Monolayers of the HSA dimer on polymeric microparticles-electrokinetic characteristics.

Human serum albumin dimer (dHSA) enhances the accumulation and retention of anti-tumor drugs. In this work, monolayers of dHSA on polystyrene microparticles were prepared and thoroughly characterized. The changes in the electrophoretic mobility of microparticles upon the addition of controlled amounts of dHSA were measured using Laser Doppler Velocimetry (LDV) technique. These dependencies were quantitatively interpreted in terms of the 3D electrokinetic model. This allowed to determine the coverage of dHSA on microparticles under in situ conditions. Additionally, the maximum coverage of dHSA was precisely determined by the concentration depletion method. At physiological ionic strength, the maximum coverage of dHSA monolayer on microparticles was 1.05mg m-2. This agrees with the theoretical value predicted from the random sequential adsorption approach by assuming a side-on orientation of molecules. A high stability of the monolayers under pH cycling was confirmed, which proved irreversibility of the protein adsorption on the microparticles. The obtained results can be exploited to prepare and characterize polymeric drug-capsule conjugated with albumin dimer.

New 3'-O-aromatic acyl-5-fluoro-2'-deoxyuridine derivatives as potential anticancer agents.

New aromatic and aliphatic 3'-O-acyl-5-fluoro-2'-deoxyuridine derivatives were synthesized and evaluated as candidates for prodrugs against various cancer cell lines. As the most promising candidate for antimalignant therapeutics was found a dual-acting acyl derivative 7h, which apparently released not only the known anticancer nucleoside, 5-fluoro-2'-deoxyuridine (FdU), but also an additional active metabolite, acetylsalicylic acid, reinforcing thus therapeutic effect of FdU. Promising therapeutic indices showed also some aromatic dicarboxylic acids derivatives decorated with FdU esters (11 and 12).

Revealing deposition mechanism of colloid particles on human serum albumin monolayers.

Colloid particle deposition was applied in order to characterize human serum albumin (HSA) monolayers on mica adsorbed under diffusion transport at pH 3.5. The surface concentration of HSA was determined by a direct AFM imaging of single molecules. The electrokinetic characteristics of the monolayers for various ionic strength were done by in situ streaming potential measurements. In this way the mean-field zeta potential of monolayers was determined. It was shown that the initially negative potential changed its sign for HSA surface concentrations above 2800µm(-2) that was interpreted as overcharging effect. The monolayers were also characterized by the colloid deposition method where negatively charged polystyrene particles, 810nm in diameter were used. The kinetics of particle deposition and their maximum coverage were determined as a function of the HSA monolayer surface concentration. An anomalous deposition of particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field theoretical predictions. This effect was quantitatively interpreted in terms of the random site sequential adsorption model. It was shown that efficient immobilization of particles only occurs at adsorption sites formed by three and more closely adsorbed HSA molecules. These results can be exploited as useful reference data for the analysis of deposition phenomena of bioparticles at protein monolayers that has practical significance for the regulation of the bioadhesive properties of surfaces.

Electrokinetic characteristics of HSA dimer and its monolayers at mica.

Human serum albumin (HSA) dimer was synthesized in a reaction of the monomer with 1,6-bis(maleimido)hexane (BMH) cross-linker. Thorough physicochemical characteristics of the dimer were performed. They comprised the diffusion coefficient, hydrodynamic diameter, electrophoretic mobility as a function of pH, isoelectric point and electrokinetic charge. The adsorption of the dimer molecules at mica was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of adsorption was determined by a direct AFM imaging of single molecules over various substrate areas and interpreted in terms of the random sequential adsorption model. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow. It was also shown by these measurements that the desorption of the monomer under flow conditions was negligible. In this way, the amount of irreversibly bound dimer was quantitatively evaluated to be 0.5 mgm(-2) for ionic strength of 0.01 and pH 3.5 that is similar that to previous result obtained for the albumin monomer. This indicates that the dimer adsorption occurs mostly in the side-on orientation. Finally, the electrokinetic characteristics of the dimer monolayers on mica were performed by the streaming potential method. It was observed that for lower pHs the zeta potential of monolayers is much lower than the bulk zeta potential of the dimer molecules. This was attributed to a heterogeneous charge distribution. It was concluded that the well-characterized HSA dimer monolayers can be used for quantitatively determining ligand and drug binding that has an essential practical significance.

Recombinant albumin adsorption on mica studied by AFM and streaming potential measurements.

Recombinant human serum albumin (rHSA) in monomeric state is widely used in pharmaceutical industry as a drug excipient and for preparing coatings for medical devices. In this work the adsorption process of rHSA on model mica surface at pH 3.5 was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of albumin adsorption was determined by a direct enumeration of single molecules over various substrate areas. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow and with theoretical predictions derived from the random sequential adsorption (RSA) model. Desorption kinetics of albumin under flow conditions was also evaluated via the streaming potential measurements. In this way, the amount of irreversibly bound albumin was quantitatively evaluated to be 0.64 and 1.2 mg m(-2) for ionic strength of 0.01 and 0.15 M, respectively. This agrees with previous results obtained for HSA and theoretical calculations derived from the RSA model. Additionally, it was demonstrated that there existed a fraction of reversibly bound albumin that can be fully eluted within a few hours. The binding energy of these fraction of molecules was -18 kT that is consistent with the electrostatic controlled adsorption mechanism of albumin at this pH. It was concluded that the rHSA monolayers of well-defined coverage can find applications for quantitatively analyzing ligand binding and for performing efficient biomaterials and immunological tests.

Recombinant albumin monolayers on latex particles.

The adsorption of recombinant human serum albumin (rHSA) on negatively charged polystyrene latex micro-particles was studied at pH 3.5 and the NaCl concentration range of 10(-3) to 0.15 M. The electrophoretic mobility of latex monotonically increased with the albumin concentration in the suspension. The coverage of adsorbed albumin was quantitatively determined using the depletion method, where the residual protein concentration was determined by electrokinetic measurements and AFM imaging. It was shown that albumin adsorption was irreversible. Its maximum coverage on latex varied between 0.7 mg m(-2) for 10(-3) M NaCl to 1.3 mg m(-2) for 0.15 M NaCl. The latter value matches the maximum coverage previously determined for human serum albumin on mica using the streaming potential method. The increase in the maximum coverage was interpreted in terms of reduced electrostatic repulsion among adsorbed molecules. These facts confirm that albumin adsorption at pH 3.5 is governed by electrostatic interactions and proceeds analogously to colloid particle deposition. The stability of albumin monolayers was measured in additional experiments where changes in the latex electrophoretic mobility and the concentration of free albumin in solutions were monitored over prolonged time periods. Based on these experimental data, a robust procedure of preparing albumin monolayers on latex particles of well-controlled coverage and molecule distribution was proposed.

KfrA plasmid protein monolayers on latex particles-electrokinetic measurements.

Monolayers of KfrA, a protein assisting in bacteria plasmid segregation, on polystyrene latex particles were produced in controlled self-assembling under diffusion-controlled conditions. The coverage of the protein was quantitatively determined as a function of ionic strength (up to 0.15 M, NaCl) via micro-electrophoretic measurements and concentration depletion with the aid of AFM imaging. The maximum monolayer coverage of KfrA  monotonically increased with ionic strength attaining 2.0 mg m(-2) for 0.15 M, NaCl that corresponds to the dimensionless coverage of 0.48. This is in accordance with theoretical calculations derived from the random sequential adsorption modeling assuming a tetrameric aggregation state. A high stability of the monolayers in pH cycling experiments was confirmed, which proves the irreversibility of protein adsorption on latex. The acid base properties and the electrokinetic charge of monolayers were also determined via the electrophoretic mobility measurements carried out for various ionic strength. In this way the isoelectric point of the protein of 4.8 was determined, which is prohibitive via bulk measurements. It was concluded that the procedure used in our work is reliable and efficient for characterizing physicochemical properties if minor amounts of a protein are available.

Revealing properties of the KfrA plasmid protein via combined DLS, AFM and electrokinetic measurements.

Physicochemical characteristics of the plasmid KfrA protein in electrolyte solutions were done using a combination of dynamic light scattering (DLS), atomic force microscopy (AFM) and electrokinetic methods. The size of the protein was determined via the diffusion coefficient measurements using DLS. It was revealed from these measurements that the protein exists in an aggregated state composed of four molecules. The size of the protein was also determined via AFM imaging of single molecules adsorbed on mica from dilute solutions at pH=3.5. It was 10.6 nm in accordance with the value predicted for an aggregate composed of four monomers in a hexagonal configuration. The aggregation number was also confirmed by kinetics measurements carried out under diffusion controlled transport using AFM imaging of proteins. Further characteristics were acquired via KfrA adsorption on polystyrene latex particles (average size of 820 nm). The electrophoretic mobility of the latex and its zeta potential were determined as a function of the coverage of the protein. The maximum monolayer coverage for pH=3.5 was 1.2 mgm(-2). Additionally, from these measurements the effective charge of KfrA tetramer equal to 12 e (elementary charges) was predicted. The KfrA monolayer on latex was used to determine the isoelectric point of the protein, which was pH=4.5. As concluded, the procedures used in our work proved advantageous for a direct determination of aggregation processes and the effective charge if minor amounts of a protein are available.

Mechanism of HSA adsorption on mica determined by streaming potential, AFM and XPS measurements.

Adsorption of human serum albumin (HSA) on mica at pH 3.5 (0.15M NaCl) was studied using in situ streaming potential measurements, AFM imaging and XPS. Results obtained by streaming potential were consistent with AFM measurements and theoretical predictions based on the random sequential adsorption model. These results in combination with complementary data derived from XPS allowed one to determine both the kinetics of adsorption and the maximum coverage of irreversibly bound HSA, which was close to 1.6 mg m(-2) (dimensionless coverage 0.45). It was concluded that HSA adsorption on mica at pH 3.5 occurred under side-on, electrostatically controlled mechanisms with no tendency to multilayer formation. Such irreversible bound HSA monolayers of well-defined coverage can find applications for performing efficient immunological tests, designing biomaterials surfaces and biosensors.

Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress.

Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.

The upregulation of thiamine (vitamin B1) biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response.

BACKGROUND: Recent reports suggest that vitamin B1 (thiamine) participates in the processes underlying plant adaptations to certain types of abiotic and biotic stress, mainly oxidative stress. Most of the genes coding for enzymes involved in thiamine biosynthesis in Arabidopsis thaliana have been identified. In our present study, we examined the expression of thiamine biosynthetic genes, of genes encoding thiamine diphosphate-dependent enzymes and the levels of thiamine compounds during the early (sensing) and late (adaptation) responses of Arabidopsis seedlings to oxidative, salinity and osmotic stress. The possible roles of plant hormones in the regulation of the thiamine contribution to stress responses were also explored. RESULTS: The expression of Arabidopsis genes involved in the thiamine diphosphate biosynthesis pathway, including that of THI1, THIC, TH1 and TPK, was analyzed for 48 h in seedlings subjected to NaCl or sorbitol treatment. These genes were found to be predominantly up-regulated in the early phase (2-6 h) of the stress response. The changes in these gene transcript levels were further found to correlate with increases in thiamine and its diphosphate ester content in seedlings, as well as with the enhancement of gene expression for enzymes which require thiamine diphosphate as a cofactor, mainly α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and transketolase. In the case of the phytohormones including the salicylic, jasmonic and abscisic acids which are known to be involved in plant stress responses, only abscisic acid was found to significantly influence the expression of thiamine biosynthetic genes, the thiamine diphosphate levels, as well as the expression of genes coding for main thiamine diphosphate-dependent enzymes. Using Arabidopsis mutant plants defective in abscisic acid production, we demonstrate that this phytohormone is important in the regulation of THI1 and THIC gene expression during salt stress but that the regulatory mechanisms underlying the osmotic stress response are more complex. CONCLUSIONS: On the basis of the obtained results and earlier reported data, a general model is proposed for the involvement of the biosynthesis of thiamine compounds and thiamine diphosphate-dependent enzymes in abiotic stress sensing and adaptation processes in plants. A possible regulatory role of abscisic acid in the stress sensing phase is also suggested by these data.

Enzymes that control the thiamine diphosphate pool in plant tissues. Properties of thiamine pyrophosphokinase and thiamine-(di)phosphate phosphatase purified from Zea mays seedlings.

The pool of thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The thiamine pyrophosphokinase (TPK) activates thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to thiamine monophosphate (TMP) and then to free thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the K(m) values of 12.4microM and 4.7mM for thiamine and ATP, respectively, and the V(max) value of 360pmol TDPmin(-1)mg(-1) protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The K(m) values for TDP and TMP were 36microM and 49microM, respectively, and the V(max) value for TDP was significantly higher than for TMP (164 versus 60nmolesmin(-1)mg(-1) protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.