Biomedical subjects

M Coletta

Publications and source records attributed to M Coletta.

At least 19 recordsLinked to original sources

PubMed2006

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts.

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.

Acoustics↗

PubMed2003

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5.

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.

Animals↗

PubMed2002

Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis.

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively. The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.

Animals↗

PubMed2001

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration.

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Animals↗

PubMed2001

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Animals↗

PubMed2001

Effect of bezafibrate and clofibrate on the heme-iron geometry of ferrous nitrosylated heme-human serum albumin: an EPR study.

The effect of bezafibrate (BZF) and clofibrate (CF), two therapeutic drugs displaying anticoagulant and antihyperlipoproteinemic activities, on the EPR-spectroscopic properties of ferrous nitrosylated heme-human serum albumin (HSA-heme-NO) has been investigated. In the absence of BZF and CF, HSA-heme-NO is a five-coordinate heme-iron system, characterised by an X-band EPR spectrum with a three-line splitting in the high magnetic field region. Addition of BZF and CF to HSA-heme-NO induced the transition towards a six-coordinate heme-iron species characterised by an X-band EPR spectrum with an axial geometry. These data indicate that HSA-heme-NO is a five-coordinate heme-iron system, BZF and CF acting as allosteric effectors, and show that the primary heme binding site and the CF cleft of HSA are conformationally-linked, regardless of their different location.

Bezafibrate↗

PubMed2001

Immunodetection of lactosylated proteins as a useful tool to determine heat treatment in milk samples.

This paper reports the optimisation of a competitive immunoassay (ELISA) to detect lactosylated proteins in milk samples. The assay employs monoclonal antibodies for lactosylated proteins produced in our laboratory and requires no pre-treatment of the samples other than a dilution step. Monoclonal antibodies were fully characterised in terms of selectivity and cross-reactivity with structurally related molecules and used in a competitive assay format with lactosylated standard proteins (lactosylated ovalbumin). The detection limit for lactosylated ovalbumin was 0.015 microgram ml-1 and the working range was from 0.010 to 40 micrograms ml-1. The data obtained indicate that the ELISA developed is applicable to diluted milk samples and is able to distinguish between milk samples that have undergone different heat treatments (UHT and pasteurised milk).

Animals↗

PubMed2001

Different functional modulation by heterotropic ligands (2,3-diphosphoglycerate and chlorides) of the two haemoglobins from fallow-deer (Dama dama).

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same alpha chain, while two different beta chains are detected in the two components. The beta chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16,023 and 16,064 Da, respectively, while alpha chain has 141 residues, yielding a molecular mass of 15,142 Da. Compared with human Hb, the N-terminal region of both beta chains shows deletion of Val beta 1 and the replacement of His beta 2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between beta chains of the two haemoglobin components.

2,3-Diphosphoglycerate↗

PubMed2001

Standardized method of measuring acanthamoeba antibodies in sera from healthy human subjects.

Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P = 0.0025) and had lower mean absorbance values (P = 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities.

Acanthamoeba↗

PubMed2000

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c.

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.

Animals↗

PubMed2000

Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket.

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an 'alkaline' and an 'acid' transition), which indicates that (at least) two protonating groups change their pK(b) values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His(173), in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N(delta) of the proximal imidazole group is redox-linked and that it is crucial in regulating the 'alkaline' transition. On the other hand, none of the proximal mutants alters the 'acid' transition, suggesting that it is modulated by groups located in a different portion of the protein.

Electrochemistry↗

PubMed2000

Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates.

The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.

Animals↗

PubMed2000

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study.

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

Bacterial Proteins↗

PubMed2000

pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B.

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the neutrophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.

Amino Acid Sequence↗

PubMed2000

Anion- and pH-linked conformational transition in horseradish peroxidase.

In a previous study we have shown that bringing horseradish peroxidase to pH 3.0 induces a spectroscopic transition (G. Smulevich et al., Biochemistry 36 (1997) 640). We have extended the investigation on this pH-linked conformational change to different experimental conditions, such as (i) in phosphate alone, (ii) in HCl alone and (iii) in phosphate + NaCl. The data obtained allow a number of conclusions to be drawn, namely: (a) the exposure to pH 3.0 under all conditions brings about an alteration of the distal portion of the heme pocket, leading to the rapid formation of a hexa-coordinated species; (b) only in the presence of phosphate is the hexa-coordination followed by a slow cleavage (or severe weakening) of the proximal Fe-His bond, and (c) the rate of this second process is speeded up in the presence of Cl- ions. Such observations underline the presence of a communication pathway between the two opposite sides of the heme pocket, such that any alteration of the structural arrangement on one side of the heme cavity is transmitted to the other, inducing a corresponding conformational change.

Anions↗

PubMed2000

Skeletal myogenic potential of human and mouse neural stem cells.

Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.

Age Factors↗

PubMed2000

Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents.

New heterocyclic derivatives of ethylpyridylthiourea, quinoxalinylethylpyridylthiourea (QXPT) and analogues, inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevented HIV-1 cytopathogenicity in T4 lymphocytes. Several of these novel non-nucleoside RT inhibitors, with a substituted pyrroloquinoxalinone heteroaromatic skeleton, showed inhibitory activity against wild-type RT as well as against mutant RTs containing the single amino acid substitutions L1001, K103N, V106A, Y1811 and Y188L that was much greater than other non-nucleoside inhibitors such as nevirapine. Maximum potency in enzymatic assays was achieved with a fluoropyrroloquinoxaline skeleton linked to the ethylpyridylthiourea moiety (FQXPT). In cell-based assays on different cell lines and on human monocyte-macrophages, 6-FQXPT exhibited EC50 values in the nanomolar range, with a promising selectivity index. Moreover, 6-FQXPT showed synergistic antiviral activity with zidovudine.

Amino Acid Substitution↗

PubMed2000

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

Amino Acid Sequence↗