[Show abstract][Hide abstract] ABSTRACT: Background
In light of recent developments in nanotechnologies, interest is growing to better comprehend the interaction of nanoparticles with body tissues, in particular within the cardiovascular system. Attention has recently focused on the link between environmental pollution and cardiovascular diseases. Nanoparticles <50 nm in size are known to pass the alveolar¿pulmonary barrier, enter into bloodstream and induce inflammation, but the direct pathogenic mechanisms still need to be evaluated. We thus focused our attention on titanium dioxide (TiO2) nanoparticles, the most diffuse nanomaterial in polluted environments and one generally considered inert for the human body.Methods
We conducted functional studies on isolated adult rat cardiomyocytes exposed acutely in vitro to TiO2 and on healthy rats administered a single dose of 2 mg/Kg TiO2 NPs via the trachea. Transmission electron microscopy was used to verify the actual presence of TiO2 nanoparticles within cardiac tissue, toxicological assays were used to assess lipid peroxidation and DNA tissue damage, and an in silico method was used to model the effect on action potential.ResultsVentricular myocytes exposed in vitro to TiO2 had significantly reduced action potential duration, impairment of sarcomere shortening and decreased stability of resting membrane potential. In vivo, a single intra-tracheal administration of saline solution containing TiO2 nanoparticles increased cardiac conduction velocity and tissue excitability, resulting in an enhanced propensity for inducible arrhythmias. Computational modeling of ventricular action potential indicated that a membrane leakage could account for the nanoparticle-induced effects measured on real cardiomyocytes.Conclusions
Acute exposure to TiO2 nanoparticles acutely alters cardiac excitability and increases the likelihood of arrhythmic events.
Full-text · Article · Dec 2014 · Particle and Fibre Toxicology
[Show abstract][Hide abstract] ABSTRACT: Bis(S-citronellalthiosemicarbazonato)nickel(ii), [Ni(tcitr)2], is a compound that inhibits proliferation of tumour line U937 by inducing a G2/M block and leading the cancer cells to apoptosis. This nickel derivative shows no activity on non proliferating healthy cells. In this paper we report our studies on the action mechanisms of [Ni(tcitr)2]. Apoptosis in U937 cells exposed to [Ni(tcitr)2] takes place through activation of caspase-9, and therefore through an intrinsic triggering mechanism. Given the DNA damage observed in the Comet assay, the mutagenic activity of the metal complex was tested, including with the Ames test, micronuclei and DNA damage recovery, but neither mutagenicity nor recovery were detected. Nickel-complex-DNA interactions were analyzed by direct action of the compound on plasmidic and linear DNA by UV-vis and CD spectroscopy, gel electrophoresis and Atomic Force Microscopy. These experiments reveal that [Ni(tcitr)2] does not cause DNA breaks and does not intercalate, but significantly alters the DNA conformation creating knot-like structures and hairpins.
[Show abstract][Hide abstract] ABSTRACT: The lateral spatial resolution of modern atomic force microscopy (AFM)
is largely limited by the radius of curvature of the probe. Owing to
their extraordinary mechanical strength, large aspect-ratio, and
sub-nanometer radius, carbon nanotubes (CNTs) have emerged as the ideal
AFM probe tip material, yet existing methods for CNT-AFM probe
fabrication have not been optimized. In this work, we present a
fabrication method that yields direct control over the CNT's length,
radius, and tilt angle by using a positioning stage operated in a
transmission electron microscope (TEM) to directly attach a
single-walled CNT to the apex of an AFM probe tip. The CNT probes are
then utilized to image gold nanoparticles and DNA with tapping-mode AFM
in ambient conditions. While imaging gold nanoparticles, we report a
full-width radius dilation of 5.5 å,and nearly 8 nm resolution
enhancement compared to commercially available super sharp Si AFM
probes. We also measure a DNA fullwidth of less than 5.0 nm and observe,
in some cases, the fine structure associated with the DNA double-helix
with a pitch of 3.32 nm, which agrees well with the theoretical value of
[Show abstract][Hide abstract] ABSTRACT: Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥6log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.
Full-text · Article · Jan 2012 · Journal of microbiological methods
[Show abstract][Hide abstract] ABSTRACT: Background
Cell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria.
Results and discussion
In this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fimA or fimP) together with one or two minor pilin subunit-encoding genes (designated as fimB and/or fimQ), and a gene encoding a sortase enzyme (strA). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fimA/P and fimB/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.
Full-text · Article · Aug 2011 · Microbial Cell Factories
[Show abstract][Hide abstract] ABSTRACT: Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.
[Show abstract][Hide abstract] ABSTRACT: The atomic force microscope (AFM) is a widely used tool to image DNA and nucleoprotein complexes at the molecular level. This is because the AFM is relatively easy to operate, has the capability to image biomolecules under aqueous solutions, and, most importantly, can image mesoscopic macromolecular structures that are too complex to be studied by X-ray or NMR and too small to be visualized with the optical microscope. Although there are many AFM studies about the structure and the physical properties of DNA, only in few cases a rigorous method has been applied to analyze AFM images. This chapter describes procedures to prepare DNA and nucleoprotein complexes for AFM imaging and methods used to carry out simple image measurements to obtain structural data. In particular, methods to measure DNA contour length and the volume of free or DNA-bound proteins are presented and discussed.
No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Virulent phages are responsible for milk fermentation failures in the dairy industry, due to their ability to infect starter cultures containing strains of Lactococcus lactis. Single-strand annealing proteins (SSAPs) have been found in several lactococcal phages, among which Sak in the phage ul36. Sak has been recently shown to be a functional homolog of the human protein RAD52, involved in homologous recombination. A comparison between full-length Sak and its N- and C-terminal domains was carried out to elucidate functional characteristics of each domain. We performed HPLC-SEC, AFM and SPR experiments to evaluate oligomerization states and compare the affinities to DNA. We have shown that the N-terminal domain (1-171) is essential and sufficient for oligomerization and binding to DNA, while the C-terminal domain (172-252) does not bind DNA nor oligomerize. Modelisation of Sak N-terminal domain suggests that DNA may bind a positively charged crevice that runs external to the ring. Annealing and stimulation of RecA strand exchange indicate that only the N-terminal domain is capable of single-strand annealing and both domains do not stimulate the RecA strand exchange reaction. We propose that Sak N-terminus is involved in DNA binding and annealing while the C-terminus may serve to contact Sak partners.
No preview · Article · Jun 2010 · Journal of Structural Biology
[Show abstract][Hide abstract] ABSTRACT: The determination of the contour length of DNA imaged by either electron microscopy or atomic force microscopy is frequently required for investigating the physical properties of nucleic acids. Nevertheless, these measurements are often carried out with methods that are not optimized for the curvilinear shape of DNA or are too complex to be of practical use. The aim of this study is to provide a method for the contour length measurements of DNA that is accurate, practical, and computationally simple. Computer simulated DNA fragments were used as experimental benchmarks in order to compute the coefficients a and b of the (n(e), n(o))-characterization [L(n(e),n(o)) = an(e) + bn(o)] so as to minimize the error of the measurements. The data show that, at variance with straight lines, a DNA length estimator depends on both the DNA flexibility and the image resolution, but it is independent of the DNA contour length. A table with DNA estimators to be used for length measurements of digitized contours obtained under commonly used imaging conditions is provided. Although the method has been developed using DNA as a benchmark, its applicability can be extended to other polymers as well as to other imaging techniques.
[Show abstract][Hide abstract] ABSTRACT: Lactococcus lactis, a Gram-positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single-stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single-strand annealing protein. Two-dimensional gel electrophoresis showed that ORF34(p2) is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel-shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34(p2) interacts with single-strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34(p2) and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding-fold of SSBs. Finally, we found that ORF34(p2) is able to stimulate Escherichia coli RecA-mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34(p2) from other SSB proteins are discussed.
[Show abstract][Hide abstract] ABSTRACT: Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P(R) and P(RM) promoters of bacteriophage lambda have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P(R) [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464-4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from -36 to -59 and from -80 to -100. Likewise, RPos formed at P(RM) showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of alpha-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P(R) promoter. We find, however, that the frequency of DNA templates with both P(R) and P(RM) occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that alpha carboxy-terminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P(R) and P(RM) is proposed.
Full-text · Article · Dec 2008 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The Enterococcus faecalis conjugative plasmid pPD1 encodes proteins responsible for the mating response to the sex pheromone cPD1 secreted by a recipient cell. This response involves the respectively negative and positive determinants traA and traE, the pheromone-inhibitor determinant ipd and structural genes participating in the conjugation process. TraA is capable of binding to key sites within the regulatory gene cluster. The binding of TraA to cognate sites is modulated by the pheromone (cPD1) and the pheromone-inhibitor (iPD1) peptides. Using atomic force microscopy and classic biochemical techniques, we mapped and characterized the TraA-DNA interactions within the pPD1 regulatory gene cluster and the role of TraA in the transcription regulation of the sex pheromone response. A previous report showed that TraA binds to three adjacent sites (tab1, tab2 and tab3) located upstream of the ipd promoter region. Here, we provide direct evidence for such interactions and show that TraA alone or in the presence of iPD1 inhibits ipd transcription by preferentially binding to tab1, whereas in the presence of saturating cPD1, the overall binding to the tab sites decreases, TraA preferentially binds to tab3 and the ipd repression is relieved. Moreover, TraA alone or in the presence of iPD1 binds to two non-adjacent sites within the ipd terminators T1 and T2, an interaction that is also relieved in the presence of cPD1. The binding of TraA to the termination region of ipd may play an important role in controlling traE and traF expression via a transcriptional read-through mechanism already postulated for the pAD1 plasmid. TraA may also regulate its own expression by binding to a site in the proximity of the traA promoter, which has been relocated 200 bp downstream of the ipd gene. A model for the TraA-mediated regulation of the pPD1-encoded sex pheromone response is presented.
No preview · Article · Aug 2008 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of G-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry52:2888–2906) suggested that there are high- and low-affinity subunit conformations of G, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of G-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the “strong” allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (<10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol12:88–118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the α and β subunits. This was shown by calculating the separate α and β subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol256:775–792).
[Show abstract][Hide abstract] ABSTRACT: A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.
[Show abstract][Hide abstract] ABSTRACT: An entirely new means of printing molecular information on a planar film, involving casting nanoscale impressions of the template protein molecules in molten gallium, is presented here for the first time. The metallic imprints not only replicate the shape and size of the proteins used as template. They also show specific binding for the template species. Such a simple approach to the creation of antibody-like properties in metallic mirrors can lead to applications in separations, microfluidic devices, and the development of new optical and electronic sensors, and will be of interest to chemists, materials scientists, analytical specialists, and electronic engineers.
Full-text · Article · Oct 2007 · Biosensors & Bioelectronics
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy against the amyloid-β (Aβ) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Aβ, and yet be capable of eliciting antibodies that recognize Aβ fibrils and neurotoxic Aβ oligomers but not the physiological monomeric species of Aβ. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Aβ1-15 (Aβ15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Aβ15)n polypeptides bearing one, four, or eight copies of Aβ15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Aβ15)4 antibody, in particular, recognized Aβ42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Aβ. We have also demonstrated that anti-Trx(Aβ15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Aβ15)4 as a promising new tool for AD immunotherapy.
Full-text · Article · May 2007 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.
[Show abstract][Hide abstract] ABSTRACT: The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also
from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity
of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus
to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family,
is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited
to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range
6.5–8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert
with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts
for all the experimental observations.
Full-text · Article · Feb 2007 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Lengsin (LGS) is an abundant transcript in the human lens, encoding a predicted polypeptide similar to glutamine synthetase (GS). We show that a major alternatively spliced product of LGS codes for a 57kDa polypeptide that assembles into a catalytically inactive dodecamer, cross-reacts with anti-GS antibodies, and is expressed at high levels in transparent, but not cataractous, human lenses. Based on this characteristic oligomeric organization, preferential expression in the transparent lens, and amyloid-beta association previously reported for GS, a potential chaperone-like role of LGS has been investigated. We find that LGS has six binding sites for the hydrophobic surface probe bis-ANS and relieves cellular toxicity caused by amyloid-beta expression in a folding-impaired yeast mutant. While documenting the structural similarity between LGS and prokaryotic GS-I, the data rule out any involvement of lengsin in glutamine biosynthesis and suggest an unrelated role that may be important for lens homeostasis and transparency.
No preview · Article · Dec 2006 · Biochemical and Biophysical Research Communications