Biomedical subjects

Fabio Beltram

Publications and source records attributed to Fabio Beltram.

16 recordsLinked to original sources

PubMed2006

Dynamic regulation of ERK2 nuclear translocation and mobility in living cells.

The extracellular signal-regulated protein kinase ERK1/2 is a crucial effector linking extracellular stimuli to cellular responses: upon phosphorylation ERK [also known as mitogen-activated protein kinase P42/P44 (MAPK)] concentrates in the nucleus where it activates specific programs of gene expression. Notwithstanding the importance of this process, little is known about the modalities, time course and regulation of ERK exchange between nucleus and cytoplasm in living cells. We visualized the dynamic of nuclear translocation by expressing low levels (<150 nM) of fluorescently tagged ERK2 in living fibroblasts. Time-lapse imaging demonstrated that nuclear concentration can change bidirectionally with a time constant of a few minutes. The increase of nuclear concentration requires continuous MEK (also known as MAPK kinase) activity upstream of ERK and is rapidly reduced by the operation of phosphatases. We measured quantitatively the speed of ERK2 shuttling between nucleus and cytoplasm and determined that shuttling accelerated after ERK activation, becoming fast enough not to be rate-limiting for translocation. Finally, we demonstrated that ERK2 did not diffuse freely in the nucleus and that diffusion was further impeded after phosphorylation, suggesting the formation of complexes of low mobility. These results show that nucleocytoplasmic trafficking of ERK2 and its mobility are dynamically regulated in living cells.

Active Transport, Cell Nucleus↗

PubMed2006

Cooling electrons by magnetic-field tuning of Andreev reflection.

A solid-state cooling principle based on magnetic-field-driven tunable suppression of Andreev reflection in superconductor/two-dimensional electron gas nanostructures is proposed. This cooling mechanism can lead to very large heat fluxes per channel up to 10;{4} times greater than currently achieved with superconducting tunnel junctions. This efficacy and its availability in a two-dimensional electron system make this method of particular relevance for the implementation of quantum nanostructures operating at cryogenic temperatures.

Journal Article↗

PubMed2006

Crossed Andreev reflection-induced magnetoresistance.

We show that very large negative magnetoresistance can be obtained in magnetic trilayers in a current-in-plane geometry owing to the existence of crossed Andreev reflection. This spin valve consists of a thin superconducting film sandwiched between two ferromagnetic layers whose magnetization is allowed to be either parallelly or antiparallelly aligned. For a suitable choice of structure parameters and nearly fully spin-polarized ferromagnets, the magnetoresistance can exceed -80%. Our results are relevant for the design and implementation of spintronic devices exploiting ferromagnet-superconductor structures.

Journal Article↗

PubMed2006

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).

Animals↗

PubMed2006

Insights on HIV-1 Tat:P/CAF bromodomain molecular recognition from in vivo experiments and molecular dynamics simulations.

Structural and functional studies indicate that, through its bromodomain, the cellular acetyltransferase P/CAF binds the acetylated Tat protein of human immunodeficiency virus type 1 (HIV-1) and promotes transcriptional activation of the integrated provirus. Based on the NMR structure of P/CAF complexed with an acetylated Tat peptide, here we use molecular dynamics simulations to construct a model describing the interaction between full length Tat and the P/CAF bromodomain. Our calculations show that the protein-protein interface involves hydrophobic interactions between the P/CAF ZA loop and the Tat core domain. In particular, tyrosines 760 and 761 of P/CAF, two residues that are highly conserved in most known bromodomains, play an essential role for the binding. Fluorescence resonance energy transfer (FRET) experiments performed in this work demonstrate that P/CAF proteins in which these tyrosines are mutated into hydrophilic residues neither bind to Tat inside the cells nor mediate Tat transactivation. The combination of theoretical and in vivo studies provides new insights into the specificity of bromodomain recognition.

Binding Sites↗

PubMed2006

Two dimensional patterning of fluorescent proteins in hydrogels.

This work describes the successful micropatterning of hybrid systems consisting of hydrogel-dispersed optically active and controllable proteins on solid surfaces without degradation of the photophysical properties of the light-emitting biomolecules. It demonstrates the preservation of the luminescence properties of proteins entrapped into isolated microstructures of poly(acrylamide) gel. This way we can exploit both the structural and function-preserving properties of the hydrogels and the functionality of light-emitting proteins. We believe that this approach can open the way to the realization of nanopatterned optical memories based on photochromic biomolecules.

Animals↗

PubMed2005

Particle-hole symmetric Luttinger liquids in a quantum Hall circuit.

We report current transmission data through a split-gate constriction fabricated onto a two-dimensional electron system in the integer quantum Hall (QH) regime. Split-gate biasing drives interedge backscattering and is shown to lead to suppressed or enhanced transmission, in marked contrast to the expected linear Fermi-liquid behavior. This evolution is described in terms of particle-hole symmetry and allows us to conclude that an unexpected class of gate-controlled particle-hole-symmetric chiral Luttinger liquids (CLLs) can exist at the edges of our QH circuit. These results highlight the role of particle-hole symmetry on the properties of CLL edge states.

Journal Article↗

PubMed2005

Manipulating nonequilibrium magnetism through superconductors.

Electrostatic control of the magnetization of a normal mesoscopic conductor is analyzed in a hybrid superconductor-normal-conductor-superconductor system. This effect stems from the interplay between the nonequilibrium condition in the normal region and the Zeeman splitting of the quasi-particle density of states of the superconductor subjected to a static in-plane magnetic field. Unexpected spin-dependent effects such as magnetization suppression, diamagnetic-like response of the susceptibility, as well as spin-polarized current generation are the most remarkable features presented. The impact of scattering events is evaluated and lets us show that this effect is compatible with realistic material properties and fabrication techniques.

Journal Article↗

PubMed2004

Interedge strong-to-weak scattering evolution at a constriction in the fractional quantum Hall regime.

Gate-voltage control of interedge tunneling at a split-gate constriction in the fractional quantum Hall regime is reported. Quantitative agreement with the behavior predicted for out-of-equilibrium quasiparticle transport between chiral Luttinger liquids is shown at low temperatures at specific values of the backscattering strength. When the latter is lowered by changing the gate voltage, the zero-bias peak of the tunneling conductance evolves into a minimum, and a nonlinear quasiholelike characteristic emerges. Our analysis emphasizes the role of the local filling factor in the split-gate constriction region.

Journal Article↗

PubMed2004

Nuclear organization and the control of HIV-1 transcription.

The regulation of transcription of the human immunodeficiency virus (HIV) is a complex event of significant pathological relevance, which recapitulates general concepts of cellular transcription with some peculiarities. The viral promoter is embedded in a chromatin structure that exerts powerful repression on transcription; activation of gene expression relies on the combined activity of a series of cellular factors that respond to different external stimuli, and on the function of a single viral regulatory protein, the Tat transactivator. Transcriptional activation is consequent to both chromatin remodeling and to the recruitment of elongation-competent RNA polymerase II complexes onto the integrated promoter, two events that require the coordinate, but transient, assembly of different protein complexes. Application of optical imaging techniques now allows us to appreciate the spatial and temporal evolvement of these reactions in vivo. The picture that is emerging is not only descriptive, but also relevant to the understanding of the regulation of the process. In particular, it appears that the confinement of biomolecules within specific subcellular compartments represents a way to control and coordinate the assembly of functional complexes that regulate viral gene expression.

Cell Nucleus↗

PubMed2003

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins.

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.

Cell Line↗

PubMed2003

Low-threshold quantum-cascade lasers at 3.5 THz (lambda = 85 microm).

Chirped-superlattice quantum-cascade lasers are reported that emit at lambda approximately 85 microm (3.6 THz), which is to the authors' knowledge the longest wavelength demonstrated so far with this technology. Collected peak output powers of 1.5 mW per facet were measured at liquid-helium temperature, and a maximum operating temperature of 45 K was reached. Record low-threshold-current densities of 95 and 115 A cm(-2) were observed in pulsed and continuous-wave operation, respectively. For the latter, output powers of a few hundred microwatts are estimated at low temperatures.

Journal Article↗

PubMed2003

Recruitment of human cyclin T1 to nuclear bodies through direct interaction with the PML protein.

Human cyclin T1, the cyclin partner of Cdk9 kinase in the positive transcription elongation factor b (P-TEFb), is an essential cellular cofactor that is recruited by the human immunodeficiency virus type 1 (HIV-1) Tat transactivator to promote transcriptional elongation from the HIV-1 long terminal repeat (LTR). Here we exploit fluorescence resonance energy transfer (FRET) to demonstrate that cyclin T1 physically interacts in vivo with the promyelocytic leukaemia (PML) protein within specific subnuclear compartments that are coincident with PML nuclear bodies. Deletion mutants at the C-terminal region of cyclin T1 are negative for FRET with PML and fail to localize to nuclear bodies. Cyclin T1 and PML are also found associated outside of nuclear bodies, and both proteins are present at the chromatinized HIV-1 LTR promoter upon Tat transactivation. Taken together these results suggest that PML proteins regulate Tat- mediated transcriptional activation by modulating the availability of cyclin T1 and other essential cofactors to the transcription machinery.

Cell Nucleus↗

PubMed2003

Nonlinear quasiparticle tunneling between fractional quantum hall edges.

Remarkable nonlinearities in the differential tunneling conductance between fractional quantum Hall edge states at a constriction are observed in the weak-backscattering regime. In the nu=1/3 state a peak develops as temperature is increased and its width is determined by the fractional charge. In the range 2/3</=nu</=1/3 this width displays a symmetric behavior around nu=1/2. We discuss the consistency of these results with available theoretical predictions for interedge quasiparticle tunneling in the weak-backscattering regime.

Journal Article↗

PubMed2003

Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.

Animals↗

PubMed2002

Terahertz semiconductor-heterostructure laser.

Semiconductor devices have become indispensable for generating electromagnetic radiation in everyday applications. Visible and infrared diode lasers are at the core of information technology, and at the other end of the spectrum, microwave and radio-frequency emitters enable wireless communications. But the terahertz region (1-10 THz; 1 THz = 10(12) Hz) between these ranges has remained largely underdeveloped, despite the identification of various possible applications--for example, chemical detection, astronomy and medical imaging. Progress in this area has been hampered by the lack of compact, low-consumption, solid-state terahertz sources. Here we report a monolithic terahertz injection laser that is based on interminiband transitions in the conduction band of a semiconductor (GaAs/AlGaAs) heterostructure. The prototype demonstrated emits a single mode at 4.4 THz, and already shows high output powers of more than 2 mW with low threshold current densities of about a few hundred A cm(-2) up to 50 K. These results are very promising for extending the present laser concept to continuous-wave and high-temperature operation, which would lead to implementation in practical photonic systems.

Journal Article↗