Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid

Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation.     

Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.     

Evidence of discrete substates and unfolding pathways in green fluorescent protein

We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules' switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.     

Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks

A 16S/23S ribosomal spacer from a Haemophilus parainfluenzae rrn locus was cloned and sequenced. Analysis of PCR-amplified genomic fragments showed that this region is strongly conserved among unrelated isolates; computer analysis of database homologies showed that the spacer consists of sequence blocks, arranged in a mosaic-like structure, with strong homologies with analogous blocks present in the spacer regions of Haemophilus influenzae, Haemophilus ducreyi and Actinobacillus spp. It also contains a tRNA^Glu gene, which is highly homologous to tRNA^Glu genes found in spacers of other species. These data strongly support the hypothesis that recombination events are involved in the organisation of the sequence of the spacer, as a result of horizontal gene transfer.     

Kinetics of Replication of a Partially Attenuated Virus and of the Challenge Virus during a Three-Year Intersubtype Feline Immunodeficiency Virus Superinfection Experiment in Cats

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4⁺ T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.     

Conformations of oligoprolines with different configurational sequences and their association complexes with alkali and alkali-earth ions

To clarify the possible molecular mechanism of cation unit conductance of poly(D ,L -proline) across lipidic membranes, the structure and conformation of tetraproline derivatives with different configurational sequences in solid state, as well as in solution and in the presence of alkali and alkali-earth ions, were investigated using x-ray analysis, CD, and nmr spectroscopy. The tetraproline derivatives Boc(D -Pro-L -Pro-L -Pro-D -Pro)OBg and Boc(L -Pro)₄OBg show very different conformational versatilities in solutions as well as different propensities to form complexes with Ca²⁺ ion. This is interpreted, based on previous evidence, as due to different abilities to form ion complexing cavities. © 1998 John Wiley & Sons, Inc. Biopoly 45: 257–267, 1998     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Silver(I) poly(1,2,3-benzotriazolyl)borate complexes containing mono- and bidentate phosphine coligands

New silver(I) derivatives [Ag{H_nB(btz)_4 − n}(PR₃)_x] (n = 1 or 2, x ranging from 1 to 3), containing monodentate tertiary phosphines and anionic poly(benzotriazol-1-yl)borates, have been prepared from the reaction of AgNO₃ with PR₃ (R = Ph, o-tolyl, m-tolyl, p-tolyl, Bns) and K[H₂B(btz)₂], or K[HB(btz)₃] (Hbtz = 1,2,3-benzotriazole). When the reaction between K[H₂B(btz)₂] and AgNO₃ was carried out in the presence of dppe (1,2-bis(diphenylphosphino)ethane), or dppf (1,1′-bis(diphenylphosphino)ferrocene), compounds [Ag{H₂B(btz)₂}]₂(L) (L = dppe or dppf) formed, the diphosphine acting as a bidentate bridging P₂-donor. Solid state and solution properties of all complexes have been investigated through analytical and spectroscopic measurements (IR, ¹H, ³¹P NMR), the ¹H and ³¹P NMR spectra being interpreted in terms of equilibria that involve mono- and di-nuclear complexes. Adducts [Ag{HB(btz)₃}(PPh₃)₃] · (1/2H₂O) and [Ag{H₂B(btz)₂}]₂ (dppf) have been characterised by single crystal X-ray studies. In the former, the HB(btz)₃ is unidentate in an NAgP₃ coordination environment; the latter is a dimer, the dppf bridging the two silver atoms, while the H₂B(btz)₂ ligand, which chelates one silver, bridges to the second also, the array having 2-symmetry.     

Silver (I) poly(1,2,4-triazolyl)borate complexes containing monodentate phosphane ligands

New silver (I) derivatives containing monodentate tertiary phosphanes and anionic poly(triazol-1-yl)borate ligands have been prepared from the reaction of AgNO₃ and PR₃ (R = Ph, Bn, o-tolyl, m-tolyl, p-tolyl) and potassium dihydrobis(1,2,4-triazolyl)borate, K[H₂B(tz)₂], or potassium hydrotris(1,2,4-triazolyl)borate, K[HB(tz)₃]; their solid state and solution properties have been investigated through analytical and spectroscopic measurements (IR, ¹H-, and ³¹P NMR). The ¹H- and ³¹P NMR solution spectra in some cases can be interpreted on the basis of a dissociation of [{H₂B(tz)₂}Ag(PR₃)₂] into [{H₂B(tz)₂}Ag(PR₃)] and PR₃. All the compounds are soluble in chlorinated solvents and are non-electrolytes in CH₂Cl₂ and acetone solutions. [{H₂B(tz)₂}Ag(PPh₃)₂] and [{H₂B(tz)₂}Ag{P(m-tolyl)₃}₂] are simple mononuclear arrays, the silver atoms lying in four-coordinate N₂AgP₂ environments. Owing to the presence of the methyl substituents on the phosphane ligand, the complex [{HB(tz)₃}Ag{P(o-tolyl)₃}], as expected, is mononuclear. In [{H₂B(tz)₂}Ag{P(p-tolyl)₃}], the silver environment is still four-coordinate but PAgN₃, utilizing the coordinating capability of one of the additional (‘exo’-) ring nitrogens not only to complete the four-coordinate array about the silver but, necessarily, to link successive asymmetric units into a single-stranded polymer.