Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Synthesis and biological evaluation of some thiazole derivatives as new cholinesterase inhibitors

In the present study, some thiazole derivatives were synthesized via the ring closure reaction of 1-[2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetyl]thiosemicarbazide with various phenacyl bromides. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR and mass spectral data and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. The most potent AChE inhibitor was found as compound 4e (IC₅₀?=?25.5?±?2.12 µg/mL) followed by compounds 4i (IC₅₀?=?38.50?±?2.12 µg/mL), 4c (IC₅₀?=?58.42?±?3.14 µg/mL) and 4g (IC₅₀?=?68?±?2.12 µg/mL) when compared with eserine (IC₅₀?=?0.025?±?0.01 µg/mL). Effective compounds on AChE exhibited weak inhibition on BuChE (IC₅₀ > 80 µg/mL). MTT assay indicated that the cytotoxic dose (IC₅₀?=?71.67?±?7.63 µg/mL) of compound 4e was higher than its effective dose.     

Isolation and biological activities of 3-hydroxy-4(1H)-pyridone

3-Hydroxy-4(4H)-pyridone (3,4-DHP), a degraded product of mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid], is known to cause goiters, loss of hair, and infertility in animals, but limits of 3,4-DHP on separation and purification have prevented efforts on investigating other toxicity and biological properties of 3,4-DHP. By this study, a novel and simple isolation of 3,4-DHP was developed either from Leucaena leaves using an ion-exchanged resin or mimosine degraded in high temperature (110°C, 6?h). The inhibition of mimosine on the growth of barnyardgrass was approximately fourfold higher (IC₅₀?=?0.04?mg?g^?1) than that of 3,4-DHP (IC₅₀?=?0.15?mg?g^?1). In general, the antifungal activity of mimosine is much stronger than that of 3,4-DHP, but it differs depending on the kind of fungi. The 1,1-diphyenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of 3,4-DHP, in contrast with the growth inhibitory activity, is about fourfold stronger [EC₅₀?=?2.4?mg?g^?1 gallic acid equivalent (GAE)] than that of mimosine [EC₅₀?=?10.3?mg?g^?1 GAE]. This study is the first to report on the herbicidal, antifungal, and antioxidant activities of 3,4-DHP.     

Probing the binding sites of resveratrol,genistein, and curcumin with milk β-lactoglobulin

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG}?=?4.4 (±?.4)?×?10⁴ M^?1, K_{resveratrol–β-LG}?=?4.2 (±?.2)?×?10⁴ M^?1, and K_{genistein–β-LG}?=?1.2 (±?.2)?×?10⁴?M^?1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of ?12.67 (curcumin–β-LG), ?12.60 (resveratrol–β-LG), and ?10.68?kcal/mol (genistein–β-LG). The order of binding was cur?>?res?>?gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.     

Discrimination between rival laccase inhibition models from data sets with one inhibitor concentration using a penalized likelihood analysis and Akaike weights

Laccase from the white-rot fungus Fomes fomentarius was used for the biodegradation of ferulic acid (FA) in the presence of chloride anions. The initial reaction rates of substrate depletion were obtained by reverse-phase HPLC determination of remaining FA since substrate and reaction products have absorption peaks at similar wavelengths. Modelling of time-course data was accomplished by discrimination of the best enzyme inhibition equation from an initial set of seven different models based on Michaelis–Menten kinetics: competitive; uncompetitive; non-competitive; mixed; mixed hyperbolic; mixed parabolic; mixed hyperbolic and parabolic. Corrected Akaike information criterion was used to evaluate the relative merit of each kinetic model in order to rank them and find the more likely one. The discrimination results showed that the models with higher probabilities were the competitive and mixed inhibition types, but Akaike weights supported the selection of competitive inhibition (CI). After optimization by nonlinear regression, laccase kinetic parameters of FA biodegradation in the presence of chloride anions were: V_max?=?0.11?μmol?min^?1?mg^?1, K_m?=?44?μmol?L^?1 and a CI constant K_ic?=?14?mmol?L^?1.     

Assessment of oxidative stress biomarkers – neuroprostanes and dihomo-isoprostanes – in the urine of elite triathletes after two weeks of moderate-altitude training

This randomized and controlled trial investigated whether the increase in elite training at different altitudes altered the oxidative stress biomarkers of the nervous system. This is the first study to investigate four F₄-neuroprostanes (F₄-NeuroPs) and four F₂-dihomo-isoprostanes (F₂-dihomo-IsoPs) quantified in 24-h urine. The quantification was carried out by ultra high pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS). Sixteen elite triathletes agreed to participate in the project. They were randomized in two groups, a group submitted to altitude training (AT, n?=?8) and a group submitted to sea level training (SLT) (n?=?8), with a control group (Cg) of non-athletes (n?=?8). After the experimental period, the AT group triathletes gave significant data: 17-epi-17-F_2t-dihomo-IsoP (from 5.2?±?1.4?μg/mL 24?h^?1 to 6.6?±?0.6?μg/mL 24?h^?1), ent-7(RS)-7-F_2t-dihomo-IsoP (from 6.6?±?1.7?μg/mL 24?h^?1 to 8.6?±?0.9?μg/mL 24?h^?1), and ent-7-epi-7-F_2t-dihomo-IsoP (from 8.4?±?2.2?μg/mL 24?h^?1 to 11.3?±?1.8?μg/mL 24?h^?1) increased, while, of the neuronal degeneration-related compounds, only 10-epi-10-F_4t-NeuroP (8.4?±?1.7?μg/mL 24?h^?1) and 10-F_4t-NeuroP (5.2?±?2.9?μg/mL 24?h^?1) were detected in this group. For the Cg and SLT groups, no significant changes had occurred at the end of the two-week experimental period. Therefore, and as the main conclusion, the training at moderate altitude increased the F₄-NeuroPs- and F₂-dihomo-isoPs-related oxidative damage of the central nervous system compared to similar training at sea level.     

Thrombin activity throughout the acute phase of acute ST-elevation myocardial infarction and the relation to outcome

Background: Thrombin and plasmin play a central role in ongoing thrombosis and platelet activation in patients with acute ST-elevation myocardial infarction (STEMI). Data of thrombin and plasmin activity in the early course of STEMI and the relation to outcome are scarce.

Methods: We included 68 consecutive patients (53 male, 59?±?11.4 years) with STEMI who underwent acute catheter-based reperfusion therapy within the first 12?h after onset of symptoms. Blood samples were taken at admission and after 4, 8, 12 and 24?h. Thrombin activity and generation was measured by changes in the thrombin/antithrombin-III complex (TAT) and prothrombin fragment (F1.2); plasmin was measured by changes in the plasmin-α₂/antiplasmin complex (PAP). A follow-up with respect to the combined primary endpoint consisting of death, acute myocardial infarction or urgent need for revascularization up to 6 weeks post-discharge was carried out.

Results: TAT values showed no significant change over time in patients with and without the primary endpoint but there was a borderline difference between these groups at 4?h after admission (event group 9.0 vs no event group 4.7?μg l^?1, p?=?0.057). F1.2 values were different between groups only after 24?h (event group 1.5 vs no event group 0.9?nmol l^?1, p?=?0.028) and did not differ in serial sampling of 24?h. PAP values were higher in patients with events after 4 and 8?h and declined over time in the group without events (p?<0.001). Odds ratios (OR) with respect to the primary endpoint were highest for TAT?>4.8?μg l^?1 at 0?h and TAT?>8.4?μg?l^?1 at 4?h (OR 7.1, 95% confidence interval (CI) 1.5–34, p?=?0.015 and OR 5.5, 95% CI 1.5–20.0, p?=?0.01, respectively). The predictive value of plasmin concentrations were equally high after 4?h (PAP?>962?μg l^?1; OR 6.8, 95% CI 1.8–26.2, p?=?0.005) and 8?h (PAP?>495?μg l^?1, OR 6.7, 95% CI 1.4–32.9, p?=?0.024). Values for F1.2 were only predictive after 24?h (F1.2?>0.85?nmol l^?1, OR 13, 95% CI 1.4–117.8, p?=?0.023).

Conclusions: Markers of thrombin and plasmin activity in acute STEMI are related to outcome. The marker for thrombin generation F1.2 becomes a significant predictor of outcome at 24?h after admission, reflecting the potentially adverse effects of ongoing thrombin generation. This underlines the potential for direct thrombin inhibition and individualization of treatment by thrombin markers in STEMI.     

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO₂ hydration reaction (k_cat?=?9.35?×?10⁵?s^?1 and k_cat/K_m?=?1.1?×?10⁸?M^?1?s^?1) which was maintained after heating the enzyme at 80?°C for 3?h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe₃O₄ nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.     

A potent peroxidase from solid cell culture of Ocimum basilicum with high sensitivity for blood glucose determination

Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020–2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. Consequently, a novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of basil in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10^–6 M) and kinetin (10^–5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg^?1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (K_m?=?21.5 mM), but obtained an exceptionally high specificity constant (k_cat/K_m?=?66,743.7 s^?1 M^?1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L^?1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

     

Design and synthesis of a biaryl series as inhibitors for the bromodomains of CBP/P300

A novel, potent, and orally bioavailable inhibitor of the bromodomain of CBP, compound 35 (GNE-207), has been identified through SAR investigations focused on optimizing al bicyclic heteroarene to replace the aniline present in the published GNE-272 series. Compound 35?has excellent CBP potency (CBP IC₅₀?=?1?nM, MYC EC₅₀?=?18?nM), a selectively index of?>2500-fold against BRD4(1), and exhibits a good pharmacokinetic profile.     

Respirometry and swimming dynamics of the giant australian cuttlefish,sepia apama (mollusca,cephalopoda)

Swimming dynamics of the giant Australian cuttlefish, Sepia apama, were investigated using swimtunnel respirometry. Relationships between jet pressure, fin frequency, swimming speed and oxygen consumption were defined. Laboratory calibration of swimming parameters is necessary to allow estimates of swimming costs in the field.

Jet pressure was the best predictor of oxygen consumption with an averaged equation of MO₂?=?722 (jet pressure)?+?107?r ²?=?0.51. Individually, fin frequency and jet pressure correlated highly to swimming speed, but due to the complicated usage of finning and jetting, the correlation between swimming speed and oxygen consumption was weaker. Cuttlefish were not optimal swimtunnel subjects and could not swim at high speeds for extended periods. At 15°C and a swimming speed of 0.06?m?s^?1, the gross cost of transport was calculated to be 10.1?kg^?1?m?^?1, with a net cost of 4.1?kg^?1?m^?1.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.     

Evaluation of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia in controlling eggs of Aedes aegypti (Diptera: Culicidae)

We evaluated the potential of a granular formulation of Metarhizium brunneum F52 containing microsclerotia (MbMSc granules) for control of Aedes aegypti by targeting eggs. MbMSc granules produced infective conidia within 14 days after application to 2.5?g moist potting soil, producing 5.9?×?10⁵, 2.08?×?10⁶ and 6.85?×?10⁶ conidia from 1, 5 and 25?mg MbMSc granules, respectively. Application of MbMSc triggered premature eclosion of eggs (EC_50?=?12?mg) with percentages as high as 31?±?2.9% and 67?±?4.3% of the eggs treated with 5 and 25?mg MbMSc granules, respectively, after 14 days on moist filter paper. Premature eclosion of eggs started at 3 days subsequent to MbMSc granule application and survival of larvae was significantly reduced for granule treated eggs (74?±?2.2%, 39?±?2.0% and 23?±?4.9% larvae survived for 1, 5 and 25?mg granule treatments, respectively, EC₅₀?=?4.9?mg). When MbMSc granules were applied in moist potting soil with mosquito eggs, rates of 1, 5 and 25?mg of MbMSc granules significantly reduced adult emergence with only 81?±?2.1%, 47?±?1.9%, and 34?±?2.1% emergence, respectively (EC₅₀?=?7?mg). Eggs treated with increasing concentrations of fungal conidia enhanced premature eclosion of eggs with an EC₅₀?=?1.6?×?10⁶ conidia/mL. Our results demonstrate that MbMSc granules are a promising candidate for control of A. aegypti and that fermentative production of Mb F52 microsclerotia as the active propagule has the potential for use for mosquito control.     

Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II) complexes as a biological active agent

Abstract

Square planar mononuclear platinum(II) complexes having general formula [Pt(Lⁿ)Cl₂], (where, Lⁿ?=?L^1–4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV–vis, ¹H NMR, ¹³C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (K_b) of ligands and complexes were observed in the range of 0.23–1.07?×?10⁵?M^?1 and 0.51–3.13?×?10⁵?M^?1, respectively. Pt(II) complexes (I–IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (K_b) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA.     

Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications

Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45?μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of K_AsA?=?3.86?±?0.01?×?10⁴?M^?1, which corresponds to the free energy of (ΔG) ?6.3?kcal?M^?1 at 25?°C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50?±?2.4 to 50%?±?2.3 and an increase in the β-turns from 25?±?0.65 to 29%?±?0.91 and random coils from 17.5%?±?0.95 to 21%?±?1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA–AsA complexes.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Hemodialysis Decreases Serum Brain-Derived Neurotrophic Factor Concentration in Humans

In the present study we have evaluated the effect of a single hemodialysis session on the brain-derived neurotrophic factor levels in plasma [BDNF]_pl and in serum [BDNF]_s as well as on the plasma isoprostanes concentration [F₂ isoprostanes]_pl, plasma total antioxidant capacity (TAC) and plasma cortisol levels in chronic kidney disease patients. Twenty male patients (age 69.8?±?2.9?years (mean?±?SE)) with end-stage renal disease undergoing maintenance hemodialysis on regular dialysis treatment for 15?C71?months participated in this study. A single hemodialysis session, lasting 4.2?±?0.1?h, resulted in a decrease (P?=?0.014) in [BDNF]_s by ~42?% (2,574?±?322 vs. 1,492?±?327?pg?ml^?1). This was accompanied by an increase (P?<?10^?4) of [F₂-Isoprostanes]_pl (38?±?3 vs. 116?±?16?pg?ml^?1), decrease (P?<?10^?4) in TAC (1,483?±?41 vs. 983?±?35 trolox equivalents, ??mol?l^?1) and a decrease (P?=?0.004) in plasma cortisol level (449.5?±?101.2 vs. 315.3?±?196.3?nmol?l^?1). No changes (P?>?0.05) in [BDNF]_pl and the platelets count were observed after a single dialysis session. Furthermore, basal [BDNF]_s in the chronic kidney disease patients was significantly lower (P?=?0.03) when compared to the age-matched control group (n?=?23). We have concluded that the observed decrease in serum BDNF level after hemodialysis accompanied by elevated [F₂-Isoprostanes]_pl and decreased plasma TAC might be caused by enhanced oxidative stress induced by hemodialysis.     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

Dual-substrate inhibition kinetic studies for recombinant human interferon gamma producing Pichia pastoris

Pichia pastoris is considered as one of the prominent host extensively used as a platform for heterologous protein production. In the present study, the growth inhibition kinetics of recombinant P. pastoris expressing human interferon gamma was studied under different initial substrate concentrations of gluconate (10–100?g?L^?1) and methanol (2–50?g?L^?1) in modified FM22 medium. The highest specific growth rate of 0.0206 and 0.019?hr^?1 was observed at 60?g?L^?1 of gluconate and 10?g?L^?1 of methanol, respectively. Various three- and four-parametric Monod-variant models were chosen to analyze the inhibition kinetics. The model parameters as well as goodness of fit were estimated using nonlinear regression analysis. The three-parameter Haldane model was found to be best fit for both gluconate (R²?=?0.95) and methanol substrate (R²?=?0.96). The parameter sensitivity analysis revealed that µ_max, K_i, and K_s are the most sensitive parameters for both methanol and gluconate. Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double Webb model was found to be the best to explain the growth kinetics of recombinant P. pastoris.     

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid–base and DNA binding properties

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy?=?2,2′-bipyridine, Hbptip?=?2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid–base properties of the complex were studied by UV–visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK _a1?=?1.31?±?0.09 and pK _a2?=?5.71?±?0.11 with the pK _a2 associated deprotonation/protonation process occurring over 3 pK _a units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip?=?2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris–HCl buffer (pH 7.1 and 50?mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV–visible and emission spectroscopy techniques of UV–visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4?, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip?=?1H-imidazo[4,5-f][1,10]phenanthroline, Htip?=?2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip?=?2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.