Structural mobility and transformations in globular proteins

Although globular proteins are endowed with well defined three-dimensional structures, they exhibit substantial mobility within the framework of the given threedimensional structure. The different types of mobility found in proteins by and large correspond to the different levels of organisational hierarchy in protein architecture. They are of considerable structural and functional significance, and can be broadly classified into(a) thermal and conformational fluctuations, (b) segmental mobility, (c) interdomain mobility and (d) intersubunit mobility. Protein crystallographic studies has provided a wealth of information on all of them. The temperature factors derived from X-ray diffraction studies provide a measure of atomic displacements caused by thermal and conformational fluctuations. The variation of displacement along the polypeptide chain have provided functionally significant information on the flexibility of different regions of the molecule in proteins such as myoglobin, lysozyme and prealbumin. Segmental mobility often involves the movement of a region or a segment of a molecule with respect to the rest, as in the transition between the apo and the holo structures of lactate dehydrogenase. It may also involve rigidification of a disordered region of the molecule as in the activation of the zymogens of serine proteases. Transitions between the apo and the holo structures of alcohol dehydrogenase,and between the free and the sugar bound forms of hexokinase, are good examples of interdomain mobility caused by hinge-bending. The capability of different domains to move semi-independently contributes greatly to the versatility of immunoglobulin molecules. Interdomain mobility in citrate synthase appears to be more complex and its study has led to an alternative description of domain closure. The classical and the most thoroughly studied case of intersubunit mobility is that in haemoglobin. The stereochemical mechanism of the action of this allosteric protein clearly brings out the functional subtilities that could be achieved through intersubunit movements. In addition to ligand binding and activation,environmental changes also often cause structural transformations. The reversible transformation between 2 Zn insulin and 4 Zn insulin is caused by changes in the ionic strength of the medium. Adenylate Kinase provides a good example for functionally significant reversible conformational transitions induced by variation in pH. Available evidences indicate that reversible structural transformations in proteins could also be caused by changes in the aqueous environment, including those in the amount of water surrounding protein molecules.

A circular dichroism study of (+) gossypol binding to proteins

The circular dichroism bands of (+) gossypol in the spectral region 300–400 nm have been shown to be sensitive to interactions with proteins. Using CD spectroscopy, gossypol has been shown to interact with lactate dehydrogenase, malate dehydrogenase, alkaline phosphatase, lysozyme, protamine and poly-L-lysine. Binding to proteins generally results in a pronounced red shift of the long wavelength CD band (not, vert, similar 380–430 nm) accompanied by a reduction in ellipticity. The changes in spectral parameters of the 1Lb binaphthyl transtion may reflect a distortion from a nearly perpendicular gossypol conformation, on binding to proteins.

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.

A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Malaria causes a worldwide annual mortality of about a million people.Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition,our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.

Abnormal levels of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in tumour tissue and blood samples from patients diagnosed with lung cancer

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

Synthesis and Antileukemic Activity of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea Derivatives

Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazoles and urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6.7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect to cancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electron-withdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH) assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.

Ketoprofeenin biologinen käytettävyys ja farmakokinetiikka sioilla eri antotavoilla ja ketoprofeeniyliannoksen farmakokinetiikka ja vaikutus munuaisiin lampailla

Ketoprofeeni on yleisesti käytetty ei-steroidinen tulehduskipulääke (NSAID) lampaiden ja sikojen kivunlievityksessä. Tietoa ketoprofeenin oikeista annosmääristä eri eläinlajeilla on saatavilla rajallisesti. Oikeaa lääkeainemäärää ei voida luotettavasti ekstrapoloida toisten eläinlajien tai ihmisten perusteella. Epäillyissä tulehduskipulääkemyrkytyksissä ongelmana on tietää, oliko eläimen saama lääkeannos toksinen. Lampailla tehdyn tutkimuksen tavoitteena oli selvittää, muuttuuko ketoprofeenin kinetiikka kymmenkertaisella yliannoksella, tutkia yliannoksen vaikutusta munuaisiin ja löytää yksinkertainen tapa diagnosoida yliannos virtsasta. Sioilla tehdyn tutkimuksen tavoitteena oli selvittää ketoprofeenin biologista käytettävyyttä ja ketoprofeenin farmakokinetiikkaa sioilla intravaskulaarisella, intramuskulaarisella ja peroraalisella annolla. Keskeiset tutkimuksessa määritettävät muuttujat olivat AUC0-_, Cmax ja tmax. Hyötyosuus laskettiin i.v. -annon perusteella. Kuudelle lampaalle annettiin 30 mg/kg i.v. -ketoprofeenia. Ketoprofeenin pitoisuuksia seurattiin 24 tunnin ajan plasmanäytteillä, joiden perusteella määritettiin farmakokineettiset parametrit. Veri- ja virtsanäytteistä tutkittiin muun muassa mahdollisesta munuaisvauriosta kertovia entsyymejä. 24 tunnin kuluttua lääkkeenannosta lampaat lopetettiin ja munuaiset tutkittiin histologisesti. Tutkittaville kahdeksalle sialle annosteltiin 3 mg/kg intravaskulaarista, intramuskulaarista ja oraalista ketoprofeenia sekä 6 mg/kg oraalista ketoprofeenia. Tutkimus suoritettiin satunnaistettuna vaihtovuorotutkimuksena. Ketoprofeenin pitoisuuksia seurattiin plasmanäytteillä 48 tunnin ajan lääkkeenannosta ja kaikille antotavoille laskettiin farmakokineettiset parametrit. Lisäksi tutkittiin valmisteiden biologinen samanarvoisuus. Molempien tutkimusten in vivo -kokeet suoritettiin Eläinlääketieteellisessä tiedekunnassa. Samoin munuaisten histologinen tutkimus ja virtsasta ja verestä tehdyt määritykset, lukuun ottamatta ketoprofeeninpitoisuuden analysointia. Plasman ketoprofeenipitoisuus analysoitiin korkean erotuskyvyn nestekromatografialla (HPLC). Ketoprofeenimääritykset ja farmakokineettinen analyysi suoritettiin Farmasian tiedekunnassa. Lampaiden kymmenkertainen ketoprofeeniyliannos oli toksinen. Seerumin urea- ja kreatiniinipitoisuus nousivat ja histologisissa näytteissä näkyi akuutti munuaistiehyen vaurio. Useiden entsyymien pitoisuus nousi virtsassa. Selvimmin ja nopeimmin nousi virtsan laktaattidehydrogenaasipitoisuus, jonka määrittäminen vaikuttaa potentiaaliselta tavalta diagnosoida ketoprofeenin toksinen annos. Ketoprofeenin eliminaation puoliintumisaika toksisella annoksella oli samaa suuruusluokkaa kuin aiemmissa tutkimuksissa terapeuttisella annoksella, joten yliannos ei muuttanut ketoprofeenin kinetiikkaa. AUC- ja Cmax -arvot olivat suhteessa suurempia kuin terapeuttisella annoksella, joten tutkimuksen perusteella kyseiset arvot eivät nousseet lineaarisesti annoksen noustessa toksiseksi. Sioille annetut ketoprofeenivalmisteet eivät olleet biologisesti samanarvoisia keskenään. Hyötyosuus oli erittäin hyvä kaikilla antotavoilla. tmax oli kaikilla antotavoilla hieman yli tunnin kuluttua lääkkeenannosta. Oraalisen 3 mg/kg -annoksen Cmax oli 5,1 mg/l ja AUC 32 mg l-1 h ja intramuskulaarisen vastaavat arvot olivat 7,6 mg/l ja 37 mg l-1 h. Oraalisen ketoprofeenin annostasojen AUC- ja Cmax -arvot korreloivat keskenään, joten ketoprofeenin kinetiikka oli lineaarista. Intravaskulaarisen ja oraalisen annon puoliintumisajoissa oli tilastollisesti merkitsevä ero. Ketoprofeenin jakautumistilavuudessa ja puhdistumassa ei ollut tilastollisesti merkitsevää eroa eri antotapojen välillä.

Synthesis and Identification of a new class of antileukemic agents containing 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole

Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahydrobenzod]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7 tetrahydrobenzod]thiazole moiety, a series of 2-arlycarboxamide substituted-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetrhydrobenzod]thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with ortho substitution showed poor activity and with meta and para substitution showed good activity. (C) 2010 Elsevier Masson SAS. All rights reserved.

Characterization of Major Zinc Containing Myonecrotic and Procoagulant Metalloprotease `Malabarin' from Non Lethal Trimeresurus malabaricus Snake Venom with Thrombin Like Activity: Its Neutralization by Chelating Agents

A major myonecrotic zinc containing metalloprotease `malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu-Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A alpha followed by B beta subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

DNA targeting polyaza macrobicyclic dizinc(II) complexes promoting high in vitro caspase dependent anti-proliferative activity against human carcinoma cancer cells

A series of macrobicyclic dizinc(II) complexes Zn2L1-2B](ClO4)(4) (1-6) have been synthesized and characterized (L1-2 are polyaza macrobicyclic binucleating ligands, and B is the N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)). The DNA and protein binding, DNA hydrolysis and anticancer activity of these complexes were investigated. The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence and CD spectroscopy. The DNA binding constant values of the complexes were found to range from 2.80 x 10(5) to 5.25 x 10(5) M-1, and the binding affinities are in the following order: 3 > 6 > 2 > 5 > 1 > 4. All the dizinc(II) complexes 1-6 are found to effectively promote the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 3 and 6 under physiological conditions give observed rate constants (k(obs)) of 5.56 +/- 0.1 and 5.12 +/- 0.2 h(-1), respectively, showing a 10(7)-fold rate acceleration over the uncatalyzed reaction of dsDNA. Remarkably, the macrobicyclic dizinc(II) complexes 1-6 bind and cleave bovine serum albumin (BSA), and effectively promote the caspase-3 and caspase-9 dependent deaths of HeLa and BeWo cancer cells. The cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme levels in cancer cell lysate and content media.

Synthesis and evaluation of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles as anti-cancer agents

A series of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles 9a-j were obtained via multistep synthesis from hydroxybenzophenones 4a-e. The cytotoxicity of compounds 9a-j was evaluated against human leukemia cell lilies (K562 and CEM). The compounds exhibited moderate to good anti-cancer activity with compounds 9b and 9i having a chloro group exhibiting the best activity (IC50 = 10 mu M). Compound 9i exhibited activity against both the cell lines and 9b only exhibited activity against CEM. Further, a lactate dehydrogenase (LDH) assay and DNA fragmentation studies of the compounds 9a-j were also performed. (C) 2013 Elsevier Masson SAS. All rights reserved.

Patterned growth and differentiation of neural cells on polymer derived carbon substrates with micro/nano structures in vitro

The growth of neuroblastoma (N2a) and Schwann cells has been explored on polymer derived carbon substrates of varying micro and nanoscale geometries: resorcinol-formaldehyde (RE) gel derived carbon films and electrospun nanofibrous (similar to 200 nm diameter) mat and SU-8 (a negative photoresist) derived carbon micro-patterns. MTT assay and complementary lactate dehydrogenase (LDH) assay established cytocompatibility of RE derived carbon films and fibers over a period of 6 days in culture. The role of length scale of surface patterns in eliciting lineage-specific adaptive response along, across and on the interspacing between adjacent micropatterns (i.e., ``on'', ``across'' and ``off'') has been assayed. Textural features were found to affect 3',5'-cyclic AMP sodium salt-induced neurite outgrowth, over a wide range of length scales: from similar to 200 nm (carbon fibers) to similar to 60 mu m (carbon patterns). Despite their innate randomness, carbon nanofibers promoted preferential differentiation of N2a cells into neuronal lineage, similar to ordered micro-patterns. Our results, for the first time, conclusively demonstrate the potential of RE-gel and SU-8 derived carbon substrates as nerve tissue engineering platforms for guided proliferation and differentiation of neural cells in vitro. (C) 2013 Elsevier Ltd. All rights reserved.

Biocompatible and Antibacterial SnO2 Nanowire Films Synthesized by E-Beam Evaporation Method

In this work, the biocompatibility and antibacterial activities of novel SnO2 nanowire coatings prepared by electron-beam (E-Beam) evaporation process at low temperatures were studied. The nanowire coatings were characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and X-ray diffraction (XRD) methods. The results of in vitro cytotoxicity and cell proliferation assays suggested that the SnO2 nanowire coatings were nontoxic and promoted the proliferation of C2C12 and L929 cells (> 90% viability). Cellular activities, cell adhesion, and lactate dehydrogenase activities were consistent with the superior biocompatibility of the nanowire materials. Notably, the nanowire coating showed potent antibacterial activity against six different bacterial strains. The antibacterial activity of the SnO2 material was attributed to the photocatalytic nature of SnO2. The antibacterial activity and biocompatibility of the newly developed SnO2 nanowire coatings may enable their use as coating materials for biomedical implants.

DNA intercalative 4-butylaminopyrimido4',5':4,5]thieno(2,3-b)quinoline induces cell cycle arrest and apoptosis in leukemia cells

DNA intercalators are one of the interesting groups in cancer chemotherapy. The development of novel anticancer small molecule has gained remarkable interest over the last decade. In this study, we synthesized and investigated the ability of a tetracyclic-condensed quinoline compound, 4-butylaminopyrimido4',5':4,5]thieno(2,3-b)quinoline (BPTQ), to interact with double-stranded DNA and inhibit cancer cell proliferation. Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of BPTQ with DNA. Cytotoxicity was studied by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Further, cell cycle analysis by flow cytometry, annexin V staining, mitochondrial membrane potential assay, DNA fragmentation, and western blot analysis were used to elucidate the mechanism of action of BPTQ at the cellular level. Spectral, topological, and docking studies confirmed that BPTQ is a typical intercalator of DNA. BPTQ induces dose-dependent inhibitory effect on the proliferation of cancer cells by arresting cells at S and G2/M phase. Further, BPTQ activates the mitochondria-mediated apoptosis pathway, as explicated by a decrease in mitochondrial membrane potential, increase in the Bax:Bcl-2 ratio, and activation of caspases. These results confirmed that BPTQ is a DNA intercalative anticancer molecule, which could aid in the development of future cancer therapeutic agents.