BBA - General Subjects (v.1725, #3)

Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS–AtoC in E. coli by Efthimia E. Lioliou; Eleni P. Mimitou; Asterios I. Grigoroudis; Cynthia H. Panagiotidis; Christos A. Panagiotidis; Dimitrios A. Kyriakidis (257-268).
Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS–AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected “H box” consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10–AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon.
Keywords: AtoC; Antizyme; AtoS; atoDAEB; Two-component system; Phosphorylation;

Analysis of glycated insulin by MALDI-TOF mass spectrometry by M. Abul Farah; Shambhunath Bose; Jeong-Heon Lee; Ho-Chul Jung; Yangsun Kim (269-282).
Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available α- or ε-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1–13 and B 22–30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.
Keywords: Non-enzymatic glycation; Insulin; Diabetes; MALDI-TOF-MS; 3-aminophenylboronic acid;

Analysis of the peanut agglutinin molten globule-like intermediate by limited proteolysis by Nivedita Mitra; Sharmistha Sinha; R. Manjunatha Kini; Avadhesha Surolia (283-289).
These studies attempt to characterize the molten globule-like intermediate in the unfolding pathway of peanut agglutinin (PNA). PNA is the only known example of a homotetrameric protein that lacks the 2,2,2 or the fourfold symmetry. Previous studies have shown that PNA describes a non two-state unfolding process populated with a clearly defined intermediate. The intermediate is monomeric and has lost most of its tertiary structure and has a substantial amount of secondary structure still intact, thus described as a molten-globule (MG)-like intermediate. It was also shown by isothermal titration calorimetry to bind to lactose and some other ligands with an affinity similar to that of the native protein. This paper describes limited protease cleavage experiments on the intermediate using trypsin and protease V8 for its structural characterization. There are two hydrophobic cores in the PNA subunit. These experiments suggest that in the MG-like intermediate, the second hydrophobic core, near the sugar-binding loop of the protein loosens up. This effect is significantly reduced by the presence of 90% saturating lactose, as deduced by a reduction in cleavage propensity. This is also supported by the gain in the tertiary structure as observed by near-UV CD.
Keywords: Legume lectin; Limited proteolysis; Molten globule; Quaternary association;

Occurrence of a unique sialyl tetrasaccharide in colostrum of a bottlenose dolphin (Tursiops truncatus) by Yusuke Uemura; Sadaki Asakuma; Tadashi Nakamura; Ikichi Arai; Michihiro Taki; Tadasu Urashima (290-297).
Crude oligosaccharides were recovered from bottlenose dolphin (Tursiops truncatus) colostrum after chloroform/methanol extraction of lipids and protein precipitation, and purified using gel filtration, anion exchange chromatography and high performance liquid chromatography (HPLC). Their chemical structures characterized by NMR spectroscopy were as follows: GalNAc(β1–4)[Neu5Ac(α2–3)]Gal(β1–4)Glc, Neu5Ac(α2–3)Gal(β1–4)Glc, Neu5Ac(α2–6)Gal(β1–4)Glc and Gal(α1–4)Gal(β1–4)Glc. The monosialyltetrasaccharide and neutral trisaccharide have not previously been found as free forms in any natural sources including milk or colostrum, although these structures have been found in the carbohydrate units of glycosphingolipids GM2 and Gb3.
Keywords: Milk oligosaccharide; Bottlenose dolphin; Colostrum; GM2 tetrasaccharide; Globotriose; NMR spectroscopy;

Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): Baicalin and genistin by Yukinobu Uchiyama; Junko Tagami; Shinji Kamisuki; Nobuyuki Kasai; Masahiko Oshige; Hiroyuki Chiku; Siroh Ibe; Osamu Koiwai; Fumio Sugawara; Kengo Sakaguchi (298-304).
Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as α, β, δ or λ polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 μM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 μM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol β core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.
Keywords: Terminal deoxyribonucleotidyltransferase (TdT); TdT inhibitor; Arctium lappa; Baicalin; Genistin;

PM-induced cardiac oxidative stress and dysfunction are mediated by autonomic stimulation by Claudia R. Rhoden; Gregory A. Wellenius; Elisa Ghelfi; Joy Lawrence; Beatriz González-Flecha (305-313).
Epidemiological studies show that increases in particulate air pollution (PM) are associated with increases in cardiopulmonary morbidity and mortality. However, the mechanism(s) underlying the cardiac effects of PM remain unknown. We used pharmacological strategies to determine whether oxidants are implicated in PM-dependent cardiac dysfunction and whether PM-induced increase in autonomic stimulation on the heart mediates cardiac oxidative stress and toxicity. Adult Sprague–Dawley rats were exposed to either intratracheal instillation of urban air particles (UAP 750 μg) or to inhalation of concentrated ambient particles (CAPs mass concentration 700 ± 180 μg/m3) for 5 h. Oxidative stress and cardiac function were evaluated 30 min after UAP instillation or immediately after exposure to CAPs. Instillation of UAP led to significant increases in heart oxidants measured as organ chemiluminescence (UAP: 38 ± 5 cps/cm2, sham: 10 ± 1 cps/cm2) or thiobarbituric acid reactive substances (TBARS, UAP: 76 ± 10, Sham 30 ± 6 pmol/mg protein). Heart rate increased immediately after exposure (UAP: 390 ± 20 bpm, sham: 350 ± 10 bpm) and returned to basal levels over the next 30 min. Heart rate variability (SDNN) was unchanged immediately after exposure, but significantly increased during the recovery phase (UAP: 3.4 ± 0.2, Sham: 2.4 ± 0.3). To determine the role of ROS in the development of cardiac malfunction, rats were treated with 50 mg/kg N-acetylcysteine (NAC) 1 h prior to UAP instillation or CAPs inhalation. NAC prevented changes in heart rate and SDNN in UAP-exposed rats (340 ± 8 and 2.9 ± 0.3, respectively). To investigate the role of the autonomic nervous system in PM-induced oxidative stress, rats were given 5 mg/kg atenolol (β-1 receptor antagonist), 0.30 mg/kg glycopyrrolate (muscarinic receptor antagonist) or saline immediately before exposure to CAPs aerosols. Both atenolol and glycopyrrolate effectively prevented CAPs-induced cardiac oxidative stress (CLATEN: 11 ± 1 cps/cm2, CLGLYCO: 10 ± 1 cps/cm2, TBARSATEN: 40 ± 6 pmol/mg protein, TBARSGLYCO: 38 ± 6 pmol/mg protein). These data indicate that PM exposure increases cardiac oxidants via autonomic signals and the resulting oxidative stress is associated with significant functional alterations in the heart.
Keywords: Reactive oxygen species; Oxidative stress; Particulate air pollution; CAPs;

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes by Tanmoy Bera; Kuruba Lakshman; Debiprasad Ghanteswari; Sabita Pal; Dharmalingam Sudhahar; Md. Nurul Islam; Nihar Ranjan Bhuyan; Pradeep Das (314-326).
An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., α-lipoic acid (ALA), 5,5′-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron–sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.
Keywords: Transplasma membrane; Electron transport chain; α-Lipoic acid reduction; 5, 5′-Dithiobis(2-nitroaniline-N-sulphonic acid) synthesis and reduction; 1,2-Naphthoquinone-4-sulphonic acid reduction; Ferricyanide reduction; Anaerobic reduction; Capsaicin; Trifluoperazine; Leishmania donovani;

High field 1H NMR spectroscopy demonstrated that the equilibration of added Al(III) ions in osteoarthritic (OA) knee-joint synovial fluid (SF) resulted in its complexation by citrate and, to a much lesser extent, tyrosine and histidine. The ability of these ligands, together with inorganic phosphate, to compete for the available Al(III) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations was probed through the use of computer speciation calculations, which considered low-molecular-mass binary and ternary Al(III) species, the predominant Al(III) plasma transport protein transferrin, and also relevant hydrolysis and precipitation processes. It was found that, at relatively low added Al(III) concentrations, citrate species were more favoured, whilst phosphate species became dominant at higher levels. The significance of these findings with regard to the in vivo corrosion of aluminium-containing metal alloy joint prostheses (e.g., TiAlV alloys) is discussed.
Keywords: Low-molecular-mass aluminium(III) ion; Osteoarthritis; 1H NMR; Speciation; Synovial fluid;

Activation of a GST-tagged AKT2/PKBβ by Kristin Baer; Ivonne Lisinski; Monika Gompert; Dominik Stuhlmann; Katja Schmolz; Helmut W. Klein; Hadi Al-Hasani (340-347).
The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (ΔPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent K m value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in K m and for a subsequent 150-fold increase in the catalytic efficiency (k cat/K m) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.
Keywords: AKT; PKB; PDK1; Autophosphorylation; Baculovirus; Protein purification;

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle by Kaïs H. Al-Gubory; Irène Ceballos-Picot; Annie Nicole; Philippe Bolifraud; Guy Germain; Marie Michaud; Camille Mayeur; François Blachier (348-357).
Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.
Keywords: Anti- and pro-oxidant enzyme; Superoxide radical; Hydrogen peroxide; DNA fragmentation; Apoptosis; Corpus luteum;

Synthesis and characterization of a novel DTPA polymerized hemoglobin based oxygen carrier by Chad R. Haney; Paul W. Buehler; Anil Gulati (358-369).
The purpose of this study was to prepare a novel polymerized hemoglobin (Hb) based oxygen carrier (HBOC) designed to minimize Hb induced hypertension, while employing a simple and inexpensive method of preparation. Cyclic-diethylenetriaminepentaacetic acid (DTPA) was used to polymerize stroma free Hb (SF-Hb).SF-Hb was isolated from red blood cells and reacted with DTPA at a constant concentration, pH, and duration. Low molar mass fractions (<100 kDa) were removed using ultrafiltration. Reactions and subsequent ultrafiltration steps were determined to be reproducible by analyzing molar mass, colloid osmotic pressure and oxygen affinity. Finally, a model of 50% exchange transfusion (ET) in rats was used to evaluate the blood pressure response to DTPA polymerized SF-Hb (Poly-DTPA-Hb).Poly-DTPA-Hb demonstrated a number averaged molar mass of 128.7 kDa and a weighted average of 223.0 kDa. Oxygen binding equilibrium indicated high oxygen affinity (P 50  = 5.1 ± 0.01 mm Hg) and little cooperativity (n  = 1.4). Poly-DTPA-Hb and a control DTPA polymerized human serum albumin (Poly-DTPA-HSA) unexpectedly caused acute hypotension during the period of ET in rats (mean arterial pressure ≈45% less than baseline). Hypotension occurring over the period of ET was determined to be mediated by calcium binding to protein associated DTPA. This effect was attenuated by the addition of calcium chloride (CaCl2) to the Poly-DTPA protein preparations.Cyclic DTPA anhydride can be used to create cross-linked and polymerized hemoglobin, using a simple and inexpensive process. However, the addition of CaCl2 to the preparation appears to be required to prevent calcium chelation and subsequent hypotension during infusion.
Keywords: Blood substitute; Polymerization; Hemoglobin; DTPA;

The role of carbohydrate side chains of plasminogen in its activation by staphylokinase by Roza Aisina; Liliya Mukhametova; Karina Gershkovich; Sergei Varfolomeyev (370-376).
Kinetic parameters (k Pg and K Pg) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin–staphylokinase (Pm–STA) complex. The K Pg values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 μM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 μM, respectively), while only minor differences in the k Pg values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k Pg/K Pg values for activation of all four plasminogens due to a decrease in the K Pg values but did not alter the k Pg values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm–STA–Pg ternary enzyme–substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.
Keywords: Staphylokinase; Plasminogen glycoform; Activation kinetics; Soluble fibrin;

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells by Jee-Young Shin; Dongchul Suh; Jung Mogg Kim; Han-Gon Choi; Jung Ae Kim; Jung Jae Ko; Yong Bok Lee; Jin-Seok Kim; Yu-Kyoung Oh (377-384).
With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800–750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.
Keywords: Nonviral gene delivery; Transfection; Hematopoietic cell; Human CD34+ cell;

Characterization of benzodiazepine binding site on human α1-acid glycoprotein using flunitrazepam as a photolabeling agent by Victor Tuan Giam Chuang; Motoki Hijioka; Masaaki Katsuki; Koji Nishi; Teppei Hara; Ken-ichi Kaneko; Megumi Ueno; Akihiko Kuniyasu; Hitoshi Nakayama; Masaki Otagiri (385-393).
The binding of flunitrazepam (FNZP) by human α1-acid glycoprotein (hAGP) and the relationships between the extent of drug binding and desialylation and the genetic variants of hAGP were examined. The photolabeling specificity of [3H]FNZP was confirmed by findings in which other hAGP-binding ligands inhibited the formation of covalent bonds between [3H]FNZP and hAGP. The photolabeling of asialo-hAGP suggested that sialic acid does not involve in the binding of [3H]FNZP. No difference in the labeling could be found between the F1 * S variants and A variant. Similarly, FNZP did not show a difference in binding affinity to the two genetic variants of hAGP. Sequence analysis of the photolabeled peptide indicated a sequence corresponding to Tyr91-Arg105 of hAGP.
Keywords: Human alpha 1-acid glycoprotein; Flunitrazepam; Binding site; Topology analysis; Photoaffinity labeling;

Investigation of Foscan® interactions with plasma proteins by Siarhei Sasnouski; Vladimir Zorin; Ivan Khludeyev; Marie-Ange D'Hallewin; François Guillemin; Lina Bezdetnaya (394-402).
The present study investigates the interaction of the second generation photosensitizer Foscan® with plasma albumin and lipoproteins. Spectroscopic studies indicated the presence of monomeric and aggregated Foscan® species upon addition to plasma protein solutions. Kinetics of Foscan® disaggregation in albumin-enriched solutions were very sensitive to the protein concentration and incubation temperature. Kinetic analysis demonstrated that two types of Foscan® aggregated species could be involved in disaggregation: dimers with a rate constant of k 1  = (2.30 ± 0.15) × 10−3 s−1 and higher aggregates with rate constants varying from (0.55 ± 0.04) × 10−3 s−1 for the lowest to the (0.17 ± 0.02) × 10−3 s−1 for the highest albumin concentration. Disaggregation considerably increased with the temperature rise from 15 °C to 37 °C. Compared to albumin, Foscan® disaggregation kinetics in the presence of lipoproteins displayed poorer dependency on lipoprotein concentrations and smaller variations in disaggregation rate constants. Gel-filtration chromatography analysis of Foscan® in albumin solutions demonstrated the presence of aggregated fraction of free, non-bound to protein Foscan® and monomeric Foscan®, bound to protein.
Keywords: Photodynamic therapy (PDT); Foscan®; Disaggregation; Bovine serum albumin (BSA); Lipoprotein; Gel-filtration chromatography;

We determined over a 3-week period some of the factors that may influence the kinetic of gene expression following in vivo gene electrotransfer. Histochemical analysis of β-galactosidase and biochemical analysis of luciferase expressions were used to determine reporter gene activity in the Tibialis anterior muscles of young Sprague–Dawley male rats. Transfection efficiency peaked 5 days after gene electrotransfer and then exponentially decreased to reach non-detectable levels at day 28. Reduction of muscle damage by decreasing the amount of DNA injected or the cumulated pulse duration did not improve the kinetic of gene expression. Electrotransfer of luciferase expression plasmids driven either by viral or mammalian promoters rather show that most of the decrease in transgene expression was related to promoter origin/strength. By regulating the amount of transgene expression, the promoter origin/strength could modulate the immune response triggered against the foreign protein and ultimately the kinetic of transgene expression.
Keywords: Electroporation; Gene therapy; In vivo; Muscle damage; Non-viral gene delivery; Plasmid DNA; Regeneration;