Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach

Cyclin‐dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508–8522) have found a ligand BS194 that has a high affinity with CDK2 (IC₅₀ = 3 nm ) but shows low affinity with CDK1 (IC₅₀ = 30 nm ). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free‐energy calculation to analyze the interactions. A rational three‐dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non‐polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1. Copyright © 2012 John Wiley & Sons, Ltd.     

Acute and chronic saturated fatty acid treatment as a key instigator of the TLR-mediated inflammatory response in human adipose tissue, in vitro

A post-prandial increase in saturated fatty acids (SFAs) and glucose (Glc) activates an inflammatory response, which may be prolonged following restoration of physiological SFAs and Glc levels — a finding referred to as ‘metabolic memory'.This study examined chronic and oscillating SFAs and Glc on the inflammatory signalling pathway in human adipose tissue (AT) and adipocytes (Ads) and determined whether Ads are subject to “metabolic memory.”Abdominal (Abd) subcutaneous (Sc) explants and Ads were treated with chronic low glucose (L-Glc): 5.6 mM and high glucose (H-Glc): 17.5 mM, with low (0.2 mM) and high (2 mM) SFA for 48 h. Abd Sc explants and Ads were also exposed to the aforementioned treatment regimen for 12-h periods, with alternating rest periods of 12 h in L-Glc.Chronic treatment with L-Glc and high SFAs, H-Glc and high SFAs up-regulated key factors of the nuclear factor-κB (NFκB) pathway in Abd Sc AT and Ads (TLR4, NFκB; P<.05), whilst down-regulating MyD88. Oscillating Glc and SFA concentrations increased TLR4, NFκB, IKKβ (P<.05) in explants and Ads and up-regulated MyD88 expression (P<.05). Both tumor necrosis factor α and interleukin 6 (P<.05) secretion were markedly increased in chronically treated Abd Sc explants and Ads whilst, with oscillating treatments, a sustained inflammatory effect was noted in absence of treatment.Therefore, SFAs may act as key instigators of the inflammatory response in human AT via NFκB activation, which suggests that short-term exposure of cells to uncontrolled levels of SFAs and Glc leads to a longer-term inflammatory insult within the Ad, which may have important implications for patients with obesity and Type 2 diabetes.     

Long period grating based biosensor for the detection of Escherichia coli bacteria

In this paper we report a stable, label-free, bacteriophage-based detection of Escherichia coli (E. coli) using ultra sensitive long-period fiber gratings (LPFGs). Bacteriophage T4 was covalently immobilized on optical fiber surface and the E. coli binding was investigated using the highly accurate spectral interrogation mechanism. In contrast to the widely used surface plasmon resonance (SPR) based sensors, no moving part or metal deposition is required in our sensor, making the present sensor extremely accurate, very compact and cost effective. We demonstrated that our detection mechanism is capable of reliable detection of E. coli concentrations as low as 10(3)cfu/ml with an experimental accuracy greater than 99%.     

Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed,paraffin embedded tissues of goats by IS900 polymerase chain reaction

The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system.     

Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease

Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.     

Study on organochlorine pesticide levels in chronic kidney disease patients: association with estimated glomerular filtration rate and oxidative stress

Nephrotoxicity of organochlorine pesticides (OCPs) has been established in experimental animal models. This study was designed to evaluate the relationship of the blood OCPs level with the estimated glomerular filtration rate (eGFR) and oxidative stress (OS) in chronic kidney disease (CKD) patients. Patients in different stages of CKD (n = 150) and age, sex matched healthy controls (n = 96) were recruited. The blood OCPs level were analyzed by gas chromatography, and plasma levels of several OS parameters such as malondialdehyde (MDA), protein carbonyl, advanced oxidation protein products (AOPP), and total thiols were quantified by standard spectrophotometric methods. We observed significantly higher levels of hexachlorocyclohexane (α, γ), endosulfan, aldrin, p,p'-dichlorodiphenyldichloroethylene (DDE), and total pesticides in CKD patients. Negative correlation was also observed for aldrin, p,p'-DDE and total pesticides (p < 0.05) with eGFR. Plasma levels of MDA and AOPP showed significant positive association with the total pesticides level, indicating augmentation of OS with increased accumulation of OCPs in CKD patients.     

Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo,partially and fully metalated forms

Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cd_i-CjMT 2 (i = 1–7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd²⁺ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd²⁺ ions and a C-terminal α-domain that binds with 2 Cd²⁺ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.     

Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp). Confocal laser scanning microscopy (CLSM) of hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when co-inoculated with an equal number (10(5) c.f.u. ml(-1)) of wild type Pantoea sp., but the converse was not true. Pantoea sp. was a more aggressive endophytic colonizer of its host than Ochrobactrum sp. The potential of using GFP reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is herewith demonstrated.     

Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis

Cultures of Haematococcus pluvialis were exposed to mutagens like u.v. and EMS (ethyl methanesulphonate). The results showed that the survival rate decreased with the increase in u.v. exposure time and increase in EMS concentration. These mutants were further screened using inhibitors of the carotenoid biosynthetic pathway viz. diphenylamine (15–90 M), nicotine (160–320 M) and compactin (1.5–3.0 M). The mutants thus obtained showed early enhanced (2.2–3.2-fold) astaxanthin accumulation and also exhibited higher lycopene cyclase activity.