A product inhibition study on adenosine deaminase by spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the inosine product on the activity of adenosine deaminase in a 50 mM sodium phosphate buffer, pH 7.5, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for inosine as a product of the enzymatic reaction. A graphical-fitting method was used for determination of the binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 140 microM by the microcalorimetry method, which agrees well with the value of 143 microM for the inhibition constant that was obtained from the spectroscopy method     

Molecular characterization of rpoB gene mutations in rifampicine-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.     

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.     

A short review on the structure-function relationship of artificial catecholase/tyrosinase and nuclease activities of Cu-complexes

The synthesis of metal complexes has vastly increased the scope of research for many scientists during the two last decades. Among these compounds, artificial tyrosinases, catecholases, proteases, and nucleases are some of the most studied due to their importance as modern tools in the fields of medicine, scientific research, and industry. Transition metals such as Zn(2+), Cu(2+), Fe(3+), Co(3+), Ni(2+), and lanthanide ions are the most commonly used. Among these ions, copper complexes have been the focus of the majority of studies thanks to their significant activity in comparison with other ions. Studies of copper-based tyrosinases, catecholases, and nucleases have revealed some of the overarching factors affecting reactions of all three types, which has led to improved activity and efficiency for all. Key factors include proper core-core distance, (Cu?Cu distance 2.90-2.99??), suitable solvent, and ligands with proper hydrophobic structure and geometry. In the present investigation, we review and introduce the proposed mechanisms and the kinetically effective factors of natural catecholase, tyrosinase, and nuclease and their Cu-based synthetic mimics.     

Estimates of economic values for traits of Arabic sheep in village system

A deterministic bio-economic model was used to estimate the economic values of different traits in Arabic sheep native to the Khuzestan province of Iran. In the studied system, variable costs accounted for about 98.5% of the total costs and among variable costs, feed costs had the highest proportion with 70.7%. Revenue sources included meat, wool, and manure, where meat was the most important one and formed 95.5% of total revenues. Economic value for a trait was estimated as the amount of change in the profit of system as its mean increased by one unit, while the means of other traits were constant. The most important trait in this system was litter size, followed by ewe survival, dressing percentage, and wool weight, respectively. Birth weight had a negative economic value but weight at older ages especially weaning weight and 12-month weight had positive economic values. The sensitivity of economic values of traits was investigated by changing feed and non-feed costs, meat and wool prices by ±10%. Results showed that economic values for dressing percentage and wool weight are not sensitive to change in costs. In addition, changes in marketing and management costs had no effect on the economic value for traits related to body weight in different ages. In general, the economic value for traits which showed sensitivity to the changes of costs, except ewe survival, decreased due to an increase in costs. The economic value for all traits, except birth and wool weight, changed because of a change in meat price. Increasing meat price meant a higher economic importance. Among different factors, meat price fluctuations had the most effect on the economic value of traits.     

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.     

Synthesis,cytotoxicity assessment,and interaction and docking of novel palladium(II) complexes of imidazole derivatives with human serum albumin

Imidazole analogs are the agents that attract both bioinorganic chemist and drug designer. Numerous methods have been proposed for synthesis of imidazole derivatives. In this study, a series of heterocyclic system with p-conjugated system such as 2-aryl-imidazo[4,5-f][1,10]phenanthroline analogs were synthesized. Then, three new palladium(II) complexes containing 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1,10]Phenanthroline (FIP) and 2-(thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (TIP) ligands were synthesized. The structures of the compounds, [Pd(Phen)(TIP)](NO₃)₂, [Pd(Phen)(FIP)](NO₃)₂, and [Pd(FIP)₂]Cl were determined by spectroscopic methods and elemental analysis. Biological activity of the complexes synthesized was assessed against chronic myelogenous leukemia cell line, K562. Also, the interactions of human serum albumin with complexes were investigated using isothermal titration in the Tris buffer, pH 7.4. According to the results obtained, it was found that there is a set of six binding sites for these complexes on HSA with positive cooperativity in the binding process. Docking technique was also applied to confirm the experimental results. The results showed that smaller complexes have higher interaction affinity.     

Comparative Studies on the Interaction Between Bovine β-lacto-globulin Type A and B and a New Designed Pd(II) Complex with Anti-tumor Activity at Different Temperatures

Abstract An new water-soluble Pd(II) complex, 2,2'-bipyridin n-butyl dithiocarbamato Pd(II) nitrate has been synthesized. The Pd(II) complex has been characterized by elemental analysis and conductivity measurements as well as spectroscopic methods such as infrared, 1H NMR, and ultraviolet-visible. The interaction between this new design Pd(II)-complex, an anti-tumor component, with carrier proteins of β-lactoglobulin-A and -B (BLG-A and -B) were studied at different temperatures of 27, 37, 42, and 47 °C by fluorescence spectroscopy and far-UV circular dichroism (CD) spectrophotometric techniques. A strong fluorescence quenching interaction of Pd(II) complex with BLG-A and -B was observed at different temperatures. The binding parameters were evaluated by fluorescence quenching method. The thermodynamic parameters, including ΔH°, ΔS°, and ΔG° were calculated by fluorescence quenching method indicated that the electrostatic and hydrophobic forces might play a major role in the interactions of Pd(II) complex with BLG-A and -B, respectively. The distances between donors (Trps of the BLG-A and -B) and acceptor (Pd(II) complex) were obtained according to the fluorescence resonance energy transfer (FRET). Far-UV CD studies showed that the Pd(II) complex did not represent any significant changes in the secondary structures of BLG- A and -B. The difference in the interaction properties observed for BLG-A and -B with Pd(II) complex is related to the difference in the amino acid sequences between these two variants.     

Investigation of effects of newly synthesized Pt(II) complex against human serum albumin and leukemia cell line of K562

The biological evaluation of a new synthesized Pt(II)-complex, 2,2'-bipyridin Butylglycinato Pt(II) nitrate, an anti-tumor component, was studied at different temperatures by fluorescence and far UV circular dichroism (CD) spectroscopic methods. Human serum albumin (HSA) and human tumor cell line K562 were as targets. The Pt(II)-complex has a strong ability to quench the intrinsic fluorescence of HSA. Binding and thermodynamic parameters of the interaction were calculated by fluorescence quenching method. Far-UV-CD results showed that Pt(II)-complex induced increasing in content of α helical structure of the protein and stabilized it. The 50% cytotoxic concentration (Cc(50)) of complex was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay at different incubation times. Also, fluorescence staining with DAPI (4,6-diamidino-2-phenylindole) revealed some typical nuclear changes, which are characteristic of apoptosis. Above results suggest that Pt (II) complex is a promising anti-proliferative agent and should execute its biological effects by inducing apoptosis.