Biomedical subjects

S Longhi

Publications and source records attributed to S Longhi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Superluminal pulse reflection in asymmetric one-dimensional photonic band gaps.

Superluminal pulse reflection is shown to occur in a class of one-dimensional asymmetric photonic band gaps in which a spectral window inside the gap is opened. By means of a coupled-mode equation analysis, we describe in detail two possible realizations of superluminal pulse reflection that can be achieved using fiber Bragg gratings. The former method is based on the introduction of a defect into the otherwise periodic dielectric structure, whereas the latter one exploits the interference of two closely-spaced resonance modes and simulates the dispersion properties of an inverted medium possessing a doublet line.

Journal Article↗

PubMed2001

An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase.

We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.

Amino Acid Substitution↗

PubMed2001

Spiral waves in a class of optical parametric oscillators.

The formation of three-armed rotating spiral waves is shown to occur in a spatially extended nonlinear optical system with broken phase invariance. These new spatial structures are found in the mean-field model of a class of optical parametric oscillators (3omega-->2omega+omega) in which the multistep process 2omega=omega+omega breaks the phase invariance of the down-conversion process. A parametrically-forced Ginzburg-Landau equation is derived to explain the existence of phase-armed spiral waves.

Journal Article↗

PubMed2001

Spectral oscillations in a frequency-modulation laser operation.

We show the existence of coherent oscillations in the optical frequency spectrum of a laser with internal frequency modulation. These oscillations arise in presence of a slight detuning between the modulation frequency and the cavity axial mode separation and are analogous to collective oscillation modes of a chain of weakly coupled pendula with resonance frequencies slightly varying along the chain. Although spectral oscillations are damped due to the finite gain bandwidth of the laser cavity, they can be observed in the transient dynamics during switch-on of the modulator, or can be resonantly excited by an external forcing.

Journal Article↗

PubMed2001

One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli.

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.

Chromatography, Affinity↗

PubMed2001

The value of a single biopsy with 12 transperineal cores for detecting prostate cancer in patients with elevated prostate specific antigen.

PURPOSE: Prostate cancer detection on standard sextant biopsy is considered inadequate. Various biopsy protocols have been introduced to improve cancer diagnosis. We report our experience with transperineal 12-core prostate biopsy. MATERIALS AND METHODS: In a prospective study 650 patients underwent prostate specific antigen (PSA) measurement during a 15-month period, of whom 141 with PSA greater than 4 ng./ml. also underwent transperineal 12-core prostate biopsy using the fan technique. Median PSA was 8 ng./ml. (range 4.1 to 5,000). RESULTS: Prostate cancer was detected in 72 of the 141 patients (51%), including 44 of the 97 (45%) with PSA between 4.1 and 10 ng./ml. This incidence is higher than previously reported in the literature using other biopsy techniques. Disease was low grade Gleason 2 to 4 in 4 cases (5%), intermediate grade Gleason 5 to 6 in 26 (35%) and high grade Gleason 7 to 10 in the remaining 42 (60%). CONCLUSIONS: A high cancer detection rate is achieved by 12-core transperineal prostate biopsy. Most tumors represent clinically significant cancer. Further randomized trials are required to confirm these data.

Aged↗

PubMed2000

The valine-to-threonine 75 substitution in human immunodeficiency virus type 1 reverse transcriptase and its relation with stavudine resistance.

The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.

Adenosine Triphosphate↗

PubMed2000

Nonadiabatic pattern formation in optical parametric oscillators.

A nonadiabatic mechanism for pattern formation in parametrically forced systems subjected to a slow periodic modulation of the excitation frequency is proposed and discussed in detail for the case of an optical parametric oscillator. It is demonstrated that nonautonomous dynamics may induce nonadiabatic off-axis emission of down-converted photons even when the signal field is blueshifted from the nearby cavity resonance.

Journal Article↗

PubMed2000

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.

Alcohol Oxidoreductases↗

PubMed2000

Excess noise in intracavity laser frequency modulation

The response to perturbations and to stochastic noise of a laser below threshold subjected to an intracavity periodic frequency modulation is theoretically studied. It is shown that, when the modulation frequency is close to the cavity axial mode separation but yet detuned from exact resonance, the laser exhibits a strongly enhanced sensitivity to external noise, which includes large transient energy amplification of perturbations in the deterministic case and enhancement of field fluctuations in presence of a continuous stochastic noise. This large excess noise is due to the nonorthogonality of Floquet laser modes which makes it possible continuous energy transfer from the forcing noise to transient growing perturbations.

Journal Article↗

PubMed2000

A navigation system for increasing the autonomy and the security of powered wheelchairs.

Assistive technology is an emerging area where some robotic devices can be used to strengthen the residual abilities of individuals with motor disabilities or to substitute their missing function thus helping them to gain a level of independence at least in the activities of daily living. This paper presents the design of a navigation system and its integration with a commercial powered wheelchair. The navigation system provides the commercial wheelchair with a set of functions which increase the autonomy of elderly and people with motor disabilities. In general, a robot device must be adapted to assistive applications in such a way as to be easily managed by the user. Users, especially young ones, prefer to directly control the robotic device and this aspect of usability has to be managed without affecting the security and efficiency of the navigation module. These aspects have been considered as specifications for the navigation module of powered wheelchairs. Different autonomy levels of the navigation module and proper user interfaces have been developed. Two autonomy levels have been designed. Simple collision avoidance is also implemented in order to stop the mobile base when an obstacle is detected. The preliminary technical tests performed on the navigation system have shown satisfactory results in terms of security and response time. A modular solution for the navigation module was considered in order to simplify the adaptation of the module to different powered wheelchairs.

Persons with Disabilities↗

PubMed1999

Structure-activity of cutinase, a small lipolytic enzyme.

Cutinase, a small lipolytic enzyme, is the smallest member of the alpha/beta-hydrolase fold family, to which the other lipases belong. Cutinase has a catalytic activity comparable to that of pancreatic lipase on short chain triglycerides, and retains a significant activity on long chain triglycerides. Cutinase has been extensively studied using site-directed mutagenesis, and we have thoroughly characterized it from a structural point of view. Besides the native enzyme, tens of mutants and several inhibitor complexes have been solved, providing a complete and precise picture of the structure, dynamics and catalytic machinery of cutinase.

Binding Sites↗

PubMed1999

Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.

A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross-reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N-terminal sequencing, and two-dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.

Amino Acid Sequence↗

PubMed1998

Packing forces in nine crystal forms of cutinase.

During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200-600 A2 range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts.

Algorithms↗

PubMed1998

Messages from ultrahigh resolution crystal structures.

The ever growing availability of macromolecular crystal structures determined at atomic resolution has now reached a critical size, making it possible to obtain statistically unbiased data on both protein stereochemistry and the validity of the parameters used in their refinement. Besides the determination of the precise geometry of proteins and their active sites, high resolution structures have made it possible to check the application of normal mode calculations, to calculate charge density distributions and to analyze hydration shells around protein molecules. Even if only a few structures involve protein complexes, either with ligands or prosthetic groups, the information obtained in these cases is of great interest for obtaining the physical parameters of these interactions.

Algorithms↗

PubMed1998

Exploring hydrophobic sites in proteins with xenon or krypton.

X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination.

Animals↗

PubMed1997

Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis.

X-ray data have been recorded to 1.0 A resolution from a crystal of Fusarium solani cutinase using synchrotron radiation and an imaging-plate scanner. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a considerable quality improvement in the electron density maps with respect to an intermediate isotropic model. The final model has an R-factor of 9.4%, with a mean coordinate error of 0.021 A, as estimated from inversion of the least-squares matrix. The availability of an accurate structure at atomic resolution and of meaningful estimates of the errors in its atomic parameters, allowed an extensive analysis of several stereochemical parameters, such as peptide planarity, main-chain and some side-chain bond distances. The hydrogen atoms could be clearly identified in the electron density, thus providing unambiguous evidence on the protonation state of the catalytic histidine residue. The atomic resolution revealed an appreciable extent of flexibility in the cutinase active site, which might be correlated with a possible adaptation to different substrates. The anisotropic treatment of thermal factors provided insights into the anisotropic nature of motions. The analysis of these motions in the two loops delimiting the catalytic crevice pointed out a "breath-like" movement in the substrate binding region of cutinase.

Anisotropy↗