C-Geranylated flavonoids from Paulownia tomentosa fruits with antimicrobial potential and synergistic activity with antibiotics.

Context C-6-Geranylated flavonoids possess promising biological activities. These substances could be a source of lead compounds for the development of therapeutics. Objective The study was designed to evaluate their antibacterial and antileishmanial activity. Materials and methods C-6-Geranylated flavanones were tested in micromolar concentrations against promastigote forms of Leishmania brazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC50 values (after 72 h) were calculated and compared with that of miltefosine. Flow cytometry and DNA fragmentation analysis were used the mechanism of the effect. Geranylated flavanones or epigallocatechin gallate were combined with oxacillin, tetracycline, and ciprofloxacin, and the effects of these two-component combinations were evaluated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were established (after 24 h), the synergy was measured by the checkerboard titration technique, and the sums of the fractional inhibitory concentrations (∑FICs) were computed. Results 3'-O-Methyl-5'-O-methyldiplacone and 3'-O-methyldiplacone showed good antileishmanial activities (IC50 8-42 µM). 3'-O-Methyl-5'-hydroxydiplacone activates the apoptotic death at leishmanias, the effect of 3'-O-methyl-5'-O-methyldiplacone has another mechanism. The test of the antibacterial activity showed good effects of 3'-O-methyldiplacol and mimulone against MRSA (MIC 2-16 µg/mL), and in six cases, the results showed synergistic effects when combined with oxacillin. Synergistic effects were also found for the combination of epigallocatechin gallate with tetracycline or oxacillin. Conclusion This work demonstrates anti-MRSA and antileishmanial potential of geranylated flavanones and uncovers their promising synergistic activities with antibiotics. In addition, the mechanism of antileishmanial effect is proposed.

Stretching the rules: monocentric chromosomes with multiple centromere domains.

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

Antimicrobial and enzyme inhibitory activities of the constituents of Plectranthus madagascariensis (Pers.) Benth.

Plectranthus madagascariensis is used as a traditional medicine in Southern Africa. In search of compounds and activities supporting the medicinal use, the chemical profile of the methanolic extract was studied by high-performance liquid chromatography with diode array detection (HPLC-DAD). Four major constituents were isolated and identified as rosmarinic acid (1), 7ß,6ß-dihydroxyroyleanone (2), 7ß-acetoxy-6ß-hydroxyroyleanone (3) and coleon U quinone (4). The two abietane diterpenoids (2 and 3) were isolated for the first time from this species. Antimicrobial, cholinesterase and α-glucosidase inhibitory activities of these compounds were studied. The compounds exhibited inhibitory activity on α-glucosidase with IC50 values from 33 to 275 µM. Abietanes showed potent antibacterial activity against Staphylococcus aureus and Enterococcus faecalis.

Survey of extrachromosomal circular DNA derived from plant satellite repeats.

BACKGROUND: Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes. RESULTS: We performed a survey of eccDNA corresponding to nine different families and three subfamilies of satellite repeats in ten species from various genera of higher plants (Arabidopsis, Oryza, Pisum, Secale, Triticum and Vicia). The repeats selected for this study differed in their monomer length, abundance, and chromosomal localization in individual species. Using two-dimensional agarose gel electrophoresis followed by Southern blotting, eccDNA molecules corresponding to all examined satellites were detected. EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size. Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths. CONCLUSION: This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution. The observed size distribution of circular molecules suggests that they are most likely generated by molecular mechanisms based on homologous recombination requiring long stretches of sequence similarity.

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula.

BACKGROUND: Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). RESULTS: Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. CONCLUSION: We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35-48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining.

Significant expansion of Vicia pannonica genome size mediated by amplification of a single type of giant retroelement.

Amplification and eventual elimination of dispersed repeats, especially those of the retroelement origin, account for most of the profound size variability observed among plant genomes. In most higher plants investigated so far, differential accumulation of various families of elements contributes to these differences. Here we report the identification of giant Ty3/gypsy-like retrotransposons from the legume plant Vicia pannonica, which alone make up approximately 38% of the genome of this species. These retrotransposons have structural features of the Ogre elements previously identified in the genomes of pea and Medicago. These features include extreme size (25 kb), the presence of an extra ORF upstream of the gag-pol region, and a putative intron dividing the prot and rt coding sequences. The Ogre elements are evenly dispersed on V. pannonica chromosomes except for terminal regions containing satellite repeats, their individual copies show extraordinary sequence similarity, and at least part of them are transcriptionally active, which suggests their recent amplification. Similar elements were also detected in several other Vicia species but in most cases in significantly lower numbers. However, there was no obvious correlation of the abundance of Ogre sequences with the genome size of these species.

PRINS on plant chromosomes.

The introduction of primed in situ labeling (PRINS) into plant cytogenetics provided a novel means for fast and highly specific visualization of DNA sequences on chromosomes and in interphase nuclei. Although the technique does not reach the sensitivity of fluorescence in situ hybridization that is needed for detection of single-copy targets, it is superior in its speed and simplicity. Thus, the main applications of PRINS include fluorescent labeling of repeated DNA sequences, such as ribosomal DNA and satellite repeats, which are used to discriminate individual chromosome types within karyotypes and to assess the purity of chromosome fractions separated by flow-sorting. Recently, an application of PRINS for the discrimination of sequence subfamilies of satellite repeats has been developed that takes advantage of the sensitivity of Taq polymerase to mismatches between 3'-end of the primer and the template sequences. This approach allows the distinguishing of sequences that differ in only a few nucleotides and has proved to be valuable for studies of satellite DNA evolution in plants.

Minor C-geranylated flavanones from Paulownia tomentosa fruits with MRSA antibacterial activity.

Exhaustive chromatographic separation of the chloroform portion of the ethanolic extract obtained from Paulownia tomentosa (Thunb). Steud. (Paulowniaceae) fruits has led to isolation of ten C-6 geranylated flavanones tomentodiplacone C-I and mimulone C-E, featured by 3'-methoxy and 4'-hydroxy or 4'-hydroxy substitution of the B-ring of the flavonoid, respectively. The structures of these compounds were determined by using mass spectrometry (including HRMS) and 1D and 2D NMR spectroscopy. The absolute configurations of the compounds at C-2 were determined using circular dichroism. The obtained compounds showed the presence of a geranyl moiety functionalized by a carbonyl, hydroxyl or methoxyl group, or by formation of tetrahydrofuran or fused-pyrane ring, respectively. All of the flavanones described were isolated for the first time from a natural source. The antibacterial activities of selected compounds isolated along with the previously isolated geranylated flavanones were evaluated against a common panel of microbes and MRSA strains. The selected isolated compounds were tested for their ability to affect eukaryotic translation initiation via dual-luciferase reporter assay (firefly and renilla).

Plant centromeric retrotransposons: a structural and cytogenetic perspective.

BACKGROUND: The centromeric and pericentromeric regions of plant chromosomes are colonized by Ty3/gypsy retrotransposons, which, on the basis of their reverse transcriptase sequences, form the chromovirus CRM clade. Despite their potential importance for centromere evolution and function, they have remained poorly characterized. In this work, we aimed to carry out a comprehensive survey of CRM clade elements with an emphasis on their diversity, structure, chromosomal distribution and transcriptional activity. RESULTS: We have surveyed a set of 190 CRM elements belonging to 81 different retrotransposon families, derived from 33 host species and falling into 12 plant families. The sequences at the C-terminus of their integrases were unexpectedly heterogeneous, despite the understanding that they are responsible for targeting to the centromere. This variation allowed the division of the CRM clade into the three groups A, B and C, and the members of each differed considerably with respect to their chromosomal distribution. The differences in chromosomal distribution coincided with variation in the integrase C-terminus sequences possessing a putative targeting domain (PTD). A majority of the group A elements possess the CR motif and are concentrated in the centromeric region, while members of group C have the type II chromodomain and are dispersed throughout the genome. Although representatives of the group B lack a PTD of any type, they appeared to be localized preferentially in the centromeres of tested species. All tested elements were found to be transcriptionally active. CONCLUSIONS: Comprehensive analysis of the CRM clade elements showed that genuinely centromeric retrotransposons represent only a fraction of the CRM clade (group A). These centromeric retrotransposons represent an active component of centromeres of a wide range of angiosperm species, implying that they play an important role in plant centromere evolution. In addition, their transcriptional activity is consistent with the notion that the transcription of centromeric retrotransposons has a role in normal centromere function.

Hypervariable 3' UTR region of plant LTR-retrotransposons as a source of novel satellite repeats.

The repetitive sequence PisTR-A has an unusual organization in the pea (Pisum sativum) genome, being present both as short dispersed repeats as well as long arrays of tandemly arranged satellite DNA. Cloning, sequencing and FISH analysis of both PisTR-A variants revealed that the former occurs in the genome embedded within the sequence of Ty3/gypsy-like Ogre elements, whereas the latter forms homogenized arrays of satellite repeats at several genomic loci. The Ogre elements carry the PisTR-A sequences in their 3' untranslated region (UTR) separating the gag-pol region from the 3' LTR. This region was found to be highly variable among pea Ogre elements, and includes a number of other tandem repeats along with or instead of PisTR-A. Bioinformatic analysis of LTR-retrotransposons mined from available plant genomic sequence data revealed that the frequent occurrence of variable tandem repeats within 3' UTRs is a typical feature of the Tat lineage of plant retrotransposons. Comparison of these repeats to known plant satellite sequences uncovered two other instances of satellites with sequence similarity to a Tat-like retrotransposon 3' UTR regions. These observations suggest that some retrotransposons may significantly contribute to satellite DNA evolution by generating a library of short repeat arrays that can subsequently be dispersed through the genome and eventually further amplified and homogenized into novel satellite repeats.

Sequence homogenization and chromosomal localization of VicTR-B satellites differ between closely related Vicia species.

Satellite sequences of the VicTR-B family are specific for the genus Vicia (Leguminosae), but their abundance varies among the species, being the highest in Vicia sativa and Vicia grandiflora. In this study, we have sequenced multiple randomly cloned VicTR-B fragments from these two species and analyzed their sequence variability, periodicity, and chromosomal localization. We have found that V. sativa VicTR-B sequences are homogeneous with respect to their nucleotide sequences and periodicity (monomers of 38 bp), whereas V. grandiflora repeats are considerably more variable, occurring in at least four distinct sequence subfamilies. Although the periodicity of 38 bp was conserved in most of the V. grandiflora sequences, one of the subfamilies was composed of higher-order repeats of 186 bp, which originated from a pentamer of the basic repeated unit. Individual VicTR-B subfamilies were preferentially located in either intercalary or subtelomeric regions of chromosomes. Interestingly, two V. grandiflora subfamilies with the highest similarity to V. sativa VicTR-B sequences were located in intercalary heterochromatic bands, showing similar chromosomal distribution as the majority of VicTR-B repeats in V. sativa. The other two V. grandiflora subfamilies showing a considerable divergence from V. sativa sequences were found to be accumulated at subtelomeric regions of V. grandiflora chromosomes.

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on Vicia sativa chromosomes.

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences.

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.