Biochemical and toxicity evaluation of Retama sphaerocarpa extracts and in-silico investigation of phenolic compounds as potential inhibitors against HPV16 E6 oncoprotein.

Cervical cancer is a type of cancer which affects the cervix cells. The conventional treatments for cervical cancer including surgery, chemotherapy, and radiotherapy are only effective in premature stages and less effective in late stages of this tumor. Therefore, the therapeutic strategies based on biologically active substances from plants are needed to develop for the treatment of cervical cancer. The aim of the present study was to assess in vivo toxicity, hematological and biochemical blood parameters in Wistar rats fed Retama sphaerocarpa aqueous leaf extract (RS-AE), as well as to perform in silico molecular docking studies and dynamic simulation of phenolic compounds against HPV16 oncoprotein E6 in order to identify potential inhibitors. RS-AE was found not to induce acute or sub-acute oral toxicity or significant alterations in hematological and biochemical blood parameters in Wistar rats. A total of 11 phenolic compounds were identified in RS-AE, including dihydrodaidzein glucuronide, chrysoperiol pentoside, genistin and vitexin, which turned out to have the highest binding affinity to HPV16 oncoprotein E6. Based on these results, these RS-AE phenolic compounds could be used as natural drugs against the HPV16 E6 oncoprotein.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues.

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water.

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.

Increasing the Detection Limit of the Parkinson Disorder through a Specific Surface Chemistry Applied onto Inner Surface of the Titration Well.

The main objective of this paper was to illustrate the enhancement of the sensitivity of ELISA titration for neurodegenerative proteins by reducing nonspecific adsorptions that could lead to false positives. This goal was obtained thanks to the association of plasma and wet chemistries applied to the inner surface of the titration well. The polypropylene surface was plasma-activated and then, dip-coated with different amphiphilic molecules. These molecules have more or less long hydrocarbon chains and may be charged. The modified surfaces were characterized in terms of hydrophilic-phobic character, surface chemical groups and topography. Finally, the coated wells were tested during the ELISA titration of the specific antibody capture of the α-synuclein protein. The highest sensitivity is obtained with polar (Θ = 35°), negatively charged and smooth inner surface.

Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress.

BACKGROUND: E. coli cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching approximately 15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress. RESULTS: In minimal M9 medium, E. coli cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 E. coli strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37 degrees C, even in the presence of metabolizable substrates (glucose or lactate) and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate). Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used. CONCLUSIONS: In E. coli, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. Both conditions prevent bacterial growth. There is no obvious link with the stringent response or catabolite repression.

Thiamine triphosphate synthesis in rat brain occurs in mitochondria and is coupled to the respiratory chain.

In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.

Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle.

In the present study we developed an enzymatic approach (through the use of collagenase and dispase) to isolate bovine intestinal epithelial cells. Using this method, freshly isolated jejunocytes could be distinguished from simultaneously isolated colonocytes, as the jejunocytes specifically exhibited the small intestinal peptidase gene transcript, as well as an active alkaline phosphatase. The transformation of both types of cell suspension was performed by retroviral infection, using reproduction-defective viruses bearing the gene coding for the large T antigen of the leukaemia simian virus (SV40). The success of the transfection was demonstrated by (1) a significant increase in cell passage numbers (52-53 vs. 7 passages for non-transfected cells), (2) the detection of both the large T transcript and the large T antigen in transformed cells. Possible contamination and progressive substitution of bovine primocultures by non-bovine lineages available in the laboratory was excluded, as the transformed cells presented a bovine typical karyotype. Most transfected cells kept an epithelial morphology after transformation. They also maintained the expression of FABP and enterocyte specific enzymes (brush-border associated maltase and IAP). However, levels of specific activity of these enzymes were low, suggesting that cell differentiation is not completely achieved under the applied culture conditions.

Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties.

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.