Synthesis of functionalized benzo[<Emphasis Type="Italic">f</Emphasis>]2<Emphasis Type="Italic">H</Emphasis>-chromenes and evaluation of their antimicrobial activities

Knoevenagel cyclocondensations of α-hydroxy naphthaldehyde with β-oxodithioesters and ketene dithioacetals yielded 2H-benzo[f]chromene-2-thiones and 2H-benzo[f]chromen-2-ones, respectively, in high yields. The newly synthesized compounds were evaluated for antifungal and antibacterial activities. Among them, compounds (2-furyl)(3-thioxo-3H-benzo[f]chromen-2-yl)methanone and phenyl(3-oxo-3H-benzo[f]chromen-2-yl)methanone exhibited excellent antifungal activity against tested fungi Curvularia lunata and Fusarium moniliforme. The highest antibacterial activity against the tested bacteria Escherichia coli and Staphylococcus aureus was observed for (4-chlorophenyl)(3-oxo-3H-benzo[f]chromen-2-yl)methanone. The results of antimicrobial screening demonstrate that (2-furyl)(3-thioxo-3H-benzo[f]chromen-2-yl)methanone, phenyl(3-oxo-3H-benzo[f]chromen-2-yl)methanone, and (4-chlorophenyl)(3-oxo-3H-benzo[f]chromen-2-yl)methanone are promising as antimicrobial drugs.     

Synthesis and Anti-Infective Activity of 2-Heteroaryl(Acyl)-9,10,12,13-Tetramethoxy-3-Methyl-4,5-Dihydro-3<Emphasis Type="Italic">H</Emphasis>-Phenanthro[1,2-<Emphasis Type="Italic">d</Emphasis>]Azepines

2-Hetaryl- and 2-acyl-9,10,12,13-tetramethoxy-3-methyl-4,5-dihydro-3H-phenanthro[1,2-d]-azepines have been synthesized from 8,9,11,12-tetramethoxy-2-methyl-3,4-dihydronaphtho[2,1-f]isoquinolinium perchlorate by pyridine-azepine recyclization. The resulting compounds have revealed a pronounced antibacterial activity against Staphylococcus aureus (strain P-209) and Escherichia coli (field strain 078). Some of these compounds have a moderate fungistatic activity against Peniciliium italicum fungi. Two compounds have shown a certain activity in relation to the Colpoda steinii protozoa.     

Synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidines

The synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-d]pyrimidine derivatives are described. The title compounds were obtained by the reaction of 1H-benzo[f]chromenes with aliphatic and aromatic amines. The structures of all newly synthesized compounds were confirmed by IR, ¹HNMR, ¹³C NMR, and NOESY experiments. The compounds exhibited potent antibacterial activity against gram-positive and gram-negative bacterial species. 10-Methyl-12-(4-hydroxyphenyl)-10,12-dihydro-11H-benzo[5,6]chromeno[2,3-d] pyrimidin-11-imine displayed greater antibacterial activity against gramnegative bacterial species than did ciprofloxacinandamoxicillin.     

Imidazo[4,5-<Emphasis Type="Italic">a</Emphasis>]quinindolines as highly effective antibacterial agents

Resistance to antimicrobial agents is a concern that exists globally and has a considerable impact on human and animal health, so that the discovery of new antibacterial compounds has become increasingly more important in combating infectious disease. In this paper, imidazo[4,5-a]quinindolines are introduced as new antibacterial agents against Gram-positive and Gram-negative bacteria. These pentacyclic compounds are synthesized by the reaction of N-alkyl-5-nitrobenzimidazoles with 2-(1-alkyl-1H-3-indolyl)acetonitrile under basic conditions in excellent yields. The structures of newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, and mass spectral data. The antibacterial activities of the synthesized compounds were screened against standard strains of two Gram-positive and two Gram-negative bacteria using the broth microdilution method. Most of the compounds studied showed promising activities against both types of bacteria.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

Multiple defaults: feminine -<Emphasis Type="Italic">et</Emphasis> and -<Emphasis Type="Italic">a</Emphasis> in Hebrew present tense

In this paper, we study the distribution of the feminine singular suffixes -et and -a in the present tense of Hebrew verb paradigms. The question we ask is which of these two suffixes is the default allomorph. The answer is not trivial. In terms of distribution, -a appears with limited environments and -et is clearly the elsewhere case, and thus the default. In terms of order, however, -et is the feminine singular suffix associated with the present tense, while -a emerges only when -et is blocked. We thus argue for multiple defaults, distinguishing between local and global default; -et is the local default, uniquely associated with feminine singular verbs in the present tense, while -a is the global default, associated with feminine singular but not specified for the present tense. We provide a formal analysis for the distribution of these suffixes within the framework of Optimality Theory, which allows the interaction of phonological constraints with constraints on morpho-syntactic feature mapping. We further study the partially unpredictable distribution of -et and -a in vowel final verbs, and present the results of an experiment where speakers employed unique strategies in order to assign the local default -et.     

Mechanisms of thaxtomin A-induced root toxicity revealed by a thaxtomin A sensitive <Emphasis Type="Italic">Arabidopsis</Emphasis> mutant (<Emphasis Type="Italic">ucu2</Emphasis>-<Emphasis Type="Italic">2/gi</Emphasis>-<Emphasis Type="Italic">2</Emphasis>)

Key message

The Arabidopsis mutant ( ucu2 - 2/gi - 2 ) is thaxtomin A, isoxaben and NPA-sensitive indicated by root growth and ion flux responses providing new insights into these compounds mode of action and interactions.

Abstract

Thaxtomin A (TA) is a cellulose biosynthetic inhibitor (CBI) that promotes plant cell hypertrophy and cell death. Electrophysiological analysis of steady-state K⁺ and Ca²⁺ fluxes in Arabidopsis thaliana roots pretreated with TA for 24 h indicated a disturbance in the regulation of ion movement across the plant cell membrane. The observed inability to control solute movement, recorded in rapidly growing meristematic and elongation root zones, may partly explain typical root toxicity responses to TA treatment. Of note, the TA-sensitive mutant (ucu2-2/gi-2) was more susceptible with K⁺ and Ca²⁺ fluxes altered between 1.3 and eightfold compared to the wild-type control where fluxes altered between 1.2 and threefold. Root growth inhibition assays showed that the ucu2-2/gi-2 mutant had an increased sensitivity to the auxin 2,4-D, but not IAA or NAA; it also had increased sensitivity to the auxin efflux transport inhibitor, 1-naphthylphthalamic acid (NPA), but not 2,3,5- Triiodobenzoic acid (TIBA), when compared to the WT. The NPA sensitivity data were supported by electrophysiological analysis of H⁺ fluxes in the mature (but not elongation) root zone. Increased sensitivity to the CBI, isoxaben (IXB), but not dichlobenil was recorded. Increased sensitivity to both TA and IXB corresponded with higher levels of accumulation of these toxins in the root tissue, compared to the WT. Further root growth inhibition assays showed no altered sensitivity of ucu2-2/gi-2 to two other plant pathogen toxins, alternariol and fusaric acid. Identification of a TA-sensitive Arabidopsis mutant provides further insight into how this CBI toxin interacts with plant cells.

     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Rnq1 protein protects [<Emphasis Type="Italic">PSI</Emphasis><Superscript>+</Superscript>] prion from effect of the <Emphasis Type="Italic">PNM</Emphasis> mutation

The interaction of [PSI ⁺] and [PIN ⁺] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [PIN ⁺] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [PSI ⁺] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [PSI ⁺] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [PSI ⁺].     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

Characterization of an IAA-glucose hydrolase gene <Emphasis Type="Italic">TaTGW6</Emphasis> associated with grain weight in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In rice, the TGW6 gene determines grain weight and encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. Its homolog in wheat, TaTGW6, is considered as a candidate gene related to grain development. To amplify this gene, we designed primers based on a homologous conserved domain of the rice TGW6 gene. Sequence analysis indicated that TaTGW6 comprises only one exon, with 1656 bp in total and an open reading frame of 1035 bp. Three alleles at TaTGW6 locus detected by the primer pair TG23 were designated as TaTGW6-a, TaTGW6-b and TaTGW6-c, respectively. Compared with TaTGW6-a, TaTGW6-b had a 6-bp InDel at the position 170 downstream of initiation codon, and TaTGW6-c was a null mutant. Both TaTGW6-b and TaTGW6-c could significantly increase grain size and weight other than TaTGW6-a; however, the former two alleles showed a low frequency distribution in modern varieties. TaTGW6 was located on chromosome 4AL using a recombinant inbred line population and a set of Chinese Spring nullisomic-tetrasomic lines. It was linked to the SSR locus Xbarc1047 with a genetic distance of 6.62 cM and explained 15.8–21.0 % of phenotypic variation of grain weight in four environments. Association analysis using a natural population and Chinese wheat mini-core collections additionally validated the relationship of TaTGW6 with grain weight; the gene could explain 7.7–12.4 % of phenotypic variation in three environments. Quantitative real-time PCR revealed that TaTGW6-b showed relatively lower expression than TaTGW6-a in immature grain at 20 and 30 days post-anthesis and in mature grain. The low expression of TaTGW6 generally associated with low IAA content, but with high grain weight. The novel functional marker, designated as TG23, can be used for marker-assisted selection to improve grain weight in wheat and also provides insights into the regulatory mechanism underlying grain weight.     

The fitness of the cotton mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis> (Tinsley) is reduced after feeding on virus-infected <Emphasis Type="Italic">Hibiscus rosa</Emphasis>-<Emphasis Type="Italic">sinensis</Emphasis>

The cotton mealybug Phenacoccus solenopsis (Tinsley) and Cotton leaf curl Multan virus (CLCuMV), serious threats to economic crops and garden plants, have invaded southern China and widely infected Hibiscus rosa-sinensis. Whether an inter-species connection has facilitated the invasion process is unclear. In this study the interaction between P. solenopsis and H. rosa-sinensis infected with CLCuMV was investigated in the laboratory. We observed that 1st and 2nd instar nymphs of P. solenopsis preferred to feed on healthy H. rosa-sinensis leaves, whereas 3rd instar nymphs and female adults had no preference between healthy and virus-infected H. rosa-sinensis leaves. The developmental time of each P. solenopsis developmental stage increased significantly after feeding on infected H. rosa-sinensis leaves (p < 0.05). In particular, the development time for 2nd instar female and male nymphs and 3rd instar female nymphs increased by approximately twofold. The generation time of female mealybugs increased from 25.84 d on healthy H. rosa-sinensis to 32.12 d when feeding on CLCuMV-infected H. rosa-sinensis, and the survival rate decreased from 71.04 % on healthy H. rosa-sinensis to 5.80 % on infected plants. Nymph survival was most affected by feeding on infected plants. Additionally, the fecundity of female mealybugs feeding on infected H. rosa-sinensis decreased by 47.8 %. Thus, feeding on CLCuMV-infected H. rosa-sinensis significantly decreased the biological fitness and invading and colonizing abilities of P. solenopsis.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.