In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.     

Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of Glutathione S-transferase Activity and Concentration in Aflatoxin-Producing and their Non-Toxigenic Counterpart Isolates

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 mumol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 mumol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.     

Study of the enhancement of a new chemiluminescence reaction and its application to determination of β‐lactam antibiotics

An enhanced thiosemicarbazide(TSC)–H₂O₂ chemiluminescence (CL) system was established and proposed as a new analytical method for determination of β‐lactam antibiotics, ampicillin sodium and amoxicillin at microgram levels. The method is based on the inhibition of CL emission accompanying oxidation of TSC by H₂O₂ in alkaline medium. The effect of anionic, cationic, and non‐ionic surfactants on the CL emission of the system was studied. Both N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB) and Triton X‐100, unlike sodium dodecyl sulfate (SDS), reinforced the CL intensity and were efficient to approximately the same level. The effect of the presence of eight non‐aqueous solvents on the CL system was also investigated. Upon addition of both of the non‐ionic surfactant, Triton X‐100, and the non‐aqueous solvent, N,N‐dimethyl formamide (DMF), the intensity of the CL reaction was increased 100‐fold. This method allows the measurement of 25–545 µg amoxicillin, and 35–350 µg ampicillin sodium. The detection limits are 8 µg for amoxicillin and 9 µg for ampicillin sodium. The relative standard deviations of six replicate measurements of 200 µg amoxicillin and 200 µg ampicillin sodium were 1.9 and 2.1%, respectively. The effect of foreign species on the determination of amoxicillin and ampicillin sodium was also examined. The proposed method was successfully applied to the determination of ampicillin sodium and amoxicillin in some pharmaceutical dosage forms. Copyright © 2008 John Wiley & Sons, Ltd.     

Neuronal apoptosis inhibitory protein,NAIP, is an inhibitor of procaspase-9

Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.     

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.     

Flow injection chemiluminescence determination of naphazoline hydrochloride in pharmaceuticals

A simple and sensitive flow injection chemiluminescence (FI‐CL) method was developed for the determination of naphazoline hydrochloride (NPZ). The method is based on the enhancing effect of NPZ on the weak CL signal from the reaction of KIO₄ with H₂O₂. Experimental parameters that affected the CL signal, including the pH of the KIO₄ solution, concentrations of KIO₄, H₂O₂ and disodium‐EDTA and flow rate were optimized. Under the optimum conditions, the increment of CL intensity was linearly proportional to the concentration of NPZ in the range 5.0 × 10^?6 to 70 × 10^?6 mol/L. The detection limit was 1.0 × 10^?6 mol/L and the relative standard deviation for 50 × 10^?6 mol/L NPZ solution was 2.8% (n = 11). In addition, a high throughput of 120 samples/h was achieved. The utility of this method was demonstrated by determining NPZ in pharmaceuticals. Copyright © 2013 John Wiley & Sons, Ltd.     

Oscillating chemiluminescence systems: state of the art

Oscillating chemiluminescence (CL) was reported for the first time about 30 years ago. Since then several systems based on addition of a chemiluminescent reagent to a known oscillator system or based on the light emitting features of one component of the oscillating system, have been described. This information, scattered in the scientific literature, is compiled in the present paper. Several oscillating CL systems, including those based on Belousov–Zhabotinskii and Orban oscillators, or horseradish peroxidase‐catalyzed reactions, among others, are critically presented. The application of this type of oscillatory systems is also discussed, in analytical chemistry and for educational purposes. Copyright © 2010 John Wiley & Sons, Ltd.     

Oscillating chemiluminescence with thiosemicarbazide in a batch reactor

Oscillating chemical reactions are complex systems involving a large number of chemical species. In oscillating chemical reactions, some species, usually reaction intermediates, exhibit fluctuations in their concentration. In this report, a novel slowly‐damped oscillating chemiluminescence produced by the addition of thiosemicarbazide (TSC) to the oscillating system H₂O₂–KSCN–CuSO₄–NaOH was investigated. Narrow and slightly asymmetric light pulses of 1.5 s half‐width are emitted at 440 nm, with an oscillation period of 22–363 s, an induction period of 9–397 s and an emitted light time of 700–1500 s, depending on reagent concentrations. In this study the dependence of the induction period and the oscillation period on the reagent concentrations was investigated and both parameters were plotted with respect to reagent concentrations. Copper concentration showed a significant effect on the oscillation period. A possible mechanism for the oscillating chemiluminescence reaction is discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Bone morphogenetic protein-7 incorporated polycaprolactone scaffold has a great potential to improve survival and proliferation rate of the human embryonic kidney cells

Renal failures treatment has been faced with several problems during the last decades. Kidney tissue engineering has been created many hopes to improve treatment procedures with scaffold fabrication that can modulate kidney cells/stem cells migration to the lesion site and increase the survival of these cells at that site with imitating the role of the kidney extracellular matrix. In this study, bone morphogenetic protein-7 (BMP7) as a vital factor for kidney development and regeneration was incorporated in the polycaprolactone (PCL) nanofibers and after morphological, mechanical, and biocompatible characterization, proliferation, and survival of the human embryonic kidney cells (HEK) were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and gene expression while cultured on scaffolds. Mechanical properties of the PCL nanofibers modulated after combining with BMP7 and hydration degree, protein adsorption and cell adhesion were enhanced in PCL-BMP7 compared to the pure PCL. Proliferation rate and growth increased significantly in HEK cells cultured on PCL-BMP7 when compared with that of PCL and tissue culture plate, whereas these data were also confirmed via significant decrease in apoptotic genes expression level in HEK cell cultured on PCL-BMP7. According to the results, PCL-BMP7 demonstrated positive effects on the survival and proliferation rate of the kidney cells and showed has also a great potential to use as a bioimplant for kidney tissue engineering applications.