Yongmin Ma

Efficient bifunctional gallium-68 chelators for positron emission tomography: tris(hydroxypyridinone) ligands

A new tripodal tris(hydroxypyridinone) bifunctional chelator for gallium allows easy production of (68)Ga-labelled proteins rapidly under mild conditions in high yields at exceptionally high specific activity and low concentration.

Iron chelation as a potential therapy for neurodegenerative disease.

Neurodegenerative disorders include a variety of pathological conditions, which share similar critical metabolic processes such as protein aggregation and oxidative stress, both of which are associated with the involvement of metal ions. Chelation therapy could provide a valuable therapeutic approach to such disease states, since metals, particularly iron, are realistic pharmacological targets for the rational design of new therapeutic agents.

Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4+/-1.3 microM.

Monitoring the efficiency of iron chelation therapy: the potential of nontransferrin-bound iron.

The major ligands of nontransferrin‐bound iron (NTBI) are suggested to be citrate and albumin. The proportion of iron binding to albumin is influenced by the degree of oxidation and glycation of the protein. LC‐ICP‐MS is demonstrated to be a useful technique for the speciation of NTBI, with unprocessed serum being subjected to analysis. Ferritin iron, citrate iron, and ferrioxamine can be quantified using this technique. This review describes the use of a new fluorescent probe for NTBI quantification.

Genetic epidemiology of induced CYP3A4 activity

Aim The cytochrome P450 3A4 (CYP3A4) enzyme is implicated in the metabolism of more than 50% of all prescribed medications and its activity – including induced or inhibited activity – is deemed to be a crucial determinant of interindividual variability in drug disposition, poor therapeutic efficacy, and adverse response to medication. Methods We used the classical twin model in conjunction with an induction experiment to uncover the relative contribution of genetic and environmental factors to interindividual variation in induced CYP3A4 activity. A total of 367 healthy twins participated in the study. Each volunteer was administered a potent inducer of CYP3A4 (St John’s Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate. Results Baseline and induced CYP3A4 activity were highly variable with a seven-fold and 11-fold difference among our population, respectively. Alcohol consumption, BMI, and smoking were significantly associated with induced CYP3A4 activity, collectively explaining 20% of the variation (P<1×10−4). The narrow-sense heritability of induced CYP3A4 activity was estimated at 66%, whereas the remainder of the variation was attributed to unique environmental factors. Conclusion To our knowledge, this is the first genetic epidemiological study of induced CYP3A4 activity. Our results motivate further research to identify common and rarer genetic variants that underpin the heritable component of variation in induced CYP3A4 activity.

Poly(vinyl alcohol) nanoparticle stability in biological media and uptake in respiratory epithelial cell layers in vitro

The influence of the size and surface properties of nanoparticles (NP) upon respiratory epithelial cell uptake and translocation is difficult to study, because NP properties are often modified upon suspension in biological fluids. However, a recently developed novel fluorescently labelled poly(vinyl alcohol) (PVA) NP, which does not aggregate in simple biological fluids, is suitable for drug delivery and can be produced with a range of surface properties is a pertinent advance in this field. The aim of this study was to employ the PVA NP to investigate how surface properties influence particle uptake and translocation across Calu-3 epithelial cell layers. Several grades of PVA were synthesised, characterised and labelled covalently with carboxyfluorescein. The labelled PVA was used to fabricate trackable NP that displayed either neutral or positive charge when suspended in Hanks Balanced Salt Solution. The NP were applied to the apical surface of Calu-3 cell layers which internalised up to 11% of the applied particle dose. The maximum fraction that translocated the Calu-3 barrier in 14 h was 1.3%.

Design and Synthesis of Fluorinated Iron Chelators for Metabolic Study and Brain Uptake

A range of fluorinated 3-hydroxypyridin-4-ones has been synthesized where fluorine or fluorinated substituent was attached at 2- or 5- position of the pyridine ring in order to improve chemical and biological properties of 3-hydroxypyridin-4-ones. The synthetic route is different from conventional counterparts where a functional group is introduced to a preformed 3-hydroxypyridin-4-one ring. Herein, we introduce a novel method which starts with a fluorine containing precursor and the two hydroxyl groups at 3- and 4- positions of the pyridine ring are introduced at a later stage. The pK(a) values of the free ligands and the affinity constants of their iron complexes demonstrate that the presence of fluorine dramatically alters the values. The distribution coefficient values of the free ligands and corresponding iron(III) complexes between 1-octanol and MOPS buffer (pH 7.4) are also influenced. Glucuronidation and oxidation studies of selected fluorinated 3-hydroxypyridin-4-ones demonstrate that some such fluorinated compounds have clear advantage over deferiprone in that they are metabolized more slowly. Blood-brain barrier permeability studies indicated that although lipophilicity influences the permeability it is not the only factor. Two of the selected seven fluorinated 3-hydroxypyridin-4-ones have improved brain distribution when compared with deferiprone.

Prediction of 3-hydroxypyridin-4-one (HPO) hydroxyl pKa values

As an aid in optimising the design of 3-hydroxypyridin-4-ones (HPOs) intended for use as therapeutic Fe(3+) chelating agents, various quantum mechanical (QM) and semi-empirical (QSAR) methods have been explored for predicting the pK(a) values of the hydroxyl groups in these compounds. Using a training set of 15 HPOs with known hydroxyl pK(a) values, reliable predictions are shown to be obtained with QM calculations using the B3LYP/6-31+G(d)/CPCM model chemistry (with Pauling radii, and water as solvent). With this methodology, the observed hydroxyl pK(a) values for the training set compound are closely matched by the predicted pK(a) values, with the correlation between the observed and predicted values giving r(2) = 0.98. Predictions subsequently made by this method for a test set of 48 HPOs of known hydroxyl pK(a) values (11 of which were determined experimentally in this study), gave predicted pK(a) values accurate to within ±0.2 log units. In order to further investigate the predictive power of the method, two novel HPOs were synthesised and their hydroxyl pK(a) values were determined experimentally. Comparison of these predicted pK(a) values against the measured values gave absolute deviations of 0.13 (10.18 vs. 10.31) and 0.43 (5.58 vs. 5.15).

A poly(vinyl alcohol) nanoparticle platform for kinetic studies of inhaled particles

The lack of a well defined nanosystem that retains its physicochemical properties and can be tracked in complex biological environments is one reason why the study of NP transport across biological barriers is currently so difficult. As a result, surprisingly little is known about the fate of sub-micron particles once they deposit in the airways of the lung. The aim of this study was to design and manufacture a novel nanoparticle (NP) core that would be physically stable, i.e., not aggregate in biological fluids, and act as a tracking system to investigate NP distribution in the lung. Accordingly, covalent fluorescent labeling (to allow particle tracking) of 40% hydrolyzed poly(vinyl alcohol) was undertaken by inducing dissociation of the carboxylic acid group (ArCOO−) of 5(6)-carboxyfluorescein (CF) which then reacted with the hydroxyl group of poly(vinyl alcohol) (PVA) to produce a covalently linked PVA-CF ester. Polymer purification was followed by NP manufacture and characterization in biological media. In contrast to commercial latex particles which aggregated in both cell culture medium and Hank’s balanced salt solution (HBSS), the PVA nanoparticles retained their original size (ca. 220 nm), maintained a neutral surface charge in cell culture medium for 24 h and were not acutely toxic to respiratory cells in vitro.

Determination of the Labile Iron Pool of Human Lymphocytes using the Fluorescent Probe, CP655

Background: We have recently developed a new technique for quantitatively measuring protein-bound 3-nitrotyrosine (3-NT), a footprint of nitrosative stress, utilizing high-performance liquid chromatography with an electrochemical detection (HPLC-ECD) system. Using this system, we showed that 3-NT formation was upregulated in the sputum of both COPD and asthmatic patients. However, in order to improve the accuracy of the measurement system, We have to resolve some problems which were the influence of free amino acid form of 3-NT and of salivary contamination. Objectives: We initially investigated the amount of the free amino acid form of 3-NT in induced sputum and compared with that of protein-bound 3-NT. Next, we evaluated the concentration of protein-bound 3-NT in saliva and compared with that in induced sputum by means of HPLC-ECD. Methods: Five male COPD patients were enrolled. Induced sputum and saliva were obtained from the patients. The free amino acid form of 3-NT in sputum and saliva was measured by HPLC-ECD, and the protein-bound 3-NT and tyrosine in sputum and saliva were enzymatically hydrolyzed by Streptomyces griseus Pronase and measured for the protein hydrolysate by HPLC-ECD. Results: The mean value of the amount of protein-bound 3-NT was 65.0 fmol (31.2 to 106.4 fmol). On the other hand, the amount of the free amino acid form of 3-NT was under the detection limit (<10 fmol). The levels of both 3-NT (sputum: 0.55 ± 0.15 pmol/ml, saliva: 0.02 ± 0.01 pmol/ml, p < 0.01) and tyrosine (sputum: 0.81 ± 0.43 μmol/ml, saliva: 0.07 ± 0.04 μmol/ml, p < 0.01) in saliva were significantly lower than in sputum. The percentage of 3-NT in saliva to that in sputum was about 3.1%, and that of tyrosine was about 9.0%. Conclusion: The free amino acid form of 3-NT does not affect the measurement of protein-bound 3-NT. Furthermore, the influence of salivary contamination on the measurement of protein-bound 3-NT in induced sputum by means of HPLC-ECD was very small and could be negligible.A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins.This research shows a novel method for hazard identification of a chemical and UV light on a single cell level with a laser probe beam. The laser probe beam was passed through interface of cell membrane/culture medium of a cultured human hepatoblastoma cell line HepG2. Deflection of the laser probe beam, which was induced by changes in concentration gradients due to the active materials movement across the cell membrane, was monitored. When a toxic hazard existed, a living cell was expected to be killed or injured, or cellular behaviors to be changed greatly. Then, the changing deflection signal from the living cell would become unchanged or altered in a different way. This was successfully demonstrated with cytotoxity of UV light and H2O2. Most of the cultured HepG2 cells showed changing deflection signals after 10 min illumination of UV-visible light longer than 370 nm, while almost all HepG2 cells showed unchanged deflection signal after 10 min illumination of UV-visible light with wavelength longer than 330 nm. The results suggested that UV light between 330–370 nm could kill the cells. Additions of H2O2 solution with different concentrations to the cell cultures caused the changing deflection signal from a living cell either unchanged or changed in different trend, suggesting toxicity of H2O2 to the cells. The results from the beam deflection detection agreed well with those obtained by the conventional trypane blue method.Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 107 per μg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.We have determined and quantified spectrophotometrically the capacity of producing reactive oxygen species (ROS) as 1O2 during the photolysis with UV-A light of 5 new synthesized naphthyl ester derivates of well-known quinolone antibacterials (nalidixic acid (1), cinoxacin (2), norfloxacin (3), ciprofloxacin (4) and enoxacin (5)). The ability of the naphthyl ester derivatives (6–10) to generate singlet oxygen were detecting and for the first time quantified by the histidine assay, a sensitive, fast and inexpensive method. The following tendency of generation of singlet oxygen was observed: compounds 7 > 10 > 6 > 8 > 9 >> parent drugs 1–5.High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol ml−1/μMol ml−1)], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.Microalbuminuria is associated with hypertension and is a strong risk factor for subsequent chronic disease, both renal and coronary heart disease (CHD), Presently there are several methods available for measurement of microalbuminuria. The aim of this study was to evaluate if the three different methods gave similar information or if one of the assays were superior to the others. Blood pressure, inflammatory markers and cardiovascular mortality and morbidity were correlated with urine albumin analysed with a point-of-care testing (POCT) instrument, nephelometric determination of albumin and albumin/creatinine ratio in elderly males. The study population consisted of 103 diabetic and 603 nondiabetic males (age 77 years) in a cross-sectional study. We analyzed urine albumin with a HemoCue® Urine Albumin POCT instrument and a ProSpec® nephelometer and albumin/creatinine ratio. There were strong correlations between both systolic and diastolic blood pressure and all three urine albumin methods (p < 0.0001). There were also significant correlations between the different urine albumin measurements and serum amyloid A component, high-sensitivity C-reactive protein and interleukin-6. The three different urine albumin methods studied provided similar information in relation to cardiovascular disease. There was a strong correlation between systolic and diastolic blood pressure and microalbuminuria in both the whole study population and in nondiabetic males emphasizing the role of hypertension in glomerular damage. The good correlation between the studied urine albumin measurements show that all three methods can be used for monitoring urine albumin excretion.Chromium is an important constituent widely used in different industrial processes for production of various synthetic materials. For evaluation of workers’ exposure to trace toxic metal of Cr (III), environmental and biological monitoring are essential processes, in which, preparation of samples is one of the most time-consuming and error-prone aspects prior to analysis. The use of solid-phase extraction (SPE) has grown and is a fertile technique of sample preparation as it provides better results than those produced by liquid-liquid extraction (LLE). SPE using mini columns filled with XAD-4 resin was optimized regarding to sample pH, ligand concentration, loading flow rate, elution solvent, sample volume, elution volume, amount of resins, and sample matrix interferences. Chromium was retained on solid sorbent and was eluted with 2 M HNO3 followed by simple determination of analytes by using flame atomic absorption spectrometery. Obtained recoveries of metal ion were more than 92%. The optimized procedure was also validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as six within-day experiments. Through this study, suitable results were obtained for relative standard deviation, therefore, it is concluded that, this optimized method can be considered to be successful in simplifying sample preparation for trace residue analysis of Cr in different matrices for evaluation of occupational and environmental exposures. To evaluate occupational exposure to chromium, 16 urine samples were taken, prepared, and analyzed based on optimized procedure.Mistletoe Extracts (ME) are of growing interest to pharmacological research because of their apoptosis-inducing/cytostatic and immunomodulatory effects. The standardization of the three different groups of Mistletoe Isolectins (ML-I, II and III) is often rendered more difficult since the primary structures are nearly identical. Their classification is based on their Galactose- and N-acetyl-D-galactosamine (GalNAc)-specificity which was measured by various inhibitory assays. The aim of the present study was to improve the characterization of the direct binding activity of the isolectins from ME to immobilized lactose, GalNAc and to the oligosaccharide asialofetuin. After careful ultrafiltration of fresh ME, affinity chromatography was carried out using lactose- agarose, GalNAc—agarose and asialofetuin—affigel 15 columns. MLs were further purified by Sephadex G-100 or by cation exchange chromatography which was adapted to a Fast Protein Liquid Chromatography (FPLC) system. Proteins from both fresh plants and commercial ME were able to bind immobilized lactose to a considerable extent. The majority of this lectin has a B-chain with a Molecular Weight (MW) of 34kD and an A-chain with a MW of 29 kD (ML-I). Only a minor part of the lactose-binding proteins has a lower MW, namely 32kD and 27kD (MLII). However, neither MLs which were eluted from lactose columns, nor the proteins from fresh plant or ME showed a direct binding to the immobilized GalNAc. In spite of this deficiency, GalNAc was able to induce a considerable (25% and 32%) inhibitory effect on their binding to immobilized asialofetuin indicating a discrepancy between the lectin binding and inhibiting effects of GalNAC. Consequently, for an improved standardization of ME more specific sugar molecules are necessary.Pentavalent technetium-99m dimercaptosuccinic acid (99mTc-(V)DMSA) is a tumor-seeking agent which was introduced to evaluate, image, and manage many types of cancers. In this review, the beginning of, and the most recent applications of using this agent was appraised. The relation with tumor cell detection and proliferation was reported and several mechanisms of uptake of 99mTc-(V)DMSA in tumor cells are described.We studied the near UV absorption spectrum of canine plasminogen. There are 19 tryptophans, 19 phenylalanines and 34 tyrosines in the protein. 4th derivative spectra optimized for either tryptophan or tyrosine give a measure of the polarity of the environments of these two aromatic amino acids. Plasminogen at temperatures between 0 °C and 37 °C exists as a mixture of four conformations: closed-relaxed, open-relaxed, closed-compact, and open-compact. The closed to open transition is driven by addition of ligand to a site on the protein. The relaxed to compact transition is driven by increasing temperature from 0 °C to above 15–20 °C. When the conformation of plasminogen is mainly closed-relaxed, the 4th derivative spectra suggest that the average tryptophan environment is similar to a solution of 20% methanol at the same temperature. Under the same conditions, 4th derivative spectra suggest that the average tyrosine environment is similar to water. These apparent polarities change as the plasminogen is forced to assume the other conformations. We try to rationalize the information based on the known portions of the plasminogen structure.Chirasil-β-Dex containing an undecamethylene spacer (C11-Chirasil-Dex) was synthesized and used as chiral stationary phase (CSP) in enantioselective gas chromatography (GC). The versatility of the new stationary phase in the simultaneous enantiomeric separation of a set of N-alkylated barbiturates is demonstrated.We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a “cytosolic” medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 ± 0.27 μM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.The aim of this study is to adopt the approach of metabolic fingerprinting through the use of Fourier Transform Infrared (FTIR) technique to understand changes in the chemical structure in Padina tetrastromatica (Hauck). The marine brown alga under study was grown in two different environmental conditions; in natural seawater (P. tetrastromatica (c)) and in seawater suplemented with 50 ppm of cadmium (P. tetrastromatica (t)) for a three-week period in the laboratory. The second derivative, IR specrum in the mid-infrared region (4000–400 cm−1) was used for discriminating and identifying various functional groups present in P. tetrastromatica (c). On exposure to Cd, P. tetrastromatica (t) accumulated 412 ppm of Cd and showed perturbation in the band structure in the mid-IR absorption region. Variation in spectral features of the IR bands of P. tetrastromatica (untreated and treated) suggests that cadmium ions bind to hydroxyl, amino, carbonyl and phosphoryl functionalities. This was attributable to the presence of the following specific bands. A band at 3666 cm−1 in untreated P. tetrastromatica (c) while a band at 3560 cm−1 in Cd-treated P. tetrastromatica (t) due to non bonded and bonded O-H respectively. Similarly, non bonded N-H for P. tetrastromatica (c) showed two bands at 3500 cm−1 and 3450 cm−1 due to the N-H stretching vibrations and a band at 1577 cm−1 due to N-H bending vibrations, while an intense band at 3350 cm−1 due to bonded N-H stretching vibrations and at 1571 cm−1 due to bending vibrations was observed for Cd-treated P. tetrastromatica (t). Involvement of ester carbonyl group is characterized by the presence of a band at 1764 cm−1 in untreated P. tetrastromatica (c) while the Cd-treated P. tetrastromatica (t) showed the band at 1760 cm−1. The intensity of the band at 1710 cm−1 in the control samples decreased drastically after cadmium treatment indicating carbonyl of COOH to be involved in metal chelation. A band at 1224 cm−1 for untreated P. tetrastromatica (c) and at 1220 cm−1 for Cd-treated P. tetrastromatica (t) is indicative of the involvement of phosphoryl group in metal binding. Several other such changes were also evident and discussed in this paper. Based on our observation, FTIR technique proves to be an efficient tool for detecting structural changes and probable binding sites induced by the presence of a metal pollutant, cadmium, in the marine environment.A high-performance liquid chromatographic (HPLC) method has been developed for the separation and determination of S- and R-enantiomers of betaxolol in tablets and ophthalmic preparations. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with fluorescence detection at excitation/emission wavelengths 275/305 nm. The polar ionic mobile phase (PIM) consists of methanol-glacial acetic acid-triethylamine, (100:0.020:0.025, v/v/v) has been used at a flow rate of 1.5 ml/min. All analytes with S-(–)-atenolol as internal standard were conducted at ambient temperature. The method is highly specific where another coformulated compounds did not interfere. The stability of betaxolol enantiomers under different degree of temperature also studied. The results showed that it is stable for at least 7 days at 70°C. The method validated for its linearity, accuracy, precision and robustness. Experimental design was used during validation to evaluate method robustness. Using the chromatographic conditions described, S- and R-betaxolol were well resolved with mean retention times of 11.3 and 12.6 min, respectively. Linear response (r > 0.997) was observed over the range of 10–500 ng/ml of betaxolol enantiomers, with detection limit of 5 ng/ml. The recoveries of S- and R-betaxolol from tablets and ophthalmic preparation ranged from 97.4 to 101.4% and 98.0 to 102.0%, respectively. The mean relative standard deviation (R.S.D.%) for both enantiomers were 1.1–1.4% and 1.3–1.7% in tablets and ophthalmic solution, respectively.Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today’s drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec™) in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK) Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (Sprycel™) or bosutinib (SKI-606). Our work has led to the development of NS-187 (INNO-406), a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profiling of NS-187, including site-directed mutagenesis experiments.