Phorate triggers oxidative stress and mitochondrial dysfunction to enhance micronuclei generation and DNA damage in human lymphocytes

Herein, we studied phorate for its toxicological effects in human lymphocytes. Phorate treatment for 3 h has induced significant increase in the lymphocytic DNA damage. Compared to control, comet data from highest concentration of phorate (1000 µM) showed 8.03-fold increase in the Olive tail moment (OTM). Cytokinesis blocked micronucleus (CBMN) assay revealed 6.4-fold increase in binucleated micronucleated (BNMN) cells following the exposure with phorate (200 µM) for 24 h. The nuclear division index (NDI) in phorate (200 µM) treated cells reduced to 1.8 vis-à-vis control cells showed NDI of 1.94. Comparative to untreated control, 60.43% greater DCF fluorescence was quantitated in lymphocytes treated with phorate (500 µM), affirming reactive oxygen species (ROS) generation and oxidative stress. Flow cytometric data of phorate (200 µM) treated lymphocytes showed 81.77% decline in the fluorescence of rhodamine 123 (Rh123) dye, confirming the perturbation of mitochondrial membrane potential (ΔΨm). Calf thymus DNA (ct-DNA) treated with phorate (1000 µM) exhibited 2.3-fold higher 8-Hydroxy-2′-deoxyguanosine (8-oxodG) DNA adduct formation, signified the oxidative DNA damage. The alkaline unwinding assay revealed 4.0 and 6.5 ct-DNA strand breaks when treated to phorate and phorate-Cu (II) complex. Overall, the data unequivocally suggests the cyto- and genotoxic potential of phorate in human lymphocytes, which may induce comparable toxicological consequences in persons occupationally or non-occupationally exposed to insecticide phorate.     

Cortical State Fluctuations across Layers of V1 during Visual Spatial Perception

1. Download high-res image (162KB)
2. Download full-size image

     

Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding

By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.     

2-Amino-5-substituted-1,3,4-oxadiazole as chemosensor for Ni(II) ion detection: antifungal,antioxidant, DNA binding,and molecular docking studies

An oxadiazole derivative 2 was prepared by condensation reaction through cyclization of semicarbazone in the presence of bromine; the structural confirmation was supported by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, Fourier transform-infrared spectroscopy, and liquid chromatography-mass spectrometry. Its sensing ability towards Ni²⁺ ion was examined showing a binding constant of 1.04 × 10⁵ compared with other suitable metal cations (Ca²⁺, Co²⁺, Cr³⁺, Ag⁺, Pb²⁺, Fe³⁺, Mg²⁺, and K⁺) using ultraviolet–visible (UV–vis) and fluorescence spectroscopic studies. The minimum concentration of Ni²⁺ ions and limit of detection was found to be 9.4 μM. A job's plot gave the binding stoichiometry ratio of oxadiazole derivative 2 vs Ni²⁺ ions as 2:1. Furthermore, the intercalative binding mode of oxadiazole derivative 2 with calf thymus DNA was supported by ultraviolet–visible (UV–vis) and fluorescent light, viscosity, cyclic voltammetry, time-resolved fluorescence, and circular dichroism measurements. The molecular docking result gave the binding score for oxadiazole derivative 2 as −6.5 kcal/mol, which further confirmed the intercalative interaction. In addition, the antifungal activity of oxadiazole derivative 2 was also screened against several fungal strains (C. albicans, C. glabrata, and C. tropicalis) by broth dilution and disc diffusion methods. In antioxidant studies, the oxadiazole derivative 2 showed potential scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and H₂O₂ free radicals.     

Effect of Environmental Factors on Cyanobacteria Richness in Some Agricultural Soils

The physicochemical variations of soil, such as temperature, pH, nutrients, and the type of plant cultivation, affect the diversity of cyanobacteria, whether heterocystous or not. The aim of this study was to identify the species of cyanobacteria in a soil and the effect of environmental characteristics on cyanobacteria. Soil samples collected from six different agricultural sites in Al Diwaniyah city/Iraq during September 2016 in the autumn season were analyzed, and the physicochemical characteristics of the samples were measured using approved methods.

The results showed significant correlation and differences between cyanobacteria composition, distribution, and physicochemical factors among soil sites. The Agricultural soil was slightly alkaline and moderately saline and contained abundant nutrients, cations and a high percentage of organic matter. All these characteristics influenced the distribution and diversity of cyanobacteria. Ninety-six species were identified, including four heterocystous species represented by Anabaena, Calothrix, Cylnidrospermum, and Nostoc. However, the non-heterocystous were represented by 13 species: Aphanocapsa, Aphanothece, Arthrospira, Chroococcus, Gloeocapsa, Lyngbya, Merismopedia, Microcystis, Microcoleus, Oscillatoria, Phormidium, Schizothrix, and Spirulina. The dominant species of cyanobacteria was Oscillatoria, followed by Phormidium, Chroococcus, Gleocapsa and Lyngbya. The highest value of Shannon’s and Simpson’s diversities were registered in the Ghammas site, which is a paddy field, but the lowest was registered in the Afak site, cultivated with the alfalfa plant. Soil was classified as finely textured with silty clayey characterization, favorable for cyanobacteria growth.     

Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase

A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.     

Die Verwertung phenolischer Verbindungen durch Weißfäulepilze

Zusammenfassung Phenolcarbonsäuren, weniger Phenolaldehyde, wie sie als Spaltstücke des Lignins auftreten können, werden durch Weißfäulepilze entweder zusammen mit Glucose oder als alleinige Kohlenstoff-und Energiequelle verwertet. Eine zentrale Stellung beim Metabolismus dieser Verbindungen nimmt die Protocatechusäure ein, da die verschiedenen Verbindungen wahrscheinlich in diese überführt werden. Bei der Einwirkung von Polystictus versicolor auf Protocatechusäure entsteht als intermediäres Abbauprodukt. -Ketoadipinsäure. Es lassen sich aus den bebrüteten Lösungen dieses Pilzes Enzymsysteme isolieren, die nicht mit der Laccase identisch sind und die Spaltung von Protocatechusäure unter Aufnahme von Sauerstoff und Bildung von -Ketoadipinsäure katalysieren. Der Weg der Spaltung ist ähnlich den bisher für andere Mikroorganismen formulierten Abbauschritten der Protocatechusäure.     

Misfolded proteins and human diseases

Though protein folding is a regular phenomenon inside the cellular milieu, slight differences in the folding pattern of proteins may lead to disease-causing forms. Many diseases have been identified that are caused by these misfolded macromolecules and a considerable amount of focus is still directed towards better understanding of the processes that lead to these misfolded structures. Further progress in the field of how soluble proteins begin to misfold and how resultant oligomers begin dysfunction offers exciting prospects for specific molecular intervention.     

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.