Design, Synthesis, Characterization, and Cytotoxicity of New Pyrazolylmethylene-2-thioxoimidazolidin-4-one Derivatives towards Androgen-Sensitive LNCaP Prostate Cancer Cells.

A new class of pyrazolylmethylene-2-thioxoimidazolidin-4-one derivatives 3a-p were rationally designed and synthesized with the aim of exploring their potential as treatments for prostate cancer. The synthesized compounds 3a-p were biologically analyzed for their anticancer effects against AR+LNCaP, AR-PC-3, and Wi38 cell lines. The observed IC50 values against AR+LNCaP ranged between 10.27 ± 0.14 and 109.72 ± 2.06 µM after 24 h of incubation. Compounds 3i-k, 3m, and 3o-p recorded IC50 values of 05.22 ± 0.12 to 11.75 ± 0.07 µM after 48 h incubation in the presence of 1 nM DHT, with higher selectivity towards AR+LNCaP. Moreover, compounds 3i and 3k significantly induced Caspase 3 accumulation, reduced DNA content at the various stages of the cell cycle, and ultimately caused AR+LNCaP cell growth arrest, as confirmed by cell apoptosis assays. These findings suggest that these analogues of androgen receptor blockers have promising potential for further investigation as effective treatments for prostate cancer.

Polysaccharides from Spirulina platensis (PSP): promising biostimulants for the green synthesis of silver nanoparticles and their potential application in the treatment of cancer tumors.

Photosynthetic cyanobacterial components are gaining great economic importance as prospective low-cost biostimulants for the green synthesis of metal nanoparticles with valuable medical and industrial applications. The current study comprises the biological synthesis of silver nanoparticles (Ag-NPs) using soluble polysaccharides isolated from Spirulina platensis (PSP) as reducing and capping agents. FTIR spectra showed major functional groups of PSP and biogenic silver nanoparticles including O-H, C-H (CH2), C-H (CH3), C=O, amide, and COO- groups. The UV/Vis spectroscopy scan analyses of the extracted PSP showed absorption spectra in the range of 200-400 nm, whereas the biogenic Ag-NPs showed a maximum spectrum at 285 nm. Transmission electron microscopy (TEM) analysis of the synthesized Ag-NPs showed spherical nanoparticles with mean size between 12 and 15.3 nm. The extracted PSP and Ag-NPs exhibited effective cytotoxic activity against Hep-G2 (human hepatocellular carcinoma). The IC50 for PSP and Ag-NPs were 65.4 and 24.5 µg/mL, respectively. Moreover, cell apoptosis assays for PSP and Ag-NPs against the growth of Hep-G2 cells revealed superior growth inhibitory effects of the green synthesized Ag-NPs that encouraged tracing the apoptotic signalling pathway. In conclusion, the current study demonstrated an unprecedented approach for the green synthesis of silver nanoparticles (NPs), using the polysaccharide of Spirulina platensis as reducing and capping agents, with superior anticancer activity against a hepatocellular carcinoma cell line.

A Biotechnological Approach for the Production of Pharmaceutically Active Human Interferon-α from Raphanus sativus L. Plants.

Human interferon (IFN) is a type of cytokine that regulates the immune system's response to viral and bacterial infections. Recombinant IFN-α has been approved for use in the treatment of a variety of viral infections as well as an anticancer medication for various forms of leukemia. The objective of the current study is to produce a functionally active recombinant human IFN-α2a from transgenic Raphanus sativus L. plants. Therefore, a binary plant expression construct containing the IFN-α2a gene coding sequence, under the regulation of the cauliflower mosaic virus 35SS promoter, was established. Agrobacterium-mediated floral dip transformation was used to introduce the IFN-α2a expression cassette into the nuclear genome of red and white rooted Raphanus sativus L. plants. From each genotype, three independent transgenic lines were established. The anticancer and antiviral activities of the partially purified recombinant IFN-α2a proteins were examined. The isolated IFN-α2a has been demonstrated to inhibit the spread of the Vesicular Stomatitis Virus (VSV). In addition, cytotoxicity and cell apoptosis assays against Hep-G2 cells (Human Hepatocellular Carcinoma) show the efficacy of the generated IFN-α2a as an anticancer agent. In comparison to bacterial, yeast, and animal cell culture systems, the overall observed results demonstrated the efficacy of using Raphanus sativus L. plants as a safe, cost-effective, and easy-to-use expression system for generating active human IFN-α2a.

Engineering Chlamydomonas reinhardtii for Expression of Functionally Active Human Interferon-α.

Human interferon (IFN) are secreted cytokines that play a major regulatory role in response to various infections. Commercially, IFN-α has been approved to treat many chronic viral diseases as well as a variety of cancers and different types of leukemia. In this study, a binary vector containing human IFN-α2a gene under the regulation of the cauliflower mosaic virus 35S promoter was constructed. IFN-œ2a expression cassette was transferred to Chlamydomonas reinhardtii cells via Agrobacterium-mediated transformation method. Three independent transgenic C. reinhartii lines were generated and reported to produce a biologically active IFN-œ2a. The expressed IFN-œ2a was partially purified and tested for their antitumor and antiviral properties. Cytotoxicity and cell apoptosis assays involving the usage of the recombinant C. reinhardtii IFN-œ2a (Cr. IFN-œ2a) against the growth of Hep-G2 cells (human hepatocellular carcinoma), EAC-induced tumors (Ehrlich Ascites Carcinoma) in mice prove the functionality of the produced IFN-œ2a as an anticancer drug. Moreover, Cr.IFN-œ2a is shown to have significant inhibitory effects on the propagation of the vesicular stomatitis virus (VSV). The overall observed results support the application of C. reinhardtii expression system as a cost effective, eco-friendly, safe, and easy to employ compared to plant, bacterial and animal cell culture systems.

Expression of the human gene CYP1A2 enhances tolerance and detoxification of the phenylurea herbicide linuron in Arabidopsis thaliana plants and Escherichia coli.

The phenylurea herbicide, linuron (LIN), is used to control various types of weeds. Despite its efficient role in controlling weeds, it presents a persistent problem to the environment. In the current study, phytoremediation properties of transgenic CYP1A2 Arabidopsis thaliana plants to LIN were assessed. CYP1A2 gene was firstly cloned and expressed in bacteria before proceeding to plants. In presence of LIN, The growth of CYP1A2 expressing bacteria was superior compared to control bacteria transformed with the empty bacterial expression vector pET22b(+). No clear morphological changes were detected on CYP1A2 transgenic plants. However, significant resistance to LIN herbicide application either via spraying the foliar parts of the plant or via supplementation of the herbicide in the growth medium was observed for CYP1A2 transformants. Plant growth assays under LIN stress provide strong evidence for the enhanced capacity of transgenic lines to grow and to tolerate high concentrations of LIN compared to control plants. HPLC analyses showed that detoxification of LIN by bacterial extracts and/or transgenic plant leaves is improved as compared to the corresponding controls. Our data indicate that over expression of the human CYP1A2 gene increases the phytoremediation capacity and tolerance of Arabidopsis thaliana plants to the phenylurea herbicide linuron.

Coumarin impairs redox homeostasis in wheat aleurone layers.

Many plant families produce coumarin (COU) and its derivatives as secondary metabolites via the phenylpropanoid biosynthetic pathway. This ubiquitous group of phytochemicals was shown to have diverse physiological effects on cellular, tissue, and organ levels. So far, research dealing with the hormonal like behavior of COU and its interaction with the activity and/or transport of phytohormones is very limited. In the current study, the impact of COU on redox homeostasis in aleurone layers of wheat grains was investigated. Aleurone layers were incubated in either 1000 µM COU or 5 µM gibberellic acid (GA3) alone or in combination with 5 µM abscisic acid (ABA). Results revealed that both COU and GA3 treatments induced the production of α-amylase but inhibited the activities of superoxide dismutase, catalase and ascorbate peroxidase. The downregulation of antioxidant enzymes that is provoked by COU and GA3 was accompanied by significant accumulation of both H2O2 and malondialdehyde. In contrast with the effect of ABA, both COU and GA3 treatments resulted in a significant reduction in cell viability as revealed by trypan blue staining. These results suggest that COU could disrupt the redox balance in aleurone layers through downregulation of the enzymatic antioxidant system. Therefore, the current study provides evidence for the gibberellin like activity of COU.

Expression of the cyanobacterial enzyme cyanase increases cyanate metabolism and cyanate tolerance in Arabidopsis.

Cyanate and its derivatives are considered as environmental hazardous materials. Cyanate is released to the environment through many chemical industries and mining wastewater. Cyanase enzyme converts cyanate into CO2 and NH3 in a bicarbonate-dependent reaction. At low cyanate concentrations, the endogenous plant cyanases play a vital role in cyanate detoxification. However, such cyanate biodegradation system is probably insufficient due to the excess cyanate concentrations at contaminated sites. In this study, we have transferred the activity of the cyanobacterial cyanase into Arabidopsis thaliana plants in order to enhance plant resistance against cyanate toxicity. The enzyme was shown to be active in planta. Transgenic plants exposed to cyanate, either applied by foliar spray or supplemented in growth medium, showed less reduction in pigment contents, antioxidant enzymes, carbohydrate contents, and reduced levels of plant growth retardation. Plant growth assays under cyanate stress showed enhanced growth and biomass accumulation in cyanase overexpressors compared to control plants. Results of this study provide evidence for developing novel eco-friendly phytoremediation systems for cyanate detoxification.

Engineering the metabolism of the phenylurea herbicide chlortoluron in genetically modified Arabidopsis thaliana plants expressing the mammalian cytochrome P450 enzyme CYP1A2.

Transgenic Arabidopsis thaliana plants were generated by introduction of the human P450 CYP1A2 gene, which metabolizes a number of herbicides, insecticides and industrial chemicals. Transgenic A. thaliana plants expressing CYP1A2 gene showed remarkable resistance to the phenylurea herbicide chlortoluron (CTU) supplemented either in plant growth medium or sprayed on foliar parts of the plants. HPLC analyses showed a strong reduction in CTU accumulation in planta supporting the tolerance of transgenic lines to high concentrations of CTU. Besides increased herbicide tolerance, expression of CYP1A2 resulted in no other visible phenotype in transgenic plants. Our data indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. Moreover, these transgenic plants appear to be useful for herbicide resistance as well as phytoremediation of environmental contaminants.

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice.

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.

Constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana.

The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

Photorespiration.

Photorespiration is initiated by the oxygenase activity of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RUBISCO), the same enzyme that is also responsible for CO(2) fixation in almost all photosynthetic organisms. Phosphoglycolate formed by oxygen fixation is recycled to the Calvin cycle intermediate phosphoglycerate in the photorespiratory pathway. This reaction cascade consumes energy and reducing equivalents and part of the afore fixed carbon is again released as CO(2). Because of this, photorespiration was often viewed as a wasteful process. Here, we review the current knowledge on the components of the photorespiratory pathway that has been mainly achieved through genetic and biochemical studies in Arabidopsis. Based on this knowledge, the energy costs of photorespiration are calculated, but the numerous positive aspects that challenge the traditional view of photorespiration as a wasteful pathway are also discussed. An outline of possible alternative pathways beside the major pathway is provided. We summarize recent results about photorespiration in photosynthetic organisms expressing a carbon concentrating mechanism and the implications of these results for understanding Arabidopsis photorespiration. Finally, metabolic engineering approaches aiming to improve plant productivity by reducing photorespiratory losses are evaluated.

Metabolic engineering towards the enhancement of photosynthesis.

Photosynthetic capacity is a promising target for metabolic engineering of crop plants towards higher productivity. Crop photosynthesis is limited by multiple factors dependent on the environmental conditions. This includes photosynthetic electron transport, regeneration of CO2 acceptor molecules in the reductive pentose phosphate cycle, the activity and substrate specificity of the CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase, and the associated flow through the photorespiratory pathway. All these aspects of the photosynthetic network have been the subject of recently published metabolic engineering approaches in model species. Together, the novel results raise hopes that engineering of photosynthesis in crop species can significantly increase agricultural productivity.

Mitochondrial glycolate oxidation contributes to photorespiration in higher plants.

The oxidation of glycolate to glyoxylate is an important reaction step in photorespiration. Land plants and charophycean green algae oxidize glycolate in the peroxisome using oxygen as a co-factor, whereas chlorophycean green algae use a mitochondrial glycolate dehydrogenase (GDH) with organic co-factors. Previous analyses revealed the existence of a GDH in the mitochondria of Arabidopsis thaliana (AtGDH). In this study, the contribution of AtGDH to photorespiration was characterized. Both RNA abundance and mitochondrial GDH activity were up-regulated under photorespiratory growth conditions. Labelling experiments indicated that glycolate oxidation in mitochondrial extracts is coupled to CO(2) release. This effect could be enhanced by adding co-factors for aminotransferases, but is inhibited by the addition of glycine. T-DNA insertion lines for AtGDH show a drastic reduction in mitochondrial GDH activity and CO(2) release from glycolate. Furthermore, photorespiration is reduced in these mutant lines compared with the wild type, as revealed by determination of the post-illumination CO(2) burst and the glycine/serine ratio under photorespiratory growth conditions. The data show that mitochondrial glycolate oxidation contributes to photorespiration in higher plants. This indicates the conservation of chlorophycean photorespiration in streptophytes despite the evolution of leaf-type peroxisomes.

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana.

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.

A glycolate dehydrogenase in the mitochondria of Arabidopsis thaliana.

The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants.