Analysis of bioactive compounds in seven citrus cultivars.

Chemical content in peels of fruits belonging to cultivars of Clementine mandarin (Fino, Loretina and Marisol), Satsume mandarin (Owari), Navel orange (Navelate and Navelina) and Common orange (Valencia Late) groups have been analyzed. The influence of the variety on the content of bioactive constituents at the varietal level is discussed. Rind contents showed similar tendencies for the majority of compounds although Satsume group presented the highest amounts on the flavanone glycosides hesperidin and narirutin, as well as the highest amounts of the carotenoid and ß-cryptoxanthin, comparing with the remaining varieties studied. Both mandarin and orange varieties studied showed similar tendencies concerning to other phenolic compounds and total ascorbic acid concentrations. Limonene was the most abundant peel essential oil in all cultivars studied, followed by myrcene. Calcium and potassium were the dominant macronutrients for each cultivar studied, although the proportion of the individual nutrients ranged among different cultivars.

Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock Poncirus trifoliata (L.) Raf.

Iron chlorosis is one of the major abiotic stresses affecting fruit trees and other crops in calcareous soils and leads to a reduction in growth and yield. Usual remediation strategies consist of amending iron to soil, which is an expensive practice, or using tolerant cultivars, which are difficult to develop when not available. To understand the mechanisms underlying the associated physiopathy better, and thus develop new strategies to overcome the problems resulting from iron deficiency, the differential gene expression induced by iron deficiency in the susceptible citrus rootstock Poncirus trifoliata (L.) Raf. have been examined. The genes identified are putatively involved in cell wall modification, in determining photosynthesis rate and chlorophyll content, and reducing oxidative stress. Additional studies on cell wall morphology, photosynthesis, and chlorophyll content, as well as peroxidase and catalase activities, support their possible functions in the response to iron deficiency in a susceptible genotype, and the results are discussed.

[Urinary incontinence and other pelvic floor damages: ethilogy and prevention strategies]. / Incontinencia urinaria y otras lesiones del suelo pelviano: etiología y estrategias de prevención.

Urinary incontinence, as well as additional pelvic floor damage, such as third and fourth degree muscular lacerations, as well as fecal incontinence, genital prolapse or dyspareunia, result from obstetric trauma, and are generally linked to the first delivery. The purpose of this study is to analyze, from a physiotherapeutic point of view, and therefore from the perspective of muscular physiology and biomechanics, why this damage occurs, while studying the birth process and the way it is currently performed in most hospitals in our country. Analysis of the birth process and, in short, of the different types of positions used for the first and second stage of labor, as well as of the care provided for women in the puerperium, leads us to propose a global prevention strategy to be carried out in three stages: --Ante-natal prevention: specific preparation of the pelvic floor and abdominal musculature during pregnancy, using massage techniques and manual stretching of the perineum. In addition, the pregnant woman learns these positions and methods of pushing, which makes the first and second stage of labour easier. An osteopathic treatment of the pelvis joints is performed in order to facilitate their mobility or to liberate blockades, if they exist. --Prevention during labour: During this stage, physiology is respected and manual, position-based and breathing techniques are implemented in order to enhance the protection of the baby and of the pelvic floor. --Postpartum prevention: The action is focused on the pelvic floor, through diaphragmatic and abdominal exercises or postures and, if necessary, osteopathic treatment in the early puerperium, in order to facilitate the correct involution of all soft tissues and the pelvic joints involved in labor. Early specific physiotherapeutic treatment will be proposed for women with functional pathology six weeks after delivery.

The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis.

Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation.

Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation.

Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.

oriT-processing and regulatory roles of TrwA protein in plasmid R388 conjugation.

TrwA protein was purified from an overproducing Escherichia coli strain and characterized as a 53 kDa tetrameric DNA-binding protein. Gel shift assays showed that TrwA bound specifically to the oriT sequence of plasmid R388. DNAse I footprinting analysis defined two DNA regions within oriT (sites A and B) that were protected by TrwA. At low TrwA concentrations only region A was protected (K(D) = 4 x 10(-8) M) while region B required higher TrwA concentrations (K(D) = 4 x 10(-7) M). As a result of its binding to oriT, TrwA was found to perform two biochemical activities related to its role in R388 conjugation. First, TrwA binding to oriT resulted in transcriptional repression of the trwABC operon as indicated by its effect on the beta-galactosidase activity of transcriptional fusions in trwB and trwC, and by direct measurement of the trwA mRNA levels by hybridization. This result was further confirmed by the fact that TrwA overexpression resulted in lowered conjugation frequencies. Second, TrwA enhanced the relaxation activity of TrwC in vitro. This effect was correlated to a 10(5)-fold increase in the frequency of conjugation in vivo and was shown to be independent of the regulation of transcription. Thus, TrwA shows functional similarities to protein TraY of F-like plasmids, that could be correlated to a structural similarity in their DNA-binding motifs.

VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1.

During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.

Functional domains in protein TrwC of plasmid R388: dissected DNA strand transferase and DNA helicase activities reconstitute protein function.

TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities. We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate. The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency. Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer. It can be assumed that the covalent linkage increases the efficiency of conjugation by increasing the effective concentration of one component (presumably the DNA helicase) at its site of action.

The beta-tubulin monomer release factor (p14) has homology with a region of the DnaJ protein.

p14 is a molecular chaperone involved in beta-tubulin folding which catalyzes the release of beta-tubulin monomers from intermediate complexes. Here we demonstrate that active p14 protein which we have purified from an overproducing Escherichia coli strain can also release beta-tubulin monomers from tubulin dimers in the presence of an additional cofactor (Z). Analysis of p14 secondary structure suggests that this protein may belong to a family of conserved proteins which share structural similarities with the J-domain of DnaJ. We have constructed deletions and site-directed mutations in the p14 gene. A single D to E mutation in the region shown in DnaJ to be an essential loop for its function affected the monomer-release activity of p14. These results support the hypothesis that this p14 loop interacts with beta-tubulin in a similar fashion as DnaJ interacts with DnaK and suggest a possible role of p14 in the folding process.

Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388.

TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity. Here we report that it also has in vitro oriT-endonuclease activity. TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.

Purification and biochemical characterization of TrwC, the helicase involved in plasmid R388 conjugal DNA transfer.

TrwC is an essential protein in conjugative DNA transfer of the broad-host-range plasmid R388. TrwC was purified in two chromatographic steps from TrwC-overproducing bacteria. The purification procedure resulted in > 90% pure TrwC protein, which was free of contaminating nuclease activities. TrwC behaved as a dimer in gel-filtration chromatography in the presence of 550 mM NaCl, and had a pI of 10.1. The purified protein showed in-vitro ssDNA-dependent nucleoside-5'-triphosphatase and DNA helicase activities. ATP was the preferred substrate for the NTP hydrolysis reaction, which required Mg2+. The helicase activity was dependent on ATP and Mg2+. The efficiency of the unwinding reaction catalyzed by TrwC ranged from > 90% of fragment displaced for a 93-nucleotide sequence to < 5% for a 365-nucleotide sequence. Unwinding was unidirectional in the 5' to 3' direction. The enzyme turned over very slowly from one DNA substrate molecule to another. TrwC is only the second DNA helicase to be described which is involved in conjugative DNA transfer. The biochemical properties of TrwC described here confirm its functional relatedness to helicase I (TraI) encoded by plasmid F of E. coli.

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC.

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.

Genetic organization of the conjugal DNA processing region of the IncW plasmid R388.

The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOBw) has been analyzed by Tn5tac1 insertion mutagenesis, genetic complementation and DNA sequencing. Three genes (trwA, trwB and trwC) were mapped within MOBw. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13.5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases. It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.

Tn5tac1 insertion polarity in Escherichia coli.

The Tn5-derived transposon Tn5tac1 carries the strong tac promoter (Ptac) facing outward at one of its ends. Expression of Ptac is under the control of the lacIq gene, also contained within the transposon. By inserting Tn5tac1 upstream from a promoterless galK gene we determined the basal level of transcription from both ends of the transposon in the absence of IPTG to be about 4% relative to the lactose promoter (Plac). As a result, derivatives of strain N100 containing these plasmids produce red colonies in MacConkey-galactose plates. Deletion of the BamHI fragment including Ptac causes galactokinase levels to drop to less than 1% of Plac, enough to render white colonies in MacConkey-galactose plates. Thus, Tn5tac1 can be used for genetic analysis under conditions in which it shows no polarity (+ IPTG), low polarity (- IPTG), or strong polarity (delta Ptac).

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.