Morphological and molecular characterization of L-methioninase producing Aspergillus species

Six species of L-methioninase producing Aspergillus species, isolated from Egyptian soil, were selected for comprehensive morphotypic and molecular characterization. Based on morphological and physiological features, these isolates were identified as Aspergillus flavipes, Aspergillus carneus, Aspergillus flavus, Aspergillus tamari, Aspergillus oryzae, and Aspergillus parasiticus. Regarding to the maximum enzyme productivity by A. flavipes, it was selected as authentic strain for ribosomal ribonucleic acid (rRNA) primer design. Using primer combinations for 18S rRNA and internal transcribed spacers (ITS)1 amplification, these isolates gave the same polymerase chain reaction (PCR) amplicon size, revealing the relative molecular identity. Moreover, using ITS2 primers, among the six isolates, Aspergillus flavipes EK and A. carneus displayed PCR products on agarose gel, approving the actual morphological and biochemical similarities of these two isolates, A. flavipes group. By sequencing of ITS1-5.8S-ITS2 region, blasting and alignment from the data base, A. flavipes EK showed a typical identity to gene bank deposited A. flavipes isolates. The rRNA sequence of A. flavipes EK was deposited to genbank under accession number JF831014.Key words: Aspergillus, morphological descriptions, 18 S rRNA, internal transcribed spacers (ITS) regions

Определение эффективности способов борьбы с асфальтеносмолопарафиновыми отложениями при эксплуатации нефтяных месторождений

В качестве объекта исследования – рассматривается нефтяные месторождения, а именно Талаканского месторождения, а предметом является технологии предупреждение асфальтеносмолопарафиновых отложений (АСПО) на нефтепромысловых оборудованиях. Цель работы – определение наиболее эффективного метода борьбы с асфальтеносмолопарафиновыми отложениями, и применение технологий удаления отложений на различных нефтяных месторождениях. В процессе исследования были раскрыты причины образования парафиновых отложений и эффективность применения некоторых методов борьбы с АСПО на месторождениях. Область применения: месторождения нефти и газа, имеющие осложнения в виде асфальтосмолопарафиновых отложений.The object of the study is the oil fields, namely the Talakan field, and the subject is the technologies for the prevention of asphaltene-tar-paraffin deposits (ASPO) on oilfield equipment. The purpose of the work is to determine the most effective method of controlling asphaltene-tar-paraffin deposits, and to apply technologies for removing deposits in various oil fields. In the course of the study, the reasons for the formation of paraffin deposits and the effectiveness of the use of some methods of combating ASPO in the fields were revealed. Field of application: oil and gas fields with complications in the form of asphalt-resin-paraffin deposits

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 CO2 release and a lower CO2 compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO2, 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

Construction and molecular analysis of genetically modified C 3 plants expressing a glycolate oxidizing pathway inside the chloroplast

Metabolism of glycolate via the photorespiratory pathway in C3 plants consumes not only ATP and reducing equivalents but results also in approximately 25% loss of the carbon from glycolate. In the present study, a novel biochemical pathway for the metabolism of glycolate was established in the chloroplast of Arabidopsis thaliana plants. The new pathway aims to increase the CO2 concentration in the vicinity of Rubisco thereby suppressing photorespiration in C3 plants. The pathway is derived from E. coli and converts the glycolate formed during photorespiration into glycerate. Three enzymatic activities are required: glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL), and tartronic semialdehyde reductase (TSR). The minimal E.coli glycolate dehydrogenase enzyme is formed from three different polypeptides. As an alternative, a glycolate dehydrogenase (AtGDH) derived from A. thaliana was used. Transgenic A. thaliana plants containing the necessary genes for the novel pathway were generated. Variable amounts of foreign proteins as well as RNA were detected by Western blot and RT-PCR, respectively. Enzymatic assays showed that the proteins are active in planta. Biochemical, physiological and biophysical analyses were performed under ambient and enhanced photorespiratory conditions using different transgenic lines for evaluating the impact of the novel pathway in planta. By measuring the Gly/Ser ratio, a clear reduction in photorespiration was observed in transgenic plants expressing the novel pathway genes compared to wild type plants. A clear decrease in the amount of CO2 released in the plant mitochondria during photorespiration was also obvious in transgenic lines. The ammonia release bioassay provides an additional evidence for the partial suppression of photorespiration in some of the transgenic lines. Furthermore, establishment of the glycolate pathway in the plant chloroplasts results in a decrease in the CO2 compensation point (Gamma*). The CO2 assimilation rates in transgenic plants were also enhanced under photorespiratory conditions. Finally, plant growth measurements revealed that the transgenic plants expressing the glycolate pathway in their chloroplasts have bigger leaf area as well as bigger rosette diameter compared to the control plants. Moreover, the total fresh and dry weight measurements showed that the total plant productivity was enhanced. Interestingly, most of the described effects were also observed in plants that only overexpressed a functional GDH. However, these effects were stronger in plants overexpressing all necessary elements of the glycolate pathway. Moreover, the phenotypical effects were much stronger when the bacterial GDH was compared to the plant GDH. Taken together, it can be concluded that expression of the novel pathway in C3 plant chloroplast does not only result in a reduction of photorespiration but it also enhances plant growth

Construction and molecular analysis of genetically modified C 3 plants expressing a glycolate oxidizing pathway inside the chloroplast

Metabolism of glycolate via the photorespiratory pathway in C3 plants consumes not only ATP and reducing equivalents but results also in approximately 25% loss of the carbon from glycolate. In the present study, a novel biochemical pathway for the metabolism of glycolate was established in the chloroplast of Arabidopsis thaliana plants. The new pathway aims to increase the CO2 concentration in the vicinity of Rubisco thereby suppressing photorespiration in C3 plants. The pathway is derived from E. coli and converts the glycolate formed during photorespiration into glycerate. Three enzymatic activities are required: glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL), and tartronic semialdehyde reductase (TSR). The minimal E.coli glycolate dehydrogenase enzyme is formed from three different polypeptides. As an alternative, a glycolate dehydrogenase (AtGDH) derived from A. thaliana was used. Transgenic A. thaliana plants containing the necessary genes for the novel pathway were generated. Variable amounts of foreign proteins as well as RNA were detected by Western blot and RT-PCR, respectively. Enzymatic assays showed that the proteins are active in planta. Biochemical, physiological and biophysical analyses were performed under ambient and enhanced photorespiratory conditions using different transgenic lines for evaluating the impact of the novel pathway in planta. By measuring the Gly/Ser ratio, a clear reduction in photorespiration was observed in transgenic plants expressing the novel pathway genes compared to wild type plants. A clear decrease in the amount of CO2 released in the plant mitochondria during photorespiration was also obvious in transgenic lines. The ammonia release bioassay provides an additional evidence for the partial suppression of photorespiration in some of the transgenic lines. Furthermore, establishment of the glycolate pathway in the plant chloroplasts results in a decrease in the CO2 compensation point (Gamma*). The CO2 assimilation rates in transgenic plants were also enhanced under photorespiratory conditions. Finally, plant growth measurements revealed that the transgenic plants expressing the glycolate pathway in their chloroplasts have bigger leaf area as well as bigger rosette diameter compared to the control plants. Moreover, the total fresh and dry weight measurements showed that the total plant productivity was enhanced. Interestingly, most of the described effects were also observed in plants that only overexpressed a functional GDH. However, these effects were stronger in plants overexpressing all necessary elements of the glycolate pathway. Moreover, the phenotypical effects were much stronger when the bacterial GDH was compared to the plant GDH. Taken together, it can be concluded that expression of the novel pathway in C3 plant chloroplast does not only result in a reduction of photorespiration but it also enhances plant growth

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

Abstract 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 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

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