Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

An Analysis of the Electron Spin Resonance of Low Spin Ferric Heme Compounds

The electron spin resonance spectra of low spin ferric heme proteins are calculated and compared with experimental spectra recently obtained for a large number of heme proteins. On the basis of observed g values, these heme proteins have been categorized into five prototypes. Since electron spin resonance spectra are quite sensitive to small changes in the local environment about the paramagnetic ion, we have explored in some detail, the possibility that the g value variation in these different types of proteins is due to systematic environmental changes. To this end, we have calculated the g values as a function of tetragonal and rhombic distortions and compared our results with observed behavior. In addition we have also calculated the energy intervals between the low spin states in zero magnetic field and the temperature dependent magnetic moments and studied the effect on these properties also of small symmetry changes.     

Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters

The seeding of cells onto biocompatible scaffolds is a determinant step in the attainment of functional properties of engineered tissues. Efficient, fast and spatially uniform cell seeding can improve the clinical potential of engineered tissue templates. One way to approach these cell seeding requirements is through bioreactor design. In the present study, bovine chondrocytes were seeded (2.5, 5.0 or 10.0 million cells per scaffold) onto polyglycolic acid scaffolds within the hydrodynamic environments of wavy-walled and spinner flask bioreactors. Previous characterizations of the hydrodynamic environment in the vicinity of constructs cultivated in these bioreactors suggested decreased flow-induced shear stress as well as increased recirculation and magnitude of the axial fluid velocities in the wavy-walled bioreactor. Here we report more efficient and spatially uniform cell seeding in the wavy-walled bioreactor, and at intermediate initial cell densities (5 million cells per scaffold). This study constitutes an important step towards the achievement of functional tissue-engineered implants by (i) increasing our understanding of the influence of hydrodynamic parameters on the efficiency and spatial distribution of cell attachment to scaffolds and the production of extracellular matrix and (ii) introducing a comprehensive approach to the investigation of the effects of bioprocessing conditions on tissue morphology and composition.     

Impact of biogenic substrates on sulfamethoxazole biodegradation kinetics by <Emphasis Type="Italic">Achromobacter denitrificans</Emphasis> strain PR1

Pure cultures have been found to degrade pharmaceutical compounds. However, these cultures are rarely characterized kinetically at environmentally relevant concentrations. This study investigated the kinetics of sulfamethoxazole (SMX) degradation by Achromobacter denitrificans strain PR1 at a wide range of concentrations, from ng/L to mg/L, to assess the feasibility of using it for bioaugmentation purposes. Complete removal of SMX occurred for all concentrations tested, i.e., 150 mg/L, 500 µg/L, 20 µg/L, and 600 ng/L. The reaction rate coefficients (k_bio) for the strain at the ng/L SMX range were: 63.4 ± 8.6, 570.1 ± 15.1 and 414.9 ± 124.2 L/g\({\text{X}}_{\text{SS}}\)·day), for tests fed without a supplemental carbon source, with acetate, and with succinate, respectively. These results were significantly higher than the value reported for non-augmented activated sludge (0.41 L/(g \({\text{X}}_{\text{SS}}\)·day) with hundreds of ng/L of SMX. The simultaneous consumption of an additional carbon source and SMX suggested that the energetic efficiency of the cells, boosted by the presence of biogenic substrates, was important in increasing the SMX degradation rate. The accumulation of 3-amino-5-methylisoxazole was observed as the only metabolite, which was found to be non-toxic. SMX inhibited the Vibrio fischeri luminescence after 5 min of contact, with EC₅₀ values of about 53 mg/L. However, this study suggested that the strain PR1 still can degrade SMX up to 150 mg/L. The results of this work demonstrated that SMX degradation kinetics by A. denitrificans PR1 compares favorably with activated sludge and the strain is a potentially interesting organism for bioaugmentation for SMX removal from polluted waters.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

In vitro α-glucosidase inhibition by Brazilian medicinal plant extracts characterised by ultra-high performance liquid chromatography coupled to mass spectrometry

Aiming at finding natural sources of antidiabetics agents, 15 extracts from Brazilian medicinal plants of the Atlantic Forest and Amazon region were tested against α-glucosidase enzyme. Plants were selected based on the taxonomic relationships with genera including several species with antidiabetic activity. In this screening, the extracts obtained from the flowers of Hyptis monticola and the leaves of Lantana trifolia and Lippia origanoides resulted endowed with promising anti-α-glucosidase activity. The extracts from H. monticola and from L. origanoides collected in two different areas, were characterised by ultra-high performance liquid chromatography coupled to mass spectrometry. Bioassay-guided fractionation led to the identification of several enzyme inhibiting compounds, among them the mechanism of action of naringenin and pinocembrin was investigated. The two L. origanoides extracts showed differences in bioactivity and in the phytochemical profiles. The fractionation of the extract from H. monticola led to a partial loss of the inhibitory effect.     

Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501

We have sequenced the chloramphenicol resistance determinant (cat) of plasmid pIP501 from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC221 and pUB112 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions. These results demonstrate that the cat determinant of plasmid pIP501 belongs to the pC221 subgroup of CAT variants.     

The Synergistic Effect of Everolimus and Chloroquine on Endothelial Cell Number Reduction Is Paralleled by Increased Apoptosis and Reduced Autophagy Occurrence

Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.