Papers by Konstantinos Grintzalis
Analytical biochemistry, Jan 30, 2015
In this protocol we present a rapid and sensitive assay for the accurate determination of protein... more In this protocol we present a rapid and sensitive assay for the accurate determination of protein concentration. The assay is a modification of a previous method, and measures minimum 0.2 μg protein.
Protocol Exchange, 2009
Several methods for protein determination have been developed [1] but the ones most commonly used... more Several methods for protein determination have been developed [1] but the ones most commonly used today are based on the reaction of proteins with Commassie Brilliant Blue G-250 (CBB) [2] and alkaline Cu(II) . The most recent modifications of these methods are the Sedmak assay [4] and the bicinchoninic acid (BCA) assay . Another sensitive method for measuring proteins is the colloidal gold protein assay [6] which has been modified to quantify specific proteins of interest . The present protocol is a very simple CBB-based protein assay which is more sensitive and less prone to interference than the commonly used Bradford, Sedmak and BCA assays . It uses the hydrophobic reagents ammonium sulfate (AS) and trichloroacetic acid (TCA) to increase the binding of more CBB molecules per protein molecule (for mechanism see ). The protocol consists of three independent sub-protocols: general assay, microplate assay and microassay, with minimum detectable protein 100, 50 and 150 ng, respectively, which make it 100/40 and 200/5 fold more sensitive than the Sigma/Pierce Bradford and BCA assays, respectively .
Laboratory Protocols in Fungal Biology, 2012
The presented protocols describe methodologies for the accurate quanti fi cation of dsDNA concent... more The presented protocols describe methodologies for the accurate quanti fi cation of dsDNA concentration and fragmentation status of fungal DNA (and that of any organism). The protocols can be used to quantify dsDNA concentration, even up to picogram level, using the PicoGreen fl uorescent dye, and to discriminate the fragmentation status of a DNA sample as fragmented (0-23 kb) and intact (> 23 kb) or as small size (0-1 kb) fragmented DNA, using DNA samples as low as 2.5 m g mL -1 . Keywords dsDNA quanti fi cation • DNA fragmentation • Small size (0-1 kb) fragmented DNA • PEG precipitation • Fluorescence • PicoGreen • Fungi
Gastroenterology Research, 2008
Background: Several studies have investigated the potential role of oxidative stress in the evolu... more Background: Several studies have investigated the potential role of oxidative stress in the evolution of colorectal cancer. In most of these studies, oxidative stress was assessed indirectly by measurements of indices like lipid peroxidation, protein oxidation or antioxidant status. The present study was undertaken to directly assess systemic oxidative stress by measuring plasma superoxide radical (O 2 -•) in patients with non-metastatic colorectal cancer.
The presented protocol for superoxide radical in vivo quanti fi cation in fungal tissues is based... more The presented protocol for superoxide radical in vivo quanti fi cation in fungal tissues is based on the quanti fi cation of 2-hydroxyethidium (2-OH-E + ) after its isolation and fl uorometric quantitation. The protocol is ultrasensitive (i.e., <1 pmol) and applicable to any kind of fungal tissue; it can also be used for in vitro studies.
Journal of Cardiothoracic Surgery, 2009
Background: Paraplegia is the most devastating complication of thoracic or thoraco-abdominal aort... more Background: Paraplegia is the most devastating complication of thoracic or thoraco-abdominal aortic surgery. During these operations, an ischemia-reperfusion process is inevitable and the produced radical oxygen species cause severe oxidative stress for the spinal cord. In this study we examined the influence of Amifostine, a triphosphate free oxygen scavenger, on oxidative stress of spinal cord ischemiareperfusion in rabbits.
Aquatic Toxicology, 2011
Certain metals, like Hg, Cu and Cd, are capable of down-regulating protein synthesis in several m... more Certain metals, like Hg, Cu and Cd, are capable of down-regulating protein synthesis in several marine organisms, including Mytilus galloprovincialis. Nevertheless, due to the complexity of the environmental stress, it is difficult to evaluate the influence of individual metals on protein synthesis via field studies. To bypass this difficulty, experimental studies were carried out on M. galloprovincialis exposed in aquarium
Nature Communications, 2015
The combination of intense solar radiation and soil desiccation creates a short circuit in the bi... more The combination of intense solar radiation and soil desiccation creates a short circuit in the biogeochemical carbon cycle, where soils release significant amounts of CO 2 and reactive nitrogen oxides by abiotic oxidation. Here we show that desert soils accumulate metal superoxides and peroxides at higher levels than non-desert soils. We also show the photogeneration of equimolar superoxide and hydroxyl radical in desiccated and aqueous soils, respectively, by a photo-induced electron transfer mechanism supported by their mineralogical composition. Reactivity of desert soils is further supported by the generation of hydroxyl radical via aqueous extracts in the dark. Our findings extend to desert soils the photogeneration of reactive oxygen species by certain mineral oxides and also explain previous studies on desert soil organic oxidant chemistry and microbiology. Similar processes driven by ultraviolet radiation may be operating in the surface soils on Mars.
Redox Report, 2008
Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology... more Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology of organ injury not only in the liver, but also in several extrahepatic tissues. The present study was designed to assess directly oxidative stress in vital organs of experimentally jaundiced rats by measuring the key oxidative stress marker superoxide radical (O 2
We show here that oxidative stress is involved in both sclerotial differentiation (SD) and aflato... more We show here that oxidative stress is involved in both sclerotial differentiation (SD) and aflatoxin B1 biosynthesis in Aspergillus flavus. Specifically, we observed that (i) oxidative stress regulates SD, as implied by its inhibition by antioxidant modulators of reactive oxygen species and thiol redox state, and that (ii) aflatoxin B1 biosynthesis and SD are comodulated by oxidative stress. However, aflatoxin B1 biosynthesis is inhibited by lower stress levels compared to SD, as shown by comparison to undifferentiated A. flavus. These same oxidative stress levels also characterize a mutant A. flavus strain, lacking the global regulatory gene veA. This mutant is unable to produce sclerotia and aflatoxin B1. (iii) Further, we show that hydrogen peroxide is the main modulator of A. flavus SD, as shown by its inhibition by both an irreversible inhibitor of catalase activity and a mimetic of superoxide dismutase activity. On the other hand, aflatoxin B1 biosynthesis is controlled by a wider array of oxidative stress factors, such as lipid hydroperoxide, superoxide, and hydroxyl and thiyl radicals.
Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. Howeve... more Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)-and cysteine (CSH)-based nonprotein (NP) thiols/ mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NP x SH NPSSNP , NP x SH NPSH ), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NP x SH PSSNP ). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4 0 -dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Exceladapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NP x SH NPSSNP , and NP x SH NPSH is 1 nmol -SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.
Redox Report, 2008
Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology... more Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology of organ injury not only in the liver, but also in several extrahepatic tissues. The present study was designed to assess directly oxidative stress in vital organs of experimentally jaundiced rats by measuring the key oxidative stress marker superoxide radical (O 2
Neurochemical Research, 2008
The study aimed to directly measure in vivo superoxide radical (O 2 -) a direct indicator of oxid... more The study aimed to directly measure in vivo superoxide radical (O 2 -) a direct indicator of oxidative stress, in the brain of rats with experimentally induced obstructive jaundice by employing a new quantitative ultrasensitive fluorescent assay requiring minimum sample. O 2 anion is specific for dihydroethidine (DHE) and upon reaction gives a characteristic product, namely 2-OHethidium. Ten male rats underwent laparotomy and were divided into two groups: I, sham operated and II bile duct ligation. Ten days later, following injection with DHE (a O 2 trap), all animals were killed and samples from cerebral cortex, midbrain and cerebellum were removed for analysis. It was shown that compared to group I, in group II the O 2 was increased by 67% in the cerebral cortex and by 37% in the midbrain as a consequence of experimental obstructive jaundice, while its levels were unaffected in the cerebellum. The data in this experimental obstructive jaundice model imply a region-specific increase of O 2 -formation rate, being higher in cerebral cortex, less so in the midbrain and not at all in cerebellum.
Nature Protocols, 2009
An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked ... more An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0-1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 lg ml À1 . The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively. ACKNOWLEDGMENTS This work was financially supported by the Greek Ministry of Education, University of Patras, Greece.
Marine Environmental Research, 2012
This laboratory study describes phenanthrene (Ph) and/or anthracene (An) ability to alter the tot... more This laboratory study describes phenanthrene (Ph) and/or anthracene (An) ability to alter the total thiol redox status (TRS), via depletion of protein free thiols (PSH) and glutathione (GSH) levels, in gills of mussel Mytilus galloprovincialis, after a short-term (7 days) exposure period to each contaminant (at a final concentration of 0.1 mg L(-1)) or in a mixture (ration 1:1, at a final concentration of 0.2 mg L(-1)). A number of observable changes, like lysosomal membrane impairment (as detected via the neutral red retention time assay, primarily performed in haemocytes), enhancement of lipid peroxidation byproducts, increased nuclear abnormalities, inhibition of AChE and ALP activity, as well as a significant depletion of PSH and GSH were detected in gills of exposed mussels, in any case. Significant relationships occurred among TRS parameters with each change/stress indices measured in tissues of mussels, could reinforce the use of PSH as a potent biomarker.
Journal of Applied Toxicology, 2013
We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage ... more We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified.
Free Radical Research, 2009
Free Radical Biology and Medicine, 2013
A simple and sensitive method is presented for the simultaneous quantification (spectrophotometri... more A simple and sensitive method is presented for the simultaneous quantification (spectrophotometric and spectrofluorimetric) of the main lipid and protein peroxidation products after their initial fractionation: free malondialdehyde (FrMDA), protein-bound malondialdehyde (PrMDA), total hydroperoxides (LOOH), and protein hydroperoxides (PrOOH). FrMDA and PrMDA (released from proteins by alkaline hydrolysis) are measured after the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions, by the specific fluorimetric quantification of the resulting MDA-(TBA) 2 adduct chromophore. The measurement of LOOH and PrOOH is based on the reaction of Fe 3 þ (resulting from the reaction of LOOH and PrOOH with Fe 2 þ ) with xylenol orange (XO) and the photometric quantification of the resulting XO-Fe complex. The sensitivity of the assays for FrMDA/PrMDA and LOOH/PrOOH is 20 and 100 pmol, respectively. The method was applied successfully on human plasma and can be used for the evaluation of oxidative stress in both basic and clinical research.
Brain Research, 2010
The development of increased oxidative stress in the context of obstructive cholestasis has been ... more The development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study aimed to detect alterations of tight junction-associated occludin in rat brain capillaries after bile duct ligation (BDL). In experiment 1, occludin expression was evaluated by Western blot analysis in 5 animals 10 days after BDL and compared with 5 sham-operated ones. In experiment 2, groups of 9 animals each were used to assess occludin levels on the 1st, 5th, and 10th days after BDL and to associate these measurements with the in vivo superoxide radical production measured by means of an ultrasensitive fluorescent assay. The results indicated that occludin expression in BDL animals, as opposed to sham-operated, was significantly reduced at every time point studied, being lowest in the rats remaining on BDL condition for 10 days. Moreover, it was demonstrated that the time-dependent downregulation of occludin expression in the brain endothelial was significantly correlated with the timedependent increase of brain superoxide radical level, implying a relationship between these two abnormalities. In conclusion, the evidence presented herein suggests the implication of occludin and, therefore, of blood-brain barrier in the pathophysiology of extrahepatic cholestasis.
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Papers by Konstantinos Grintzalis