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PubMed2006

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.

Aging↗
PubMed2005

A modified embryo cryopreservation method increases post-thaw survival with a concomitant increase in implantation.

OBJECTIVE: To achieve maximum post-thaw survival of frozen embryos. DESIGN: Historical controlled study. SETTING: Hospital-based fertility center. PATIENT(S): One hundred forty-five patients whose embryos were frozen and thawed according to the standard method, and 56 patients whose embryos were frozen and thawed according to a modified method. INTERVENTION(S): Modifications were made to the various steps of cryopreservation: freezing and thawing solutions, loading of embryos into the straws, and warming rates. MAIN OUTCOME MEASURE(S): Post-thaw survival, implantation, and pregnancy rates. RESULT(S): With the modified method, 138 (93%) of the 149 embryos thawed for 56 patients survived freezing, and 79.8% had all their blastomeres intact, which is almost double the result obtained (41.8%) for patients whose embryos were thawed with the standard method. The implantation and pregnancy rates were also significantly higher with the modified method compared with the standard method. CONCLUSION(S): Greater post-thaw embryo survival was achieved, with a concomitant increase in implantation and pregnancy rates, by modifying the various steps in the standard cryopreservation methodology. This has important implications in IVF practice.

Adult↗
PubMed2006

Oxidatively modified autoantigens in autoimmune diseases.

Free radical-mediated oxidative damage and consequent protein modification by the end products of oxidative damage are important mediators of cell toxicity and disease pathogenesis. Aldehydic products, mainly the 4-hydroxy-2-alkenals, form adducts with proteins and make them highly immunogenic. Oxidative modification of proteins has been shown to elicit antibodies in a variety of diseases including systemic lupus erythematosus (SLE), alcoholic liver disease, diabetes mellitus (DM), and rheumatoid arthritis (RA). Oxidatively modified DNA (8-oxodeoxyguanine) and low-density lipoproteins (LDL) occur in SLE, a disease in which premature atherosclerosis is a serious problem. In addition, immunization with 4-hydroxy-2-nonenal (HNE)-modified 60-kDa Ro autoantigen elicits an accelerated epitope spreading in an animal model of SLE. Advanced glycation end product (AGE) pentosidine and AGE-modified IgG have been shown to correlate with RA disease activity. Oxidatively modified glutamic acid decarboxylase is important in type 1 DM, while autoantibodies against oxidized LDL are prevalent in Behcet's disease. The fragmentation of scleroderma-specific autoantigens occurs as a result of oxidative modification and is thought to be responsible for the production of autoantibodies through the release of cryptic epitopes. In the face of overwhelming evidence for the involvement of oxidative damage in autoimmunity the administration of antioxidants is a viable untried alternative for preventing or ameliorating autoimmune disease, although results in cardiovascular disease are disappointing.

Autoantigens↗
PubMed2006

Enhancement of transfection activity of lipoplexes by complexation with transferrin-bearing fusogenic polymer-modified liposomes.

We previously developed complexes of lipoplexes containing 3beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-chol) and succinylated poly(glycidol)-modified liposome, which becomes fusogenic under weakly acidic condition, for use as a novel gene delivery system. This study explored the effect of lipoplex structures--the type of cationic lipid and cationic lipid/DNA charge ratio--on the transfection activity of those complexes. Three types of cationic lipid with different polar groups were used for the preparation of lipoplexes: DC-chol, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), and 3,5-dipentadecyloxybenzamidine (TRX-20) with dimethylamino group, trimethylammonium group, and benzamidine group, respectively. Complexation with the SucPG-modified transferrin-bearing liposomes affected transfection activity of these lipoplexes differently. The TRX-20 lipoplexes exhibited the most marked enhancement of transfection activity upon complexation with the SucPG-modified liposomes among these lipoplexes. The cationic lipid/DNA charge ratio of the lipoplex and the amount of the transferrin-bearing SucPG-modified liposomes associated to the lipoplex also affected the transfection activity of the resultant complexes. Highly potent gene vectors were obtained by adjusting these factors.

Genetic Vectors↗
PubMed2006

A modified process for preparing cationic polylactide-co-glycolide microparticles with adsorbed DNA.

We have previously shown that cationic polylactide-co-glycolide (PLG) microparticles can be effectively used to adsorb DNA and generate potent immune responses in vivo. We now describe a modified and easier process containing a single lyophilization step to prepare these cationic PLG microparticles with adsorbed DNA. Cationic PLG microparticle formulations with adsorbed DNA were prepared using a modified solvent evaporation technique. Formulations with a fixed CTAB content and DNA load were prepared. The loading efficiency and 24h DNA release was evaluated for each formulation and compared to the earlier method of preparation. Select formulations were tested in vivo. The modified cationic PLG microparticle preparation method with a single lyophilization step, showed comparable physico-chemical behaviour to the two lyophilization steps process and induced comparable immune. The modified process with a single lyophilization step is a more practical process and can be utlized to prepare cationic PLG microparticles with adsorbed DNA on a large scale.

Adsorption↗
PubMed2005

Single B or T-cell epitope-based DNA vaccine using modified vector induces specific immune response against hepadnavirus.

Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.

Animals↗
PubMed2005

Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity.

BACKGROUND: We have performed a double-blind, placebo-controlled injection immunotherapy study with genetically modified derivatives of the major birch pollen allergen, Bet v 1 (Bet v 1-trimer, Bet v 1-fragments). OBJECTIVE: To investigate whether vaccination with genetically modified allergens induces allergen-specific antibodies in nasal secretions and to study whether these antibodies affect nasal allergen sensitivity. METHODS: A randomly picked subgroup of patients (n = 23; placebo, n = 10; trimer, n = 10; fragments, n = 3) was subjected to an extensive analysis of serum samples and nasal lavage fluids and to nasal provocation testing. Bet v 1-specific IgG(1-4) and IgA antibodies were determined in serum samples obtained before and after vaccination, after the birch pollen season, and 1 year after start of vaccination as well as in nasal lavage fluids obtained after the birch pollen season and 1 year after start of vaccination by ELISA. Nasal sensitivity to natural, birch pollen-derived Bet v 1 was determined by active anterior rhinomanometry after the birch pollen season and 1 year after start of vaccination. RESULTS: Vaccination with genetically modified Bet v 1 derivatives, but not with placebo, induced Bet v 1-specific IgG1, IgG2, and IgG4, and low IgA antibodies in serum, which also appeared in nasal secretions, but no IgG3 antibodies. The levels of therapy-induced Bet v 1-specific IgG4 antibodies in nasal secretions were significantly (P < .05) associated with reduced nasal sensitivity to natural, birch pollen-derived Bet v 1 as objectively determined by controlled nasal provocation experiments. CONCLUSION: Our data demonstrate that vaccination with genetically modified allergens induces IgG antibody responses against the corresponding natural allergen not only in serum but also in mucosal fluids, where they may protect against allergen-induced inflammation.

Adult↗
PubMed2005

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces.

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.

Coated Materials, Biocompatible↗
PubMed2005

Disease modifying genes in cystic fibrosis.

The variation in cystic fibrosis (CF) lung disease and development of CF related complications correlates poorly with the genotype of the CF transmembrane regulator (CFTR) and with environmental factors. Increasing evidence suggests that phenotypic variation in CF can be attributed to genetic variation in genes other than the CFTR gene, so-called modifier genes. In recent years, multiple candidate modifier genes have been investigated in CF, especially genes that are involved in the control of infection, immunity and inflammation. Some of these genes have been rather conclusively identified as modifiers of the CF phenotype, whereas associations found in other genes have not been confirmed or are conflicting. Identification of genetic variation in modifier genes, obtained by genotype-phenotype studies in well-defined patient populations, may be used as an aid to prognosis and may provide the possibility of new therapeutic interventions.

Cystic Fibrosis↗
PubMed2005

Hydrothermal synthesis, characterization, and photocatalytic performance of silica-modified titanium dioxide nanoparticles.

Silica-modified titanium dioxides were prepared by a hydrothermal method and then characterized by XRD, FT-IR, XPS, TEM, and UV-visible spectroscopy. The silica-modified titanium dioxides were in anatase phase and had large surface areas. There was strong interaction between SiO2 and TiO2, and TiOSi bonds formed during the hydrothermal process. The addition of silica in TiO2 particles could effectively suppress the formation of the rutile phase and the growth of titanium dioxide crystals. DRS spectra proved an increase in the band-gap transition with the increase of silica. The silica-modified TiO2 nanoparticles exhibited better photocatalytic activity, which increased with the silica amount, in comparison with pure TiO2 nanoparticles. Due to better thermal stability, the photocatalytic activity of the silica-modified TiO2 sample held good photocatalytic activity even after calcined at 1273 K.

Journal Article↗
PubMed2005

The interaction at the solid/liquid interface of 2,4-dichlorophenoxyacetic acid with silica modified by reaction with ammonia gas.

A method for the attachment of ammonia to modified silica gel has been developed. In the first step, tetraethylorthosilicate and (3-glycidoxypropyl)trimethoxysilane were co-condensed in the presence of n-dodecylamine, a neutral surfactant template, to produce a modified mesoporous silica. The epoxy group incorporated into the mesoporous silica was opened by ammonia gas thus introducing amine chelate groups covalently bound to the inorganic surface. The modified material contained 1.13+/-0.06 mmol of amine per gram of silica, exhibited a surface area of 831+/-29 m(2)g(-1) and a porous diameter of 1.95 nm. Infrared, 29Si and 13C NMR spectra were in agreement with the proposed structure of the modified mesoporous silica in the solid state. This ordered organic-inorganic hybrid presented a high capacity for the removal of the agrochemical 2,4-dichlorophenoxyacetic acid from water: the DeltaH and DeltaG values for the interaction were determined to be -110.61 and -9.37 kJ mol(-1), respectively.

Journal Article↗
PubMed2004

Tumor-targeted liposomes: doxorubicin-loaded long-circulating liposomes modified with anti-cancer antibody.

Commercially available doxorubicin-loaded long-circulating liposomes (Doxil, Alza Pharmaceuticals) were modified with the monoclonal nucleosome (NS)-specific 2C5 antibody (mAb 2C5) that recognizes a broad variety of tumors via the tumor cell surface-bound NSs. For incorporation into liposomes, mAb 2C5 was modified with poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEG-PE) with the free PEG terminus activated with the p-nitrophenylcarbonyl group (pNP-PEG-PE). Derivatives of mAb 2C5 containing a variable number of PEG-PE residues (10-32) per protein molecule were prepared with a reasonably good preservation of the antibody specific activity even at the highest degree of modification. PEG-PE-modified antibody quantitatively incorporated into the liposomal membrane of doxorubicin-loaded liposomes with a loss of not more than 20% of the encapsulated doxorubicin. 2C5-targeted Doxil liposomes acquired the ability to recognize NSs and specifically bind to various tumor cells. Doxorubicin-loaded long-circulating liposomes modified with the mAb 2C5 kill various tumor cells in vitro with the efficiency higher than non-targeted doxorubicin-loaded liposomes.

Animals↗
PubMed2006

DNA nano-carriers from biodegradable cationic branched polyesters are formed by a modified solvent displacement method.

DNA nano-carriers were formulated relying on biodegradable polyesters consisting of amine-modified poly(vinyl alcohol) (PVAL) backbones grafted with PLGA, based on the Marangoni effect thus avoiding detrimental shear or ultrasonic forces. These amine modified high molecular weight biodegradable polyesters combine specific characteristics, such as electrostatic interactions between DNA and cationic branched polyesters facilitating loading of NP with DNA. The resulting DNA containing NP showed hydrodynamic diameters in the range of 175-285 nm and highly positive xi-potentials, depending on the nitrogen to phosphate (N/P) ratio used for the particle formation. Atomic force microscopy (AFM) demonstrated well-defined spherical particle morphologies. DNA was released from NP upon incubation in PBS buffer in its intact supercoiled form. Agarose gel electrophoresis demonstrated that DNA within the NP was protected from enzyme degradation. The biological efficiency of the DNA delivery by this nano-carrier was demonstrated by an in vitro transfection assay using four cell lines. Reporter gene delivery of the amine-modified polymers was higher than naked DNA (Control) and raised with increasing degree of amine substitution. Also type of amine and distance of cationic charge from the backbone play an important role. Further, this feature was shown by Luciferase expression of the pCMV-Luc plasmid with PEI 25 kDa/DNA polyplexes and NP prepared with amine modified polyesters with a grafted PLGA chain length of 10 monomers compared at equal N/P ratios. DNA loaded NP from P(68)-10 showed 8x higher transfection efficiencies than the PEI 25 kDa at an N/P ratio of 9 for both preparations. These novel DNA nano-carriers merit further investigations in particular for DNA vaccination under in vivo conditions.

Amines↗
PubMed2006

Stabilizing of plasmid DNA in vivo by PEG-modified cationic gold nanoparticles and the gene expression assisted with electrical pulses.

This study aimed to investigate the benefits of combining the use of PEG-modified cationic gold nanoparticles with electroporation for in vivo gene delivery. PEG-modified cationic gold nanoparticles were prepared by NaBH(4) reduction of HAuCl(4) in the presence of 2-aminoethanethiol and mPEG-SH. Zeta-potential of the particles was nearly neutral (+0.1 mV). After forming complexes with plasmid DNA at a w/w ratio of 8.4, nanoparticle complexes were 90 nm for at least 60 min and showed a negative zeta-potential. After intravenous injection of DNA-nanoparticle complexes, 20% of gold were detected in blood at 120 min after injection and 5% of DNA were observed in blood after 5 min, suggesting that PEG-modified nanoparticles were stably circulating in the blood flow, but some of the DNA bound to particles degraded during circulation. When electroporation was applied to a lobe of the liver following injection of DNA-nanoparticle complexes, significant gene expression was specifically observed in the pulsed lobe. We concluded that PEG-modified nanoparticles maintained DNA more stably in the blood flow than in the case of naked DNA and electroporation assisted in restricted gene expression of circulating DNA in limited areas of the liver.

Animals↗
PubMed2006

Removal of phenol from aqueous solution by adsorption onto OTMAC-modified attapulgite.

The potential of octodecyl trimethyl ammonium chloride (OTMAC)-modified attapulgite (AT) for phenol adsorption from aqueous solutions was studied. The comparison of natural AT and modified AT showed that it is possible to utilize the sonication-modified OTMAC-AT in the treatment of phenol-contaminated wastewaters. Batch sorption studies were carried out to evaluate the effect of contact time, shaking frequency, temperature and the amount of AT. The results showed that in a lab-scale reactor, at room temperature, with an amount of the modified AT added (2.5 g), and a shaking frequency of 140 rev/min, the adsorption rate of phenol could be 60.4% for a duration of 60 min. The sorption kinetics were described by a pseudo-second-order model, and the values of k and q(e) were 1.367 mg/ig min and 0.7901 ig/mg, respectively. The analysis of equilibrium data showed that the Freundlich isotherms were found to be applicable for the adsorption equilibrium data. K and 1/n were estimated to be 14.53 and 0.8438, respectively.

Adsorption↗
PubMed2005

Adsorption of Acid Red 57 from aqueous solutions onto surfactant-modified sepiolite.

The adsorption of Acid Red 57 (AR57) onto surfactant-modified sepiolite was investigated in aqueous solution in a batch system with respect to contact time, pH and temperature. The surface modification of surfactant-modified sepiolite was controlled using the FTIR technique. The pseudo-first-order, pseudo-second-order kinetic models and the intraparticle diffusion model were used to describe the kinetic data and the rate constants were evaluated. The experimental data fitted very well the pseudo-second-order kinetic model and also followed the intraparticle diffusion model up to 90 min, whereas diffusion is not only the rate controlling step. The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms and the isotherm constants were also determined. The Freundlich model agrees with experimental data well. The activation energy, change of free energy, enthalpy and entropy of adsorption were also evaluated for the adsorption of AR57 onto surfactant-modified sepiolite. The results indicate that surfactant-modified sepiolite could be employed as low-cost material for the removal of textile dyes from effluents.

Adsorption↗
PubMed2005

Total and intrasynovial work of flexion of human cadaver flexor digitorum profundus tendons after modified Kessler and MGH repair techniques.

PURPOSE: The purpose of this study was to compare directly the total work of flexion (TWOF) and the intrasynovial work of flexion (IWOF) of human flexor digitorum profundus tendons and to analyze the ratio of the IWOF to the TWOF of human flexor digitorum profundus tendons. These factors may be important clinically in understanding the role of different methods of postoperative tendon rehabilitation for different types of tendon repairs, especially at the early stage after tendon repair. METHODS: Two different tendon repairs, the modified Kessler and the Massachusetts General Hospital, were used in 18 digits from 6 freshly frozen human cadaver hands. The TWOF and the IWOF were tested by using a digit-resistance testing device. RESULTS: After tendon repair the TWOF increased 11.2% and 26.9% for the modified Kessler and MGH groups, respectively. The differences in increase between the 2 groups were significant. The IWOF increased 126.8% and 308.8% for the modified Kessler and Massachusetts General Hospital groups, respectively. The IWOF accounted for 16.4% of the TWOF for the intact tendon; this percentage was 28.6% and 45.0% for the modified Kessler and Massachusetts General Hospital groups, respectively. CONCLUSIONS: The IWOF accounts for 16% of the TWOF of normal human cadaver digits but it accounts for a much higher fraction after tendon repair. The ratio of the work of flexion within the synovial sheath to the TWOF varies depending on the type of repair chosen.

Aged↗
PubMed2004

Translation, adaptation and validation of the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) for an Arab population: the Sfax modified WOMAC.

OBJECTIVE: To translate into Arabic and validate the Western Ontario and McMaster Universities (WOMAC) index. METHOD: Arabic translation was obtained with use of the forward and backward translation method. Adaptations were made after a pilot study. Patients with symptomatic knee OA fulfilling the revised criteria of the American College of Rheumatology were included. Impairment outcome measures (pain as measured on a visual analog scale, the maximum distance walked, Kellgren's radiological score), Lequesne index score and Beck depression scale score were recorded. Each item was analyzed. Test-retest reliability was assessed with use of the intra-class correlation coefficient (ICC) and the Bland and Altman method. Construct validity was investigated with use of Spearman's rank correlation coefficient, and a factor analysis was performed. RESULTS: One hundred and three patients were included in the study. Eight questions of the WOMAC physical function subscale (PF) had insufficient psychometric properties and were excluded. Although test-retest reliability of the questionnaire was good (0.84, 0.84, and 0.92 for pain, stiffness, and modified PF subscales respectively), construct validity could not be demonstrated. Factor analysis of the modified form of the WOMAC extracted four factors, which differed from the a priori triple stratification. However, factor analysis of the modified PF subscales extracted two factors, which accounted for 68.4% of the total variance and could be clinically characterized (disability during activities requiring knee flexion within the first 90 degrees and activities requiring knee flexion over more than 90 degrees ). CONCLUSION: We translated and adapted the WOMAC index into Arabic to suit Tunisian people. The translated questionnaire is reliable but not valid in its original form. We propose the use of a modified version of PF subscale of the WOMAC, although the psychometric properties of this instrument must be examined in a larger population.

Aged↗