Circadian clock genes cause activation of the human PAI-1 gene promoter with 4G/5G allelic preference

Increased plasminogen activator inhibitor-1 (PAI-1) activity is associated with greater risk of myocardial infarction. PAI-1 expression is regulated by a 4G/5G promoter polymorphism. The 4G allele is associated with higher PAI-levels and greater circadian variation. Here we show that clock protein heterodimers BMAL/CLOCK cause greater activation (approximately 2-fold, P<0.05) of the 4G allele. Site-directed mutagenesis studies suggest that clock genes act on two canonical E-boxes to regulate PAI-1 promoter activity. These results identify a potential novel mechanism whereby allele-specific clock genes - mediated modulation of PAI-1 expression may contribute to circadian variation in cardiac risk.     

Decolorisation, biodegradation and detoxification of benzidine based azo dye

The present study deals with the decolorisation, biodegradation and detoxification of Direct Black-38, a benzidine based azo dye, by a mixed microbial culture isolated from an aerobic bioreactor treating textile wastewater. The studies revealed a biotransformation of Direct Black-38 into benzidine and 4-aminobiphenyl followed by complete decolorisation and biodegradation of these toxic intermediates. From cytotoxicity studies, it was concluded that detoxification of the dye took place after degradation of the toxic intermediates by the culture.     

One RNA plays three roles to provide catalytic activity to a group I intron lacking an endogenous internal guide sequence

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5′ end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.     

Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones

The physiological function of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is to detoxify potentially reactive quinones by direct transfer of two electrons. A similar detoxification role has not been established for its homologue NRH:quinone oxidoreductase 2 (NQO2). Estrogen quinones, including estradiol(E(2))-3,4-Q, generated by estrogen metabolism, are thought to be responsible for estrogen-initiated carcinogenesis. In this investigation, we have shown for the first time that NQO2 catalyzes the reduction of electrophilic estrogen quinones and thereby may act as a detoxification enzyme. ESI and MALDI mass spectrometric binding studies involving E(2)-3,4-Q with NQO2 clearly support the formation of an enzyme-substrate physical complex. The problem of spontaneous reduction of substrate by cofactor, benzyldihydronicotinamide riboside (BNAH), was successfully overcome by taking advantage of the ping-pong mechanism of NQO2 catalysis. The involvement of the enzyme in the reduction of E(2)-3,4-Q was further supported by addition of the inhibitor quercetin to the assay mixture. NQO2 is a newly discovered binding site (MT3) of melatonin. However, addition of melatonin to the assay mixture did not affect the catalytic activity of NQO2. Preliminary kinetic studies show that NQO2 is faster in reducing estrogen quinones than its homologue NQO1. Both UV and liquid chromatography-tandem mass spectrometry assays unequivocally corroborate the reduction of estrogen ortho-quinones by NQO2, indicating that it could be a novel target for prevention of breast cancer initiation.     

Novel derivatives of ISO-1 as potent inhibitors of MIF biological function

A series of novel 1,2,4-oxadiazole, phthalimide, amide and other derivatives of ISO-1 were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds inhibited MIF enzymatic activity at levels better than ISO-1. Of note, compounds 7, 22, 23, 24, 25 and 27 inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells and, more importantly, inhibited the MIF-induced production of interleukin-6 (IL-6) and/or interleukin-1β (IL-1β) significantly better than ISO-1.     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

Application of LIBS in Detection of Antihyperglycemic Trace Elements in Momordica charantia

The present study exploits the information based on concentration of trace elements and minerals in understanding the role/mechanism of action of freeze-dried fruit powder suspended in distilled water of Momordica charantia (family: Cucurbitaceae) in diabetes treatment. Laser-induced break down spectroscopy (LIBS) spectra of plant product was recorded under optimized experimental conditions and analyzed. Several atomic lines such as Na, K, Mg, Ca, Fe, Al, etc. have been observed in the LIBS spectra of the above plant product. The concentrations of these minerals are determined by using calibration-free LIBS method. Correlation between the concentration of these elements/minerals and their defined role in diabetes management was studied in normal as well as diabetic animal models.     

Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-<Emphasis Type="Italic">Cereus pterogonus</Emphasis> sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively,. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 μmol min⁻¹ mg⁻¹. In the presence of metal ions (1 mM) such as Co²⁺,Mn²⁺, Ni²⁺, Ca²⁺ and Fe³⁺ the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg²⁺, Cd²⁺, Cu²⁺, while partial inhibition was noted with Zn²⁺ and Mg²⁺. The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.     

Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups

Nucleoside triphosphates having a 3'-ONH? blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH? group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH? blocking group in "next generation sequencing."