Micro-NMR elucidates altered metabolites in the Parkinson’s disease-related <Emphasis Type="Italic">catp-6</Emphasis> genotype of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Introduction

A severe form of Parkinson’s disease (PD) is the Kufor-Rakeb syndrome. Here mutations in the ATP13A2 (PARK9) gene lead to an early juvenile-onset Parkinsonism often accompanied by dementia. ATP13A2 encodes a lysosomal P-type ATPase. Its ortholog in Caenorhabditis elegans is the catp-6 gene where phenotypes with mutations in the alleles ok3473 and tm3190 show high mortality and low reproduction.

Objectives

Since PD is difficult to study in humans we wanted to investigate the potential to use C. elegans as model for the Kufor-Rakeb syndrome. As it is difficult to obtain enough catp-6 mutant worms for standard NMR metabolic profiling, we explored focused ultrasonication extraction and miniaturized NMR as techniques to overcome this limitation.

Methods

One- and two-dimensional NMR experiments (¹ H, JRES, TOCSY) were performed with a commercial high-resolution magic angle spinning (HR-MAS) probe (25 µL sample volume). Significant features were identified through analysis of variance (ANOVA, p?<?0.05), volcano plots (p?<?0.05, fold change?>1.5), PCA, and PLS-DA.

Results

Assignment of statistically relevant peaks resulted in the identification of twenty altered metabolites. Previous studies on catp-6 mutants identified strong morphological and functional changes in their mitochondria. Our findings of altered TCA metabolites (fumarate, succinate), branched-chain amino acids (leucine, isoleucine and valine) and nucleotides (AMP, ATP and GTP), formate and hypoxanthine appear to support these findings. Highest fold changes (< ?5) in wildtype relative to both catp-6 strains were found for GTP. Formic acid is known to inhibit the mitochondrial respiratory chain complex IV and high hypoxanthine in catp-6 indicates an increased nucleotide salvage pathway. Alterations in most of the remaining metabolites may be the result of the recently discovered activation of AMPK (AMP-activated protein kinase) and inhibition of mTOR (mechanistic target of rapamycin) pathways together with a catabolic response to recover energy production.

Conclusions

If the effect of the catp-6 mutation in C. elegans at the level of metabolites is correlated to the metabolic dysfunction in the human PARK9 ortholog, then it may be possible to uncover the molecular mechanism behind Parkinsonism and the Kufor-Rakeb syndrome.

     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

Shengmai Formula Ameliorates Pathological Characteristics in AD <Emphasis Type="Italic">C.</Emphasis> <Emphasis Type="Italic">elegans</Emphasis>

Shengmai (SM) formula, a classical traditional Chinese medicine formula, is composed of Panax ginseng (Pg), Ophiopogon japonicus (Oj), and Schisandra Chinesis (Sc). SM has been clinically used to treat heart failure and ischemic heart disease. Although SM formula has been reported to be potential for fighting against Alzheimer’s disease (AD) by previous works, there are many gaps in our knowledge on its usage in AD treatment on an organism level and will then need to be further clarified. In this study, transgenic Caenorhabditis elegans expressing human Aβ_1–42 are used to evaluate SM formula efficacy to treat AD phenotype and to investigate its underlying mechanism. The results showed that SM formula ameliorated AD pathological characteristics of paralysis behavior and chemotaxis defect in transgenic C. elegans. With SM treatment, the number of Aβ deposits decreased, the levels of gene expressions of hsp16-2, hsp16-41, ace-1, ace-2, and TNFA1P1 homolog genes were down-regulated. Our results also showed that Oj exhibited more stronger effect on delaying paralysis in worms than Pg and Sc did, and synergistic action was observed between Pg and Oj, and Sc further enhanced the activity of Pg/Oj combination on delaying paralysis behavior. Further, SM with herbs of Pg, Oj, and Sc at a dose proportion of 9:9:6 exhibited superior therapeutic efficacy in comparison with herbs at other dose proportions. After SM formula extracted by ethanol, it delayed AD symptoms on a wider dose from 0.2 to 10.0 mg/mL with no toxic effect. These results provided more evidence for SM formula being potential to be used to treat AD.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">Z</Emphasis>, <Emphasis Type="Italic">N</Emphasis>′-Formyl α-Amino Acid Derived Gem-Diamines

A variety of N-carbobenzoxy, N′-formyl gem-diaminoalkyl derivatives have been obtained through Goldsmith-Wick reaction of Z-α-amino acid/peptide acid derived isocyanates with 96% HCOOH in presence of 4-dimethylaminopyridine (DMAP) as catalyst. The reaction proceeds to completion within 2–4 h and results in good yields of the products isolated as stable solids.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Effect of Phenolic Compounds Against A<Emphasis Type="Italic">β</Emphasis> Aggregation and A<Emphasis Type="Italic">β</Emphasis>-Induced Toxicity in Transgenic <Emphasis Type="Italic">C. elegans</Emphasis>

Substantial evidence suggests that the aggregation of amyloid-β (Aβ) peptide into fibrillar structures that is rich in β-sheets is implicated as the cause of Alzheimer’s disease. Therefore, an attractive therapeutic strategy is to prevent or alter Aβ aggregation. Phenolic compounds are natural substances that are composed of one or more aromatic phenolic rings and present in wine, tea, fruits, vegetables and a wide variety of plants. In this work, we investigated the effects of ferulic acid, morin, quercetin and gossypol against Aβ aggregation. From the ThT and turbidity assays, it is observed that in addition to the fibril aggregate, another type of aggregate is formed in the presence of morin, quercetin, and gossypol. On the other hand, ferulic acid did not prevent fibril formation, but it did appear to reduce the average length of fibrils compared to Aβ alone. To study the protective effects of phenolic compounds on Aβ-induced toxicity, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism, human Aβ is expressed intracellularly in the body wall muscle. We found that exposure of Caenorhabditis elegans to ferulic acid give more protection against Aβ toxicity than morin, quercetin and gossypol.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Cloning and expression of <Emphasis Type="Italic">β</Emphasis>-glucosidase gene from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> into <Emphasis Type="Italic">E. coli</Emphasis> BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. K _m and V _max were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy E_a, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pK_a1 and pK_a2 of the ionizable groups of active site residues involved in V_max were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

A Single and Robust Critical Nitrogen Dilution Curve for <Emphasis Type="Italic">Miscanthus</Emphasis> × <Emphasis Type="Italic">giganteus</Emphasis> and <Emphasis Type="Italic">Miscanthus sinensis</Emphasis>

The sustainable development of miscanthus as a bioenergy feedstock requires optimizing its fertilizer inputs and, therefore, determining its nitrogen (N) requirements. The ‘critical nitrogen dilution curve’ is a powerful tool to characterize such N requirements; it relates the N concentration ([N]) in aboveground organs to their biomass, defining two domains depending on whether the N factor limits biomass growth or not. We aimed to develop such a tool in miscanthus. Using a rhizome N depletion strategy with green cutting pre-treatment over several years before the start of the experiment, we grew, in 2014, two cultivated species, Miscanthus × giganteus (M×g) and Miscanthus sinensis (Msin), at four fertilizer levels (0, 80, 160 and 240 kg N ha^?1). We found a strong nitrogen fertilization effect. The shoot [N] decreased as the aboveground biomass increased in both species and in all of the treatments. [N] was strongly correlated with leaf/stem biomass ratio. The N treatments enabled the identification of the observed critical points, i.e. points with the maximum biomass (W) and the lowest [N], on each measurement date. These points could be fitted to the following critical dilution curve that was common between M×g and Msin: N concentration (Nc) (critical [N], g N kg^?1) = 27.0 W ^?0.48 when W > 1 t ha^?1 and Nc = 27.0 when W ≤ 1. This curve was validated by literature data, separated into N-limited or not-limited conditions. The similarity of the curves between the two species was due to compensation between leaf/stem biomass ratio and [N] in the stems. This curve is helpful to diagnose the crop N status and define the optimal fertilizer requirements of miscanthus crops.     

BacA is indispensable for successful <Emphasis Type="Italic">Mesorhizobium</Emphasis>–<Emphasis Type="Italic">Astragalus</Emphasis> symbiosis

A homologue of Sinorhizobium meliloti bacA was isolated from Mesorhizobium huakuii 7653R, which is capable of fixing atmospheric nitrogen in symbiotic association with leguminous Astragalus sinicus (Chinese milk vetch). Inactivation of the bacA gene abolished the ability of M. huakuii 7653R to establish a successful symbiosis with its host plant. Simultaneously, compared with wild-type M. huakuii 7653R, the bacA mutant was more sensitive to cell envelope-disrupting agents (acidic solution, ethanol, SDS, and crystal violet). Mass spectrometry analysis revealed that the very-long-chain fatty acid (27-OHC-28:0 and 29-OHC-30:0) contents of lipid A was reduced in the M. huakuii 7653R bacA mutant. Taken together, our data suggest that the cell envelope was altered in the M. huakuii 7653R bacA mutant, which might deteriorate bacterial adaption to acute environmental changes encountered in host cells and ultimately result in the failure of Mesorhizobium–legume symbiosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.