bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors
Authors: Nicklin Stuart A. , White Steve J. , Nicol Campbell G. , Von Seggern Dan J. , Baker Andrew H. 
Published: March 2004

AbstractBackgroundBoth viral and non‐viral gene transfer vectors transduce vascular endothelial cells (EC) with low efficiency compared with other cell types such as hepatocytes. Generation of EC‐selective vectors would enhance the clinical utility of gene therapy for diverse vascular‐targeted applications.Methods12mer peptides derived by in vitro phage display with EC binding specificity [MTPFPTSNEANL (MTP) and MSLTTPPAVARP (MSL)] were inserted at position T542 in the exposed HI loop of the adenovirus (Ad) serotype 5 fiber using overlapping oligonucleotides; in combination with a double point mutation (KO1) to ablate virus : cell binding via the coxsackie‐adenovirus receptor (CAR). The resulting modified viruses were tested in vitro and in vivo for their ability to direct endothelial‐specific gene transfer.ResultsPeptide insertion was not deleterious to fiber trimerisation or virion maturation. In vitro gene transfer studies using a panel of cell types demonstrated that both peptide‐targeted Ad vectors mediated efficient CAR‐independent gene transfer to vascular EC compared with non‐modified Ads. Neither peptide supported gene delivery to non‐EC. Upon systemic injection into mice and subsequent evaluation of transgene expression we failed to observe a reduction in hepatic Ad accumulation but observed a significant elevation in β‐galactosidase in blood vessels with the MSLTTPPAVARP‐targeted Ad vector.ConclusionsWe have genetically engineered two novel Ads that transduce human EC selectively in vitro, one of which leads to altered Ad biodistribution in vivo. The successful generation of genetically engineered tropism for EC has broad implications for cardiovascular gene therapy. Further modifications to the Ad capsid will be required to improve in vivo biodistribution profiles. Copyright © 2004 John Wiley & Sons, Ltd.
Gene transfection into fetal sheep airways in utero using guanidinium‐cholesterol cationic lipids
Authors: Luton Dominique , Oudrhiri Noufissa , de Lagausie Pascal , Aissaoui Abderrahim , Hauchecorne Michelle , Julia Sébastien , Oury Jean‐François , Aigrain Yves , Peuchmaur Michel , Vigneron Jean‐Pierre , Lehn Jean‐Marie , Lehn Pierre 
Published: March 2004

AbstractBackgroundOver the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions. We have in particular shown that bis(guanidinium)‐tren‐cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation. As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC‐mediated gene transfection into the respiratory tract of fetal sheep in utero.MethodsThus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion. The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity.ResultsCAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA. Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions.ConclusionsThese results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium. They also invite the development of optimized BGTC‐based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes. Copyright © 2004 John Wiley & Sons, Ltd.
Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region
Authors: Uray Katalin , Medgyesi Dávid , Hilbert Ágnes , Sármay Gabriella , Gergely János , Hudecz Ferenc 
Published: March 2004

AbstractThe IgG binding Fcγ receptors (FcγRs) play a key role in defence against pathogens by linking humoral and cell‐mediated immune responses. Impaired expression and/or function of FcγR may result in the development of pathological autoimmunity. Considering the functions of FcγRs, they are potential target molecules for drug design to aim at developing novel anti‐inflammatory and immunomodulatory therapies. Previous data mostly obtained by X‐ray analysis of ligand–receptor complexes indicate the profound role of the CH2 domain in binding to various FcγRs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity FcγRs, like FcγRIIb and FcγRIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231–298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble FcγRIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble FcγRIIb, as well as to FcγRs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg255–Ser267 sequence of IgG1 is implicated in the binding to FcγRIIb. In addition we found that peptides corresponding to the Arg255–Ser267, Lys288–Ser298 or Pro230–Val240 when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNFα, a pro‐inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating FcγRs, and mediate FcγR dependent function. Peptide Arg255–Ser267 can also be considered as a lead for further functional studies. Copyright © 2004 John Wiley & Sons, Ltd.
The substrate recognition mechanisms in chaperonins
Authors: Gómez‐Puertas Paulino , Martín‐Benito Jaime , Carrascosa José L. , Willison Keith R. , Valpuesta José M. 
Published: March 2004

AbstractChaperonins are a family of proteins devoted to assisting the folding of other proteins. They are large oligomers assembled into ring structures that enclose a cavity in which folding takes place. For this process to occur, the chaperonin must first recognize and interact with the unfolded polypeptide, then undergo a conformational change upon nucleotide binding that results in the closure of the cavity which in turn mediates the folding reaction inside the cavity. Although this general mechanism seems to apply to every chaperonin studied so far, there exist two different modes of interaction between the chaperonin and the substrate. The first occurs mainly through the interaction between the exposed hydrophobic residues of the unfolded polypeptides and those of the chaperonin substrate binding site, as elucidated for the chaperonin GroEL from E. coli. The second type of mechanism has been described so far only for the cytosolic chaperonin CCT (Chaperonin Containing TCP‐1) and here the interaction seems to be of a more specific nature, involving charged and polar residues in both the chaperonin and the substrate, which interacts with CCT in a structured, quasi‐native conformation. Copyright © 2004 John Wiley & Sons, Ltd.
Kinetic and thermodynamic characterization of HIV‐1 protease inhibitors
Authors: Shuman Cynthia F. , Hämäläinen Markku D. , Danielson U. Helena 
Published: March 2004

AbstractInteraction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor‐based method was used to characterize the interactions between HIV‐1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature‐dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non‐linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (ΔG, ΔH, ΔS) were determined, and the energetics of complex association (ΔGon, ΔHon, ΔSon) and dissociation (ΔGoff, ΔHoff, ΔSoff) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (ΔG < 0) due primarily to increased entropy (ΔS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the ΔGon and ΔGoff were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (ΔHoff > 0) and negative entropy (ΔSoff < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (ΔSon > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target–ligand interactions and the development of new inhibitors of HIV‐1 protease. Copyright © 2004 John Wiley & Sons, Ltd.
The role of flexibility and hydration on the sequence‐specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis
Authors: Gorfe Alemayehu A. , Caflisch Amedeo , Jelesarov Ilian 
Published: March 2004

AbstractThe N‐terminal domain of the Tn916 integrase protein (INT‐DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site‐specific association is facilitated by numerous protein–DNA contacts from the face of a three‐stranded β‐sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein–DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non‐polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein–DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT–DBD–DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site‐specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA‐bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein–DNA contacts. The protein–DNA hydrogen‐bonding network is highly dynamic. The simulation identifies protein–DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the ‘wet’ protein–DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT–DBD alone and in complex with DNA. Copyright © 2004 John Wiley & Sons, Ltd.
Identification of differences in the specificity‐determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires
Authors: Almagro Juan C. 
Published: March 2004

AbstractStudies of antibodies of known three‐dimensional structure have revealed that insertion and deletion of amino acids at the hypervariable loops change the canonical structures, thus generating differences in the antigen‐binding site topography. Such differences determine the size of the antigen with which the antibody interacts. Here, 59 unique antibodies determined at a resolution of 3.0 Å or below, including 19 in complex with proteins, 18 with peptides and 22 with haptens, were analyzed to identify and characterize differences in the residues that are directly involved in the interaction with antigen, so‐called specificity‐determining residues (SDRs). It was found that antibodies use a similar number of SDRs to recognize proteins and peptides but contact haptens with five SDRs less. By using a score of SDR usage, differences in the location of the SDRs, depending on the type of antigen recognized, were then identified with precision. An analysis of the surface generated by the SDRs usage indicates that the differences found correlate well with the size of the antigen. Anti‐protein antibodies have the largest SDR surface, with SDRs of high usage located in the edge of the surface. The SDR surface of anti‐hapten antibodies is the smallest, with hot spots of contacts in the interior of the binding surface and buried in the VL:VH interface. The SDR surface of anti‐peptide antibodies has a size in between anti‐protein and anti‐hapten antibodies, with the SDRs of high usage located in the interior of the antigen‐binding site but do not buried as in anti‐hapten antibodies. These findings led to a fine‐tuning of the model correlating differences in the antigen‐binding site topography with its preference to recognize antigens of different size. Therefore, it is discussed how this knowledge should help to design antibody repertoires biased toward the recognition of antigens of predefined size. Copyright © 2004 John Wiley & Sons, Ltd.
Comparative study of the phototoxicity of two chrolin type photosensitizers, ATX‐S10(Na) and verteporfin, on vascular endothelial and retinal pigment epithelial cells
Authors: Huang Yufang , Obana Akira , Gohto Yuko , Nakajima Susumu 
Published: March 2004

AbstractBackground and ObjectivesTo compare the phototoxicity in photodynamic therapy (PDT) of ATX‐S10(Na) and Verteporfin on human microvascular endothelial cells (HMVEC), vascular endothelial cells of monkey choroid and retina (CRVEC), and human retinal pigment epithelial cells (HRPE).Study Design/Materials and MethodsPDT was performed in two different ways. In short dye‐exposure PDT, HMVEC and CRVEC were exposed to each photosensitizer for 5 minutes followed by laser irradiation of 670 nm wavelength for ATX‐S10(Na) or 689 nm for Verteporfin without washing out the photosensitizer in the medium. In long dye‐exposure PDT, the cells were exposed to photosensitizers for times ranging from 5 minutes to 2 hours, washed out the photosensitizers, followed by laser irradiation in a fresh medium. PDT was performed on HRPE with PDT doses that resulted in damaging 90% of the HMVEC (ED90). Phototoxicity was determined by MTS Assay 1 day after PDT.ResultsThe degree of phototoxicity depended on the dye concentration, laser dose, and dye exposure time. In short dye‐exposure PDT on HMVEC with a laser dose of 50 J/cm2, the ED90 was 6.3 μg/ml of ATX‐S10(Na) and 0.04 μg/ml of Verteporfin, while in long dye‐exposure PDT the ED90 was 50.0 μg/ml of ATX‐S10(Na) and 0.04 μg/ml of Verteporfin when the medium was supplemented with 5% fetal calf serum. The phototoxic rate on HMVEC was higher when the medium contained 5% as contrasted with 10% of serum. In short dye‐exposure PDT, the ED90 of CRVEC was 100 μg/ml of ATX‐S10(Na) and an irradiance of 100 J/cm2 , and 0.08 μg/ml of Verteporfin and an irradiance of 100 J/cm2 when the medium was supplemented with 10% serum. With some doses of short dye‐exposure PDT, the ATX‐S10(Na) achieved higher phototoxic rates on HMVEC and CRVEC than on the HRPE. However, long dye‐exposure PDT with ATX‐S10(Na) and short and long dye‐exposure PDT with Vereteporfin failed to obtain higher phototoxic rates on HMVEC and CRVEC than on HRPE.ConclusionsVerteporfin had a higher phototoxicity than ATX‐S10(Na) on HMVEC and CRVEC. The CRVEC resisted more than HMVEC following PDT with both photosensitizers. In short dye‐exposure PDT, ATX‐S10(Na) had a more selective phototoxicity on HMVEC and CRVEC than on HRPE. Lasers Surg. Med. 34:216–226, 2004. © 2004 Wiley‐Liss, Inc.
Flashlamp pulsed dye laser (FPDL) did not cure papulopustular rosacea
Authors: Berg Mats , Edström Desiree Wiegleb 
Published: March 2004

AbstractBackground and ObjectivesPharmacological treatment has a fairly good effect on the papulopustular lesions in rosacea, but not as good an effect on the erythema and telangiectases. The aim was to treat rosacea patients with both erythematotelangiectatic and papulopustular lesions with flashlamp pulsed dye laser (FPDL) until telangiectases/erythema disappeared, and to evaluate whether the treatment might also be effective on papulopustular lesions.Study Design/Materials and MethodsTen patients were treated on one side of the face with FPDL. The final examination was blinded and performed on the average 10 months after the last treatment.ResultsTwo of the patients had more lesions after treatment, three were unchanged, three had only slightly less, and two had markedly less papulopustular lesions.ConclusionOur conclusion from this small sample of patients is that FPDL probably has limited value on papulopustular lesions in rosacea. This indicates that the origin of rosacea may not be only vascular. Lasers Surg. Med. 34:266–268, 2004. © 2004 Wiley‐Liss, Inc.
Effect of low power 655 nm diode laser irradiation on the neuromuscular junctions of the mouse diaphragm
Authors: Nicolau Renata Amadei , Martinez Manel Santafé , Rigau Josepa , Tomàs Josep 
Published: March 2004

AbstractBackground and ObjectivesLow level laser therapy (LLLT) in specific wavelengths and fluence maintains the electrophysiological activity of injured peripheral nerve in rats, preventing scar formation (at injury site) as well as degenerative changes in the corresponding motor neurons of the spinal cord, thus accelerating regeneration of the injured nerve. We studied the effect of LLLT on the neurotransmitter release in neuromuscular junctions of the mouse diaphragm.Study Design/Materials and MethodsThirty‐nine diaphragm muscles were studied. LLLT with GaAlAs 655 nm (1–12 J/cm2) was used. Neurotransmitter release was studied by conventional intracellular recording techniques on curarised or high magnesium media. Quantal content, amplitude, latency and rise time were analysed for end‐plate potentials (EPPs). Frequency and amplitude were evaluated for the miniature end‐plate potentials (MEPPs). Short‐term plasticity of the neurotransmitter release (fast facilitation) was also evaluated by paired pulse stimulation.Results and ConclusionsThis study showed that LLLT (655 nm) in these doses has no detectable physiological effect on the motor end‐plate neurotransmitter release in mice. Lasers Surg. Med. 34:277–284, 2004. © 2004 Wiley‐Liss, Inc.
Influence of pulse duration and pulse number in selective RPE laser treatment
Authors: Framme Carsten , Schuele Georg , Roider Johann , Birngruber Reginald , Brinkmann Ralf 
Published: March 2004

AbstractBackground and ObjectivesThe therapeutic effect of laser treatment for macular diseases is related to the damage to the retinal pigment epithelium (RPE) and the subsequent restoration of the defect due to RPE proliferation. In contrast to conventional laser treatment, it is possible to damage the RPE selectively and to spare the photoreceptors by using repetitive microsecond laser pulses. It was the aim of the study to investigate the influence of pulse duration and number of pulses on angiographically and ophthalmoscopically visible retinal damage thresholds in order to optimize treatment modalities.Study Design/Materials and MethodsIn total, 625 laser lesions with various parameters were applied to the retina in 11 eyes of 6 Chinchilla breed rabbits using an experimental laser system (Nd:YLF at 527 nm). Pulse duration (1.7 microseconds and 200 nanoseconds) and number of pulses (100, 10, and 1 pulses) were varied at a constant repetition rate of 100 Hz. Damage thresholds were determined in terms of ophthalmoscopic and fluorescein angiographic visibility, and the therapeutic window (TW; angiographic ED50 vs. ophthalmoscopic ED50) as well as the safety range (SR; angiographic ED84 vs. ophthalmoscopic ED16) between both thresholds were calculated. Selected laser lesions were evaluated by histology.ResultsGenerally, the ED50 radiant exposure for angiographic visibility decreases for shorter laser pulses and with an increase in the number of pulses. The TW for both pulse durations (1.7 microseconds and 200 nanoseconds) was wider with 100 pulses than with single pulses. The widest TW was found for 100 pulses at 200 nanoseconds pulse duration (5.9‐fold above the angiographic threshold), and the smallest TW with a factor of 1.6 was found for 1.7 microseconds single pulses. In terms of SR, only irradiation with 100 pulses at 200 nanoseconds pulse duration was associated with a ratio >2. Independently of pulse duration, histological examination of laser sites 1 hour after irradiation revealed widely intact photoreceptors, while the underlying RPE was damaged.ConclusionsPulse duration and number of pulses have a significant influence on RPE damage thresholds and consecutively on TW and SR. Because fundus pigmentation in humans may vary intra‐ and interindividually by a factor of 2, a large TW and ideally also a large SR should be ensured in a clinical treatment context. In rabbits, the safety range with 200 nanoseconds pulses is higher than with the pulse duration of 1.7 microseconds currently in clinical use. These findings suggest the need for clinical pilot studies to prove whether these results can be transposed to the situation in humans. Lasers Surg. Med. 34:206–215, 2004. © 2004 Wiley‐Liss, Inc.
Radical Copolymerization of α‐Trifluoromethylacrylic Acid with Vinylidene Fluoride and Vinylidene Fluoride/Hexafluoropropene
Authors: Souzy Renaud , Ameduri Bruno , Boutevin Bernard 
Published: March 2004

AbstractSummary: The radical copolymerization of α‐trifluoromethylacrylic acid (TFMAA) with vinylidene fluoride (VDF), initiated by tert‐butyl 2,2‐dimethyl peroxypropanoate (or tert‐butyl peroxypivalate) is presented. The kinetics of copolymerization were investigated from a series of eight reactions for which the initial [VDF]0/[TFMAA]0 molar ratios ranged between 15.0/85.0 and 89.4/10.6. The compositions of the copolymers, i.e. the molar ratios of VDF and TFMAA monomeric units, were determined mainly by 19F and 1H NMR spectroscopy. According to the Tidwell and Mortimer method, the reactivity ratios, ri, were assessed to be: rVDF = 0.33 ± 0.09 and rTFMAA = 0 at 55 °C, leading to copolymers of mainly alternating structure. Then, the radical terpolymerization of TFMAA with VDF and hexafluoropropene (HFP), initiated by 2,5‐bis(tert‐butylperoxy)‐2,5‐dimethylhexane is described and the thermal properties of the materials produced are discussed.
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