Regulation of multimeric structure of NO-synthase as a new factor of organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

Regulation of isoprene synthase promoter by environmental and internal factors

Isoprene synthase (ISPS) catalyzes the formation of isoprene, an important volatile terpenoid with strong effects on global atmospheric chemistry and protective physiological functions in plant leaves. Many terpene synthase genes including isoprene synthase, a member of the TPS-b cluster of this numerous gene family, were already functionally analysed but much less is known about regulation of their promoters. To study regulation of the PcISPS gene in detail we developed transgenic Grey poplar (Populus × canescens) and Arabidopsis thaliana plants in which the PcISPS promoter is fused to enhanced green fluorescent protein (E-GFP) and β-glucuronidase (GUS) reporter genes. We analysed these reporters during plant development, for organ specificity and in plants subjected to different light and temperature regimes. We observed low promoter activity in non-isoprene emitting tissue like roots where ISPS gene is transcribed but no active enzyme is detectable. In leaves we demonstrate that light and temperature directly modulate ISPS promoter activity. Moreover, with confocal laser scanning microscopy we show a cell specific gradient of ISPS promoter activity within the leaf parenchyma depending on light direction. Our results indicate that ISPS promoter activity, which correlates with basal isoprene emission capacity, is not uniformly distributed within leaf tissue and that it can adapt rapidly towards internal as well as external environmental stimuli.     

Nitric oxide synthase mediates regulation of cell polarity and movement during <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> morphogenesis

The effect of expression of the enzyme producing nitric oxide on Drosophila melanogaster morphogenesis was studied using ectopic expression of a truncated splice form of the enzyme that has a dominant negative effect on the full-length enzyme form. The suppressed enzyme expression was shown to affect the cell polarity and movement as well as the structural pattern of organs during Drosophila development. The effect of nitric oxide production on the intercellular distribution of significant factors of cell polarity, protein products of the Strabismus and Prospero genes, has been revealed.     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Effects of <Emphasis Type="Italic">sarah/nebula</Emphasis> knockdown on Aβ42-induced phenotypes during <Emphasis Type="Italic">Drosophila</Emphasis> development

The Down syndrome critical region 1 (DSCR1), a Down syndrome-associated protein, is an endogenous inhibitor of the Ca²⁺-dependent phosphatase calcineurin. It has been also suggested to be associated with Alzheimer’s disease (AD) but the role of DSCR1 in the pathogenesis of AD still remains controversial. In this paper, we investigated the effects of knockdown of sarah (sra), a Drosophila DSCR1 ortholog, on the Aβ42-induced developmental phenotypes of Drosophila. Knockdown of sra showed detrimental effects on the rough eye phenotype and survival of Aβ42-expressing flies without altering the Aβ42 accumulation. Furthermore, the knockdown of sra increased glial cell numbers in the larval brains and its susceptibility to oxidative stress. Overexpression of an active form of calcineurin produced similar results to sra knockdown as they both exacerbated the Aβ42-induced rough eye phenotype. However, sra knockdown did not alter apoptosis or c-Jun N-terminal kinase activation in Aβ42-expressing flies. In conclusion, our results suggest that sra does play an important role in Aβ42-induced developmental defects in Drosophila without affecting its stress responses.     

The effects of natural and hybrid lectins on the legume-rhizobium interactions

The effects of hybrid lectins—full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)—on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.     

The cloning,expression, and comparative analysis of peroxiredoxin 6 from various sources

Human, rat, Xenopus, and Drosophila (DPx2540 and DPx6005) peroxiredoxin cDNAs were cloned and expressed in Escherichia coli. The recombinant enzymes were compared with respect to enzymatic activity toward various substrates and protection of plasmid DNA from the Fenton reaction products. The activity toward H₂O₂ decreased in the following order: DPx2540 > human Prx6 > Xenopus Prx6 > rat Prx6 > DPx6005. The activity toward tret-butyl hydroperoxide decreased in the following order: DPx2540 = DPx6005 > rat Prx6 > Xenopus Prx6 > human Prx6. The efficiency of plasmid DNA protection from oxidative damage mediated by the Fenton reaction decreased in the order of DPx2540 > DPx6005 = rat Prx6 = human Prx6 > Xenopus Prx6. The optimal temperature for activity of all enzymes was 37°C. Peroxiredoxins from rat, Xenopus, and Drosophila (DPx6005) retained no less than 50% of their activity in a wider temperature range (10–50°C) as compared with the human and Drosophila (DPx2540) enzymes (25–45°C). The thermostability of the enzymes decreased in the following order: DPx6005 = rat > human > Xenopus > DPx2540. The results confirmed a negative correlation between the activity and stability of peroxiredoxin 6, especially in the case of the Xenopus and Drosophila enzymes.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analysis reveal a novel gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> trehalose metabolism

Background

The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.

Results

In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.

Conclusions

These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

     

The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene <Emphasis Type="Italic">Mth1</Emphasis> in methylotrophic yeast <Emphasis Type="Italic">Pichia methanolica</Emphasis>

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.     

Interactions Between the Circadian Clock and Heme Oxygenase in the Retina of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The Drosophila retina has an autonomous peripheral circadian clock in which the expression of the gene encoding heme oxygenase (HO) is under circadian control with the ho mRNA peaking at the beginning of the day and in the middle of the night. The function of HO in the retina is unknown, but we observed that it regulates the circadian clock and protects photoreceptors against DNA damage. The decline in HO level increases and decreases the expression of the canonical clock genes period (per) and Clock (Clk), respectively. The opposite result was observed after increasing HO expression. Among three products of HO activity—carbon monoxide (CO), ferrous ions, and biliverdin—the latter has no effect on per and Clk expressions, but CO exerts the same effect as the increase of ho expression. This suggests that HO action on the clock is mediated by CO, which may affect Clk expression during the day and the level of per expression. While ho expression is not stimulated by nitric oxide (NO), NO has the same effect on the clock as HO, increasing Clk expression and decreasing the expression of per.     

Effects of endogenous salicylic acid synthesized through PAL and ICS pathway on baicalin and baicalein accumulation in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

The aim of this study was to investigate the effect of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS) on free salicylic acid (FSA) or total salicylic acid (TSA) content, and the effect of endogenous SA on baicalin and baicalein accumulation in Scutellaria baicalensis Georgi, respectively. We amplified partial sequences of PAL and ICS genes in Scutellaria baicalensis Georgi and silenced the two genes with virus-induced gene silence (VIGS) technique, respectively. The influence of gene silence on FSA, TSA, baicalin, and baicalein accumulation in Scutellaria baicalensis Georgi were analyzed, and these parameters were also investigated under high temperature. Results indicated that PAL silence significantly affected the FSA, ICS affected TSA content. FSA significantly affected the baicalin, rather than baicalein content. Our results along with previous studies indicated PAL and ICS were different in the regulation of FSA or TSA synthesis, and FSA and TSA were different in the regulation of baicalin and baicalein synthesis in Scutellaria baicalensis Georgi.     

Application of ZFN for Site Directed Mutagenesis of Rice <Emphasis Type="Italic">SSIVa</Emphasis> Gene

Many successful studies on genome editing in plants have been reported and one of the popular genome editing technology used in plants is Zinc Finger Nucleases (ZFN), which are chimeric proteins composed of synthetic zinc finger-based DNA binding domain and a DNA cleavage domain. The objective of this research was to utilize ZFNs to induce a double-stranded break in SSIVa, a soluble starch synthase involved in starch biosynthesis pathway, leading to the regulation of the SSIVa expression. The isoform SSIVa is not yet well studied, thus, by modifying the endogenous loci in SSIVa, we can explore on the specific roles of this gene in starch biosynthesis and other possible functions it might play. In this study, we used ZFN-mediated targeted gene disruption in the coding sequence of the SSIVa rice gene in an effort to elucidate the functional role of the gene. Generation of transgenic plants carrying premature stop codons and substitution events, revealed no SSIVa mRNA expression, low starch contents and dwarf phenotypes. Remarkably, based on our analysis SSIVa gene disruption had no effect on other starch synthesis related genes as their expression remained at wild type levels. Therefore, the engineered ZFNs can efficiently cleave and stimulate mutations at SSIVa locus in rice to     

Mod(mdg4)-58.0, the product of <Emphasis Type="Italic">mod</Emphasis>(<Emphasis Type="Italic">mdg4</Emphasis>) locus,directly interacts with kermit protein of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The experiments in a yeast two-hybrid system, on co-immunoprecipitation of proteins from the D. melanogaster embryonic cell lysates, and on cell immunostaining have shown for the first time that the product of mod(mdg4) locus, Mod (mdg4)-58.0 (isoform C), directly interacts with Kermit protein (dGIPC). Kermit protein plays an important role in locomotor activity, polarization of the cells within the plane of the epithelium at the early stages of development, transfer of intercellular signals, and determining the longevity of Drosophila. These data expand the understanding of the role of Mod(mdg4)-58.0 protein in the eukaryotic cell activity.     

Evidence for a role for the putative <Emphasis Type="Italic">Drosophila</Emphasis> hGRX1 orthologue in copper homeostasis

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.     

Light regulation of succinate dehydrogenase expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.     

The insulin-like receptor gene expression in the tissues synthesizing gonadotropic hormones at sexual maturation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> females

The insulin/insulin-like growth factor signaling pathway is involved in the regulation of the synthesis of insect gonadotropic hormones, juvenile (JH) and 20-hydroxyecdysone (20E). We carried out the immunohistochemical analysis of the insulin receptor (InR) expression in the corpus allatum (the JH-producing gland) and in the ovarian follicular cells (a site for the synthesis of 20E precursor, ecdysone) in the process of sexual maturation of D. melanogaster females and examined the influence of exogenous JH on the InR expression in these tissues. For the first time, it was demonstrated that InR was expressed in follicular cells and that its expression in corpus allatum and follicular cells of Drosophila females was stage-specific, i.e., the expression intensity in young females greatly exceeded that in mature individuals. We also found a negative feedback loop in the regulation of JH levels by the insulin signaling pathway in Drosophila adults: the experimental increase in the JH titers in young females dramatically reduced the InR expression intensity in corpus allatum and follicular cells.