Isolation of a CK2α Subunit and the Holoenzyme from the Mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> and Construction of the <Emphasis Type="Italic">CK2α</Emphasis> and <Emphasis Type="Italic">CK2β</Emphasis> cDNAs

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α′) and two regulatory (β) subunits, forming a differently assembled tetramer. The free and catalytically active α/α′ subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs.     

Evolution of Crohn’s disease-associated <Emphasis Type="Italic">Nod2</Emphasis> mutations

Several lines of evidence have confirmed the importance of Nod2 mutations for disease susceptibility in Crohn’s disease. For tracing Nod2 evolution, exons 4a, 4e, 8, and 12 mutations were screened in a collection of 1,064 DNA samples from 52 worldwide populations. The overall allele frequency was 7.5% for single nucleotide polymorphism (SNP)5, 0.2% for SNP8, 0.3% for SNP12, and 0.4% for SNP13. Nod2 mutations are mainly Caucasian alleles with strong distribution dissimilarity between single populations and major geographical regions. This regional diversity of Nod2 mutations within Europe points to the regional existence of selection pressure (possibly through dairy-associated bacterial infections within Neolithic cattle farming populations). The SNP5 gradient between Africa and the Middle East and its absence in Asian and Native American populations indicate that the evolution of this variant occurred in the Middle East. As mutations in exons 4e, 8, and 12 were only found in association with SNP5, this variant may have allowed selection pressure to arise.     

Overexpression of <Emphasis Type="Italic">SNF4</Emphasis> and deletions of <Emphasis Type="Italic">REG1</Emphasis>- and <Emphasis Type="Italic">REG2</Emphasis>-enhanced maltose metabolism and leavening ability of baker’s yeast in lean dough

Maltose metabolism of baker’s yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker’s yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2?+?SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker’s yeast strains had excellent potential applications.     

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.     

Over-expressing <Emphasis Type="Italic">GLT1</Emphasis> in a <Emphasis Type="Italic">gpd2</Emphasis>Δ mutant of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to improve ethanol production

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATα ura3 gpd2Δ::RPT) strain were slower than the original strain, and the KAM-13 (MATα ura3 gpd2Δ::RPT P _PGK -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

NMR analysis of duplex d(CGCGATCGCG)<Subscript>2</Subscript> modified by <Emphasis Type="Italic">Λ</Emphasis>- and <Emphasis Type="Italic">Δ</Emphasis>-[Ru(bpy)<Subscript>2</Subscript>(m-GHK)]Cl<Subscript>2</Subscript> and DNA photocleavage study

The interaction of the diastereomeric complexes Λ-[Ru(bpy)₂(m-GHK)]Cl₂ and Δ-[Ru(bpy)₂(m-GHK)]Cl₂ (bpy is 2,2′-bipyridine, GHK is glycine–l-histidine–l-lysine) with the deoxynucleotide duplex d(5′-CGCGATCGCG)₂ was studied by means of ¹H NMR spectroscopy. At a Δ-isomer to DNA ratio of 1:1, significant shifts for the metal complex are observed, whereas there is negligible effect on the oligonucleotide protons and only one intermolecular nuclear Overhauser effect (NOE) is present at the 2D nuclear Overhauser enhancement spectroscopy spectrum. The ¹Η NMR spectrum at ratio 2:1 is characterized by a slight shift for the Δ-isomer’s bpy aromatic protons as well as significant shifts for the decanucleotide G₄ H1′ and Η2″, A₅ H2, G₁₀ H1′, T₆ NH and G₂ NH protons. Furthermore, at ratio 2:1, 11 intermolecular NOEs are observed. The majority of the NOEs involve the sugar Η2′ and Η2″ protons sited in the major groove of the decanucleotide. Increasing the Δ-isomer to d(CGCGATCGCG)₂ ratio to 5:1 results in noteworthy spectral changes. The Δ-isomer’s proton shifts are reduced, whereas significant shifts are observed for the decanucleotide protons, especially the sugar protons, as well as for the exchangeable protons. Interaction is characterized by the presence of only one intermolecular NOE. Furthermore, there is significant broadening of the imino proton signals as the ratio of the Δ-isomer to DΝΑ increases, which is attributed to the opening of the two strands of the duplex. The Λ-isomer, on the other hand, approaches the minor groove of the oligonucleotide and interacts only weakly, possibly by electrostatic interactions. Photocleavage studies were also conducted with the plasmid pUC19 and a 158-bp restriction fragment, showing that both diastereomers cleave DNA with similar efficiency, attacking mainly the guanines of the sequence probably by generating active oxygen species. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. An erratum to this article can be found at     

Theoretical study of hydrogen bond interactions of fluvastatin with <Emphasis Type="BoldItalic">ι</Emphasis>-carrageenan and <Emphasis Type="Italic">λ</Emphasis>-carrageenan

The binding of the reductase inhibitor drug fluvastatin, hydroxy-3-methylglutaryl coenzyme A, with the hydrophilic ι- or λ-carrageenan polymers, serving as potential controllers of the drug’s release rate, have been studied at the density functional level of theory with the B3LYP exchange correlation functional. Three low energy conformers of fluvastatin have been calculated. The vibrational spectroscopic properties calculated for the most stable conformer were in satisfactory agreement with the experimental data. A series of hydrogen bonded complexes of the most stable conformer of fluvastatin anion with low molecular weight models of the polymers have been fully optimized. In almost all, intermolecular H-bonds are formed between the sulfate groups of ι- or λ-carrageenan and fluvastatin’s hydroxyls, resulting in a red shift of the fluvastatin’s O − H stretching vibrations. Cooperative intramolecular H-bonds within fluvastatin or ι-, λ-carrageenan are also present. The BSSE and ZPE corrected interaction energies were estimated in the range 281–318 kJ mol⁻¹ for ι-carrageenan - fluvastatin and 145–200 kJ mol⁻¹ for λ-carrageenan - fluvastatin complexes. The electron density (ρ _bcp) and Laplacian (∇² ρ _bcp) properties at critical points of the intermolecular hydrogen bonds, estimated by AIM (atoms in molecules) calculations, have a low and positive character (∇² ρ _bcp > 0), consistent with the electrostatic character of the hydrogen bonds. The structural and energetic data observed, as well as the extent of the red shift of the fluvastatin’s O − H stretching vibrations upon complex formation and the properties of electron density show a stronger binding of fluvastatin to ι- than to λ-carrageenan.     

Novel deletion of <Emphasis Type="Italic">SLC34A2</Emphasis> in Chinese patients of PAM shares mutation hot spot with fusion gene <Emphasis Type="Italic">SLC34A2</Emphasis>–<Emphasis Type="Italic">ROS1</Emphasis> in lung cancer

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive disorder with distinctive deposition of calcium phosphate microliths in the lungs. Mutation of the SLC34A2 gene was proved to be responsible for PAM. Here, we report the study of a family affected by PAM in China. Two daughters of an inbred family whose parents are cousins and are affected by PAM. Mutation analysis of the SLC34A2 gene by polymerase chain reaction (PCR) amplification and direct sequencing in both patients revealed that exon 5 was deleted on both alleles. Both parents of the patients are proved to be carriers of the mutated allele. Gap-PCR was performed to determine the breakpoints and a homologous deletion of 1152 bp encompassing exon 5 of the SLC34A2 gene (c.IVS4+1452_IVS5+660del) was confirmed. A 4-bp fragment of TGGG was located on the edge of both upstream and downstream breakpoints. The upstream breakpoint lies in the same region as the breakpoint of a fused gene SLC34A2–ROS1, which encodes a constitutive kinase in the lung cancer cell line HCC78 and nonsmall-cell lung cancer (NSCLC), suggesting that the deletion in this family is a hot spot for recombination, not only in cancer samples with somatic mutation, but also in PAM patients with germline genetic defects of SLC34A2.     

Novel peroxidases of <Emphasis Type="Italic">Marasmius scorodonius</Emphasis> degrade <Emphasis Type="Italic">β</Emphasis>-carotene

Two extracellular enzymes (MsP1 and MsP2) capable of efficient β-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).     

Localisation of <Emphasis Type="Italic">Arabidopsis</Emphasis> NDPK2—revisited

Phytochromes are light responsive photoreceptors in plants that influence development and differentiation during the entire plant life cycle. Plant nucleoside diphosphate kinase 2 (NDPK2) has been reported to be a component of the light-mediated signalling cascade and to interact physically with phytochrome A in the cytosol. By using diverse methods as in vitro imports, in vivo localisation of GFP-fusion proteins and immuno blotting of plant cell fractions we clearly localise NDPK2 only to chloroplasts but not to the cytosol, demonstrating that although high affinity protein–protein interactions can occur in vitro, their physiological relevance can be artificial if the proteins are localised to different cell compartments in vivo.     

Differential expression of <Emphasis Type="Italic">Gnrh2</Emphasis>, <Emphasis Type="Italic">Gthβ</Emphasis>, and <Emphasis Type="Italic">Gthr</Emphasis> genes in sterile triploids and fertile tetraploids

Gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GTH), and gonadotropin hormone receptor (GTHR) are the pivotal signal molecules of the hypothalamic-pituitary-gonad (HPG) axis, which plays a crucial role in regulating gonadal development in vertebrate. In this study, we comparatively analyze the expression characteristics of Gnrh2, Gthβ, and Gthr in red crucian carp diploids, triploids, and allotetraploids. The expression patterns of these genes are similar in the three fish ploidy types: the Gnrh2 gene is expressed in midbrains, pituitaries, and gonads; the Gthβ gene is expressed in pituitaries; the Gthr gene is mainly expressed in gonads. These results indicate that the three genes participate in the regulation of gonadal development. By real-time polymerase chain reaction and in situ hybridization, we find that, among these three fish ploidy types, the expression level of Gthr in the gonads of triploids is lower than those of diploids and tetraploids; this weakens the combination of GTHR with GTH released from the pituitary and leads to the sterility of triploids, since the gonad cannot produce enough sex steroids. In addition, the low expression of Gthr in triploids may affect the down-regulation of Gthβ, which then affects the down-regulation of Gnrh2; hence, the expression levels of Gnrh2 and Gthβ genes in triploids are the highest after the breeding season. In conclusion, the differential expression of Gnrh2, Gthβ, and Gthr in triploids and tetraploids is related to their sterility and bisexual fertility, respectively.     

Optimal choice of word length when comparing two Markov sequences using a <Emphasis Type="Italic">χ</Emphasis><Superscript>2</Superscript>-statistic

Background

Alignment-free sequence comparison using counts of word patterns (grams, k-tuples) has become an active research topic due to the large amount of sequence data from the new sequencing technologies. Genome sequences are frequently modelled by Markov chains and the likelihood ratio test or the corresponding approximate χ ²-statistic has been suggested to compare two sequences. However, it is not known how to best choose the word length k in such studies.

Results

We develop an optimal strategy to choose k by maximizing the statistical power of detecting differences between two sequences. Let the orders of the Markov chains for the two sequences be r ₁ and r ₂, respectively. We show through both simulations and theoretical studies that the optimal k= max(r ₁,r ₂)+1 for both long sequences and next generation sequencing (NGS) read data. The orders of the Markov chains may be unknown and several methods have been developed to estimate the orders of Markov chains based on both long sequences and NGS reads. We study the power loss of the statistics when the estimated orders are used. It is shown that the power loss is minimal for some of the estimators of the orders of Markov chains.

Conclusion

Our studies provide guidelines on choosing the optimal word length for the comparison of Markov sequences.

     

Effect of glycosylation on the biochemical properties of <Emphasis Type="Italic">β</Emphasis>-xylosidases from <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R_f. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.