Effective Biotransformation of Ethyl 4-Chloro-3-Oxobutanoate into Ethyl (<Emphasis Type="Italic">S</Emphasis>)-4-Chloro-3-Hydroxybutanoate by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> CCZU-T15 Whole Cells in [ChCl][Gly]–Water Media

To increase the biocatalytic activity of Escherichia coli CCZU-T15 whole cells, choline chloride/glycerol ([ChCl][Gly]) was firstly used as biocompatible solvent for the effective biotransformation of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Furthermore, L-glutamine (150 mM) was added into [ChCl][Gly]–water ([ChCl][Gly] 12.5 vol%, pH 6.5) media instead of NAD⁺ for increasing the biocatalytic efficiency. To further improve the biosynthesis of (S)-CHBE (>99 % e.e.) by E. coli CCZU-T15 whole cells, Tween-80 (7.5 mM) was also added into this reaction media, and (S)-CHBE (>9 % e.e.) could be effectively synthesized from 2000 and 3000 mM COBE in the yields of 100 and 93.0 % by whole cells of recombinant E. coli CCZU-T15, respectively. TEM image indicated that the cell membrane was permeabilized and lost its integrity and when the cell was exposed to [ChCl][Gly]–water media with Tween-80. Clearly, this bioprocess has high potential for the effective biosynthesis of (S)-CHBE (>99 % e.e.).     

Effect of <Emphasis Type="Italic">Rol</Emphasis> Genes on Polyphenols Biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> and Their Effect on Antioxidant and Cytotoxic Potential of the Plant

Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8–5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.     

Structure of the N-acetylpseudaminic acid-containing capsular polysaccharide of Acinetobacter baumannii NIPH67

Capsular polysaccharide (CPS) was isolated from a nosocomial pathogen Acinetobacter baumannii (A. baumannii) NIPH67 and studied by sugar analysis, Smith degradation, and ¹H and ¹³C NMR spectroscopy. The CPS was found to contain 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac), and the structure of the linear trisaccharide repeating unit of the CPS was established as →4)-α-Psep5Ac7Ac-(2→6)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. The genetic content of the capsule biosynthesis cluster of A. baumannii NIPH67, designated KL33, is consistent with the established CPS structure, and thus the capsule of the investigated strain was assigned to K33 group. Functions of proteins including two glycosyltransferases encoded by the genes of the K33 locus were assigned based on the structure of CPS and by the comparison with related proteins of other capsular types of A. baumannii.     

Effect of donor and acceptor properties of solvents on the kinetics of photoreduction of sterically hindered <Emphasis Type="Italic">о</Emphasis>-benzoquinones

Effect of the solvent nature on the kinetics of photoreduction of 3,6-di-tert-butyl-1,2-benzoquinone and its six derivatives in the presence of N,N-dimethylaniline and 4-(N,N-dimethylamino)benzaldehyde has been investigated. It has been found that for the о-quinone—amine pair, for which the free energy change of electron transfer is ΔG_e > +0.11 eV, the rate constant of о-quinone photoreduction k_H decreases proportionally to the increase in the acceptor number of the solvent. For the о-quinone—amine pair with ΔG_e < +0.11 eV, the k_H value decreases proportionally to the increase in the donor number of the solvent. It has been established that the enhancement of the electron-acceptor properties of the solvent leads to the emergence of kinetic isotope effect for the reactant pairs of 3,6-di-tert-butyl-1,2-benzoquinone and 4,5-dimethoxy-3,6-di-tert-butyl-1,2-benzoquinone with N,N-di-methylaniline (ΔG_e = +0.11 and +0.22 eV, respectively).     

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in <Emphasis Type="Italic">Leucaena leucocephala</Emphasis>

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of β-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent K_m, k_cat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na₂S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent k_cat for Cys production is over sixfold higher than mimosine synthesis and the apparent K_m is 3.7 times lower, suggesting Cys synthesis is the favored pathway.     

Trehalose-6-Phosphate as a Potential Lead Candidate for the Development of Tps1 Inhibitors: Insights from the Trehalose Biosynthesis Pathway in Diverse Yeast Species

In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 μM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.     

<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.

     

Alkylation of 2-(methylsulfanyl)-6-polyfluoroalkylpyrimidin-4(3<Emphasis Type="Italic">H</Emphasis>)-ones with haloalkanes

The alkylation of ambident anions of 2-(methylsulfanyl)-6-(polyfluoroalkyl)pyrimidin-4(3H)-ones with 4-bromobutyl acetate leads to concurrent formation of O- and N-(4-acetoxybutyl) derivatives. Polar aprotic solvents favor formation of the O-isomer, and weakly polar dioxane favors N-alkylation. The reaction of 2-(methylsulfanyl)-6-(trifluoromethyl)pyrimidin-4(3H)-one with an equimolar amount of 1,2-dibromoethane in polar acetonitrile gives a mixture of N,N-, O,O-, and N,O-bridged bis-pyrimidines, as well as N- and O-[2-(methylsulfanyl)ethyl] derivatives, whereas in the presence of 10 equiv of 1,2-dibromoethane the N,O-isomer is formed as the only product. The reaction in weakly polar tetrahydrofuran yields N,N- and N,O-bispyrimidines.     

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.

     

Synthesis of pyranopyrazoles,benzopyrans, amino-2-chromenes and dihydropyrano[<Emphasis Type="Italic">c</Emphasis>]chromenes using ionic liquid with dual Brønsted acidic and Lewis basic sites

An efficient ionic liquid with both Brønsted acidic and Lewis basic sites, namely 1,4-dimethyl-1-(4-sulphobutyl)piperazinium hydrogen sulphate (IL1), was synthesised and characterised. IL1 is a “green”, homogeneous and reusable catalyst for: i) the synthesis of pyranopyrazoles (Va-Vj)and benzopyrans (VIa-VIj and VIIa-VIIf) at ambient temperature under solvent-free conditions and ii) the synthesis of amino-2-chromenes (VIIIa-VIIIi and IXa-IXi) and dihyropyrano[c]chromenes (Xa-Xi) at 80 °C under solvent-free conditions. The reactions were rapid with excellent product yields. In addition, the double Brønsted acid, 1,4-dimethyl-1,4-bis(4-sulphobutyl)piperazinium hydrogen sulphate (IL2), was prepared to evaluate the cooperation efficiency of their Brønsted acidic and Lewis basic sites as compared with the double Brønsted acidic sites in IL1.     

Power consumption for an agitated vessel equipped with pitched blade turbine and short baffles

Power characteristics for an agitated vessel equipped with planar short baffles of length L and pitched blade turbine of pitch β are presented in the paper. The studies were carried out in the vessel of inner diameter D = 0.6 m, where the baffles were located in the distance p from the vessel bottom (p + L = H). Torque was measured using strain gauge method within the turbulent regime of the flow of Newtonian liquid in the agitated vessel. The effects of the pitch β and geometrical parameter p/H on the power number Ne were determined mathematically. The results showed that, for the assumed value of the angle β, the function Ne = f (L/H) decreases with the decrease in the baffle length L (i.e. with the increase in the parameter p). Moreover, for the assumed value of the baffle length L, the function Ne = f (β) increases with the increase in the angle β of the inclination of the impeller blade.     

Compositions of nanoparticles of hexagonal symmetry

The formulas for calculation of the number of atoms in nanoparticles with symmetry group D _6h are reported. The numbers of atoms are determined by six structurally invariant numbers and the “quantum number” of the group order n. Eight classes of nanostructures with symmetry group D _6h are revealed: C _{? + 12z} , where z = 0, 1, 2, …, and C _? is C ₂, C ₆, C ₈, or C ₁₄. The sum rule for the coordination numbers of all atoms of subshells related to symmetry elements is established. Two-dimensional nanoparticles are considered.     

The response electron–electron repulsion energy and energy component analysis in CC/MBPT methods

Molecular properties are computed as responses to perturbations (energy derivatives) in coupled-cluster (CC)/many-body perturbation theory (MBPT) models. Here, the CC/MBPT energy derivative with respect to a general two-electron (2-e) perturbation is assembled from gradient theory for 2-e property evaluation, including the electron repulsion energy. The correlation energy (?E) is shown to be the sum of response kinetic (?T), electron–nuclear attraction (?V), and electron repulsion (?V _ee ) energies. Thus, evaluation of total V _ee for energy component analysis is simple: For total energy (E), total 1-e responses T and V, and nuclear–nuclear repulsion energy (V _NN ), V _ee  = E ? V _NN  ? T ? V is the true 2-e response value. Component energy analysis is illustrated in an assessment of steric repulsion in ethane’s rotational barrier. Earlier SCF-based results (Bader et al. in J Am Chem Soc 112:6530, 1990) are corroborated: The higher-energy eclipsed geometry is favored versus staggered in the two repulsion energies (V _NN and V _ee ), while decisively disfavored in electron–nuclear attraction energy (V). Our best quality calculations (CCSD/cc-pVQZ) attain practical Virial Theorem compliance (i.e., agreement among the kinetic energy, potential energy, and total energy representations) in assigning 2.70 ± 0.06 to the barrier height; ?195.80 kcal/mol is assigned to the drop in “steric” repulsion upon going to the eclipsed geometry. Steric repulsion is not responsible for any fraction of the ~3 kcal/mol barrier.     

Method for calculating the sizes of nucleation centers upon homogeneous crystallization from a supercooled liquid

An alternative approach to calculating critical sizes l_k of nucleation centers and work A_k of their formation upon crystallization from a supercooled melt by analyzing the variation in the Gibbs energy during the phase transformation is considered. Unlike the classical variant, it is proposed that the transformation entropy be associated not with melting temperature T_L but with temperature T < T_L at which the nucleation of crystals occurs. New equations for l_k and A_k are derived. Based on the results from calculating these quantities for a series of compounds, it is shown that this approach is unbiased and it is possible to eliminate known conflicts in analyzing these parameters in the classical interpretation.     

Directed Aminomethylation of Pyrrole,Indole, and Carbazole with <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N′</Emphasis>,<Emphasis Type="Italic">N′</Emphasis>-Tetramethylmethanediamine

Catalytic aminomethylation of pyrrole and indole with N,N,N′,N′-tetramethylmethanediamine in the presence of 5 mol % of ZrOCl₂·8H₂O proceeds selectively at the positions 2, 5 of pyrrole and 1, 3 of indole. Carbazole under the same conditions affords 3-formyl-9-aminomethyl derivative. The reaction in the presence of 5 mol % of K₂CO₃ occurs as monoaminomethylation: for pyrrole at the position 2, for indole at the position 3, and for carbazole at the nitrogen atom of the substrate. Water-soluble 1,1′-(1H-pyrrole-2,5-diyl)bis(N,N-dimethylmethanamine) exhibits a fungistatic activity with respect to phytopathogenic fungi Rhizoctonia solani.     

New approaches to the calculation and interpretation of asymmetry factors of chromatographic peaks

The suitability of the determination of the asymmetry factor of chromatographic peaks by the ratio of areas of two components separated by a perpendicular dropped from the maximum of the peak to the base-line, A _s * = S _b /S _a , where symbol a corresponds to the leading edge of the peak and b is for its tailing slope, is discussed. It is demonstrated that this method enables the estimation of the asymmetry of even partially separated chromatographic signals, including those eluted “in the tail” of intense peaks of solvents. The concepts of the asymmetry index I(A _s *) and its increment ΔI(A _s *) = (A _s *)–I(A _s *) are introduced, which ensures the characterization of the asymmetry of peaks of polar analytes with respect to the asymmetry of nonpolar reference components, that is, the separation of the effects of the polarity of analytes and their quantities injected into the chromatographic column on this parameter. For the first time we revealed a correlation of the asymmetry factors of compounds of different chemical nature with such a characteristic of their polarity as the difference in chromatographic separation temperature and the normal boiling point of analytes.     

Biosynthesis,characterization, and antimicrobial effect of silver nanoparticles obtained using <Emphasis Type="Italic">Lavandula</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis>

The use..... of aqueous leaf extract of Lavandula × intermedia for biosynthesis of silver nanoparticles (AgNPs) is presented. The plant extract was obtained by boiling dried leaves and using the obtained filtrate for the synthesis of AgNPs. The study was conducted to investigate an ecofriendly approach to metal nanoparticle synthesis and to evaluate the antimicrobial potential of both the aqueous plant extract and resulting silver nanoparticles against different microbes using the disc diffusion method. The synthesis of silver nanoparticles was monitored using ultraviolet–visible (UV–v is) spectroscopy, which showed a localized surface plasmon resonance band at 411 nm and a shift of the band to higher wavenumber of 422 nm after 90 min of reaction. Powder X-ray diffraction analysis and transmission electron microscopy of the obtained AgNPs revealed their crystalline nature, with average size of 12.6 nm. Presence of elemental silver was further confirmed by energy-dispersive X-ray spectroscopy. Fourier-transform infrared spectroscopy confirmed presence of phytochemicals from Lavandula × intermedia leaf extract on the AgNPs. The AgNPs showed good antimicrobial activity with inhibition zone ranging from 10 to 23 mm; the largest inhibition zone (23 mm) occurred against Escherichia coli. Generally, the AgNPs displayed more antimicrobial activity against all investigated pathogens compared with Lavandula × intermedia leaf extract, and were also more active than streptomycin against Klebsiella oxytoca and E. coli at the same concentration. The silver nanoparticles showed prominent antimicrobial activity with a lowest minimum inhibitory concentration (MIC) value of 15 μg/mL against E. coli, K. oxytoca, and Candida albicans.     

Function Analysis of Caffeoyl-CoA O-Methyltransferase for Biosynthesis of Lignin and Phenolic Acid in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

In this study, we cloned a full-length cDNA and the genomic DNA sequence of SmCCoAOMT (GenBank ID JQ007585) from Salvia miltiorrhiza. The 744-bp open-reading frame encodes a protein of 247 amino acids that shares 95 % similarity with one in Vitis vinifera. Real-time quantitative PCR analysis revealed that SmCCoAOMT is most highly expressed in the stems and can be induced by methyl jasmonate (MeJA) and XC-1 treatment. To evaluate its function in vivo, we generated RNA interference transgenic plants through Agrobacterium tumefaciens-mediated gene transfer. Compared with untransformed control plants, the transgenics had significantly less lignin and the expression of lignin-biosynthetic genes SmCCR and SmCOMT was depressed. In 90-day-old roots from plants of transgenic line M5, accumulations of rosmarinic acid and salvianolic acid B (Sal B) were greatly reduced by 0.89- and 0.69-fold, respectively. This low-Sal B phenotype was stable in the roots, with the level of accumulation being approximately 43.58 mg g^?1 dry weight, which was 52 % of the amount measured in the untransformed control. Our results suggest that SmCCoAOMT is involved in lignin biosynthesis and affects the accumulation of phenolic acids. This study also provides potential guidance for using lignin-related genes to genetically engineer Salvia miltiorrhiza.