in vitro antimicrobial and antibiofilm activities of the peptide alone and in combination with five different types of antibiotics were assessed against wild-type and resistant Gram-positive and Gram-negative bacterial strains. Our results showed that most of the combination groups displayed a synergistic mode of action against planktonic and biofilm forming bacteria which resulted in decreasing the effective MIC values for MelitAP-27 to the nanomolar concentrations. These effective concentrations were associated with negligible toxicities on mammalian cells. The results of our study indicate that combinations of MelitAP-27 with conventional antibiotics may be pursued as a potential novel treatment strategy against MDRB and biofilm forming bacteria.