<Emphasis Type="Italic">CrMAPK3</Emphasis> regulates the expression of iron-deficiency-responsive genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas.

Results

Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2–CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced.

Conclusion

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

     

Isoform specific gene auto-regulation via miRNAs: a case study on miR-128b and ARPP-21

In this study, we investigate whether miRNAs located within “host” protein-coding genes may regulate the expression of their host genes. We find that 43 of 174 miRNAs encoded within RefSeq genes are predicted to target their host genes. Statistical analysis of this phenomenon suggests that gene auto-regulation via miRNAs may be under positive selective pressure. Our analysis also indicates that several of the 43 miRNAs have a much lower expectation of targeting their host genes by chance than others. Among these examples, we identify miR-128b:ARPP-21 (cyclic AMP-regulated phosphoprotein, 21 kD) as a case in which both the miRNA and the target site are also evolutionarily conserved. We provide experimental support for this miRNA:target interaction via reporter silencing assays, and present evidence that this isoform-specific gene auto-regulation has been preserved in vertebrate species in order to prevent detrimental consequences of ARPP-21 over-expression in brain.     

Loss of NAD(H) from swollen yeast mitochondria

Background

The mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.

Results

NAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.

Conclusion

Loss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.     

Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae

Background

Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics [1]. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.

Results

In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.

Conclusion

Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.     

Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine

Background

Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8^th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.

Results

The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.

Conclusion

It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.     

Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway.

Results

In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae.

Conclusions

The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.

     

A β-Secretase (BACE1)-inhibiting C-Methylrotenoid Induced by Yeast Elicitation in Abronia nana Suspension Cultures

Suspension cultures of Abronia nana were established to produce C-methylisoflavones. Treatment of the A. nana cultures with yeast elicitor induced boeravinone E (1), with maximum induction at 24 h after elicitor treatment. Of the biotic and abiotic elicitors tested, yeast extract gave the strongest induction of 1. The IC₅₀ value of 1 against β-secretase (β-amyloid cleaving enzyme-1) was 5.57 μM. Other proteases such as trypsin, chymotrypsin, and elastase were not inhibited by concentrations up to 1.0 mM, indicating that inhibition of β-secretase was specific. 1 was noncompetitive in Dixon plot, and Ki value was 3.79 μM.     

Nucleic Acid Binding Behavior and Cytotoxicity Properties of a Ruthenium(II) Polypyridyl Complex

The binding of [Ru(IP)₂(dppz-11-CO₂Me)]²⁺ (1) {IP = imidazo[4,5-f][1,10]phenanthroline, dppz-11-CO₂Me = dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxylic acid methyl ester} to calf thymus DNA and yeast tRNA has been investigated by UV–Vis spectroscopy, fluorescence spectroscopy, viscosity, as well as equilibrium dialysis and circular dichroism. In addition, the antitumor activities of complex 1 have been evaluated by the MTT method. On the basis of the spectroscopic results, the binding mode of complex 1 to CT-DNA and yeast tRNA is intercalation. However, DNA binding with complex 1 is stronger than RNA binding with complex 1, and complex 1 is a better candidate for an enantioselective binder to CT-DNA than to yeast tRNA. These results indicate that the structures of DNA and RNA have significant effects on the binding behaviors of complex 1. Furthermore, complex 1 demonstrates different antitumor activities against selected cancer cell lines in vitro.     

Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Quantitative real-time PCR (qPCR) is commonly used for gene expression analyses with defined documentation guidelines to compare published results. To minimize the impact of variances from qPCR performance, sample handling and processing reference genes are used. Their selection process cannot be completely aligned due to variations in experimental conditions. Furthermore, the named sources of error are also present when determining the stability of the reference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the reference gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes. Figure

We here compare the expression of reference genes for qPCR and NanoString data and determine the value of the latter method as a bias-free mRNA quantification method     

Real-time determination of the activity of ATPase by use of a water-soluble polythiophene

This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K _m and V _max were 0.30?mmol?L^?1 ATP and 0.134?nmol ATP min^?1?μg^?1 enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ.

The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p

Background

The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex.

Results

We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to ～ 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN.

Conclusions

Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.     

Lipid bilayer composition influences small multidrug transporters

Background

Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes.

Results

The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity.

Conclusion

These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.     

Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic <Emphasis Type="Italic">Corynebacterium urealyticum</Emphasis> DSM 7109

Background

Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes.

Results

In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Conclusions

Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

     

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

Background

The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent K _m of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.

Results

We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.

Conclusions

Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of K _m associated with kinase enzymes.     

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

Background

Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants.

Results

When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide), respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property.

Conclusions

We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.     

Enhanced l-Lactic Acid Production from Biomass-Derived Xylose by a Mutant Bacillus coagulans

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient l-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for l-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l^?1? l-lactic acid was obtained from 100 g l^?1 xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l^?1? l-lactic acid was obtained in 36 h and the yield was 83.09 %.