Elimination of Endogenous Toxin, Creatinine from Blood Plasma Depends on Albumin Conformation: Site Specific Uremic Toxicity & Impaired Drug Binding

Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state

Anti- Japanese-Encephalitis-Viral Effects of Kaempferol and Daidzin and Their RNA-Binding Characteristics

Background: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an a-1 translational frameshift to synthesize the NS1 ’ protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described. Methodology/Principal Findings: The 50 % cytotoxic concentration (CC50) and 50 % effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC 50 values of Kae and Dai were 12.6 and 25.9 mM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 mM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes wit

Interaction of antioxidant flavonoids with tRNA: Intercalation or external binding and comparison with flavonoid-DNA adducts

Antioxidants are essential to good health. Flavonoids are powerful antioxidants, and prevent DNA damage. The antioxidative protections are related to their binding modes to a DNA duplex and complexation with free radicals in vivo. Recently we reported the interaction of flavonoids with DNA in vitro (Kanakis et al., J. Biomol. Struct. Dyn. 22, 719-724, 2005), where polyphenol different binding modes were discussed. The aim of this study was to examine the interaction of transfer RNA with quercetin (que), kaempferol (kae), and delphinidin (del) in aqueous solution at physiological conditions and to make a comparison with the corresponding pigment-DNA adducts. Constant tRNA concentration (6.25 mM) and various drug/RNA( phosphate) molar ratios of 1/48 to 1/8 were used. FTIR and UV-visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants, and the effects of drug complexation on the stability and conformation of tRNA duplex. Both intercalative and external binding modes were observed. Structural analysis showed que, kae, and a del intercalate tRNA duplex with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants K-que = 4.80 x 10(4) M-1, K-kae = 4.65 x 10(4) M-1, and K-del = 9.47 x 10(4) M-1. The stability of adduct formation is in the order of del > que > kae. A comparison with flavonoids-DNA adducts showed both intercalation and external bindings with the stability order K-que = 7.25 x 10(4) M-1, K-kae = 3.60 x 10(4) M-1, and K-del = 1.66 x 10(4) M-1. Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial B- to A-DNA transition occurs at high drug concentration, while tRNA remains in the A-family structure

Characterization of bioactive and nutraceutical compounds occurring in olive oil processing wastes

Rationale Several bioactive compounds, including phenolic acids and secoiridoids, are transferred from olive drupes to olive oil during the first stage of production. Here, the characterization of these low molecular weight (LMW) compounds in olive oil and in closely related processing materials, like olive leaves (OL) and olive mill wastewaters (OMW), was faced up, for the first time, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS). Methods A novel binary matrix composed of 1,8-bis(tetramethylguanidino)naphthalene (TMGN) and 9-aminoacridine (9AA) (1:1 molar ratio), displaying excellent ionization properties at low levels of laser energy, was employed in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm). MS/MS experiments were performed by using ambient air as the collision gas. Results Four major secoiridoids typically present in olive oil, i.e., the aglycones of oleuropein and ligstroside, and oleacein and olecanthal at m/z 377.1, 361.1, 319.1 and 303.1, respectively, were detected in virgin olive oil (VOO) extracts, along with some of their chemical/enzymatic hydrolysis by-products, such as elenolic (m/z 241.1), decarboxymethyl-elenolic acids (m/z 183.1) and hydroxytyrosol (m/z 153.1). Besides oleuropein aglycone and oleacein, hydroxylated derivatives of decarboxymethyl-elenolic acid and hydroxytyrosol were evidenced in OMW. Conclusions While oleuropein was confirmed in OL extracts, several interesting phenolic compounds, including hydroxytyrosol, were recognized in OMW. The proposed approach based on the use of a novel binary matrix for MALDI MS/MS analyses of LMW bioactive compounds can be considered a promising analytical tool for a rapid screening of the phenolic fraction in olive oils and related processing wastes

Geographical differentiation of saffron by GC-MS/FID and chemometrics

The volatile compounds of saffron of different origins were investigated to check their suitability as markers of geographic differentiation. A total of 247 saffron samples from Greece (40 samples), Iran (84 samples), Italy (60 samples) and Spain (63 samples) which were harvested in 2006 were analysed using ultrasound-assisted extraction, gas chromatography followed by mass spectrometry and flame ionisation. All regions were easily differentiated by canonical discriminant analysis. The percentages of correct classification and validation were 96.4 and 94.3%, respectively. These investigations showed the potential of saffron volatiles to discriminate saffron samples with different geographical origins

Crocins, the active constituents of Crocus Sativus L., counteracted ketamine-induced behavioural deficits in rats

Experimental evidence indicates that the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine impairs cognition and can mimic certain aspects of positive and negative symptoms of schizophrenia in rodents. Crocins are among the active components of the plant Crocus sativus L. and were found to be effective in different models of psychiatric disorders including anxiety and depression. The present study was designed to investigate the ability of crocins to counteract schizophrenia-like behavioural deficits produced by ketamine in rats. Crocin's ability to counteract hypermotility, stereotypies and ataxia induced by ketamine was evaluated in a motor activity cage. The ability of crocins to reverse ketamine-induced memory deficits was assessed using the novel object recognition task (NORT). The social interaction test was used in order to examine the effects of crocins on ketamine-induced social withdrawal. Crocins (50 but not 30 mg/kg, i.p.) attenuated ketamine (25 mg/kg, i.p.)-induced hypermotility, stereotypies and ataxia. In a subsequent study, post-training administration of crocins (15 and 30 mg/kg, i.p.) reversed ketamine (3 mg/kg, i.p.)-induced performance deficits in the NORT. Finally, crocins (50 but not 30 mg/kg, i.p.) counteracted the ketamine (8 mg/kg, i.p.)-induced social isolation in the social interaction test. Our findings show that crocins attenuated schizophrenia-like behavioural deficits induced by the non-competitive NMDA receptor antagonist ketamine in rats