UHPLC-ESI-ORBITRAP-MS analysis of the native Mapuche medicinal plant palo negro (Leptocarpha rivularis DC. – Asteraceae) and evaluation of its antioxidant and cholinesterase inhibitory properties

UHPLC/ESI/MS identification of organic compounds is the first step in the majority of screening techniques for the characterization of biologically active metabolites in natural sources. This paper describes a method for the fast identification and characterisation of secondary metabolites in Leptocarpha rivularis DC. (Palo negro) extracts by HPLC/UV (DAD)–Mass Spectrometry (HPLC/MS). The plant is used for the treatment of several diseases since pre-hispanic Mapuche times. Thirty-seven compounds were detected in the aqueous edible extract for the first time including 4 sesquiterpenes, 10 flavonoids, 9 oxylipins, 2 organic acids, and 11 phenolic acids. In addition, phenolic content antioxidant and cholinesterase inhibitory activities were measured for the first time using the edible infusion. The total polyphenol content of the infusion was 230.76?±?2.5?mmol GAE/kg dry weight, while the antioxidant activity was 176.51?±?28.84; 195.28?±?4.83; and 223.92?±?2.95?mmol TE/kg dry weight, for the DPPH, ABTS, and FRAP assays, respectively. The cholinesterase inhibitory activity was 7.38?±?0.03 and 5.74?±?0.06?mmol GALAE/kg, for the inhibition of acetylcholinesterase AChE and BChE, respectively, showing that this plant is a candidate for the isolation of compounds that can be useful for the treatment of neurodegenerative diseases. Furthermore, this plant could serve also as a raw material for the production of dietary supplements, due to its content of polyphenolic compounds.     

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

β-galactosidases (GUS, EC 3.2.1.23) are character- ized by their ability to hydrolyze terminal, non-re- ducing β-D-galactosyl residues from β-D-galactosides and are widely distributed in microbes, plants and animals. To date, the primary structures of …     

In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.     

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method – plant development and surfactant are important in optimizing transformation efficiency

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.     

Aristolochic acid-induced accumulation of methylglyoxal and Nε-(carboxymethyl)lysine: an important and novel pathway in the pathogenic mechanism for aristolochic acid nephropathy

Aristolochic acid, found in the Aristolochia species, causes aristolochic acid nephropathy (AAN) and can develop into renal failure. Methylglyoxal (MGO) is a highly cytotoxic compound generated from the metabolic process of glucose or fatty acids. It binds to proteins and forms N(ε)-(carboxymethyl)lysine (CML), which contributes to aging and diabetes mellitus complications. However, no relevant literature explores the relationship of MGO and CML with AAN. By injecting AA (10mg/kg BW) into C3H/He mice for 5 consecutive days, we successfully developed an AAN model and observed tubular atrophy with decreased renal function. Creatinine clearance also decreased from 10.32 ± 0.79 ml/min/kg to 2.19 ± 0.29 ml/min/kg (p<0.01). The concentration of MGO in kidney homogenates increased 12 × compared to the control group (from 18.23 ± 8.05 μg/mg of protein to 231.16 ± 17.57 μg/mg of protein, p<0.01), and CML was observed in the renal tubules of the mice by immunohistochemistry. Furthermore, compared to the control group, GSH levels decreased by 0.32 × (from 2.46 ± 0.41 μM/μg of protein to 0.78 ± 0.15 μM/μg of protein, p<0.01), whereas intra-renal antioxidant capacity decreased by 0.54×(from 6.82 ± 0.97 U to 3.71 ± 0.25 U; unit is equivalent to μM Trolox/mg of protein, p<0.01). In this study, we found that serious kidney damage induced by AA is related to an increase and accumulation of MGO and CML.     

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium,Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacteriumAnabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene inEscherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced intoAnabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-³²P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic c~anobacteriumAnabaena sp. PCC 7120.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

Inhibitory mechanism of pure curcumin on Wilms' tumor 1 (WT1) gene expression through the PKCα signaling pathway in leukemic K562 cells

The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells.     

Distal element(s) is(are) required for position-independent expression of the goat α-lactalbumin gene in transgenic mice. Potential relationship with the location of the cyclin T1 locus

We recently reported the site-independent and copy-number-related expression in mice of a goat α-lactalbumin gene with 150 kb and 10 kb of 5''- and 3''-flanking sequences, respectively. In the present study, we observed that the resection of the 5''-flanking region, leaving only 70 kb, resulted in a site-dependent expression of this milk protein-encoding transgene. This suggests that important cis-regulatory elements are located within the distal-deleted sequence. Within this region, we localised the promoter of the cyclin T1 gene, an ubiquitously expressed gene. So far, no other gene has been located between these two loci. Since these two genes are differentially expressed, our data suggest the potential location of an insulator within the deleted region that allows the two genes to be independently regulated.     

Alleviating the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticum – fenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment

Rhizobia-legume symbiosis depends on molecular dialog, which involves the production of specific plant flavonoid compounds as signal molecules. Rhizobium tibeticum was recovered from the root nodule of fenugreek and identified by sequencing the 16S rRNA gene. The effect of salinity stress on nod gene expression was measured in terms of β-galactosidase activity. R. tibeticum containing Escherichia coli lacZ gene fusions to specific nodulation (nod) genes were used to determine β-galactosidase activity. Combination of hesperetin (7.5 µM) and apigenin (7.5 µM) significantly increased β-galactosidase activity more than the single application of hesperetin or apigenin. Preincubation of R. tibeticum with hesperetin and apigenin combination significantly alleviates the adverse effect of salinity on nod gene expression and therefore, enhances nodulation and nitrogen fixation of fenugreek.     

Comparison of the Higher-Severity Fire Regime in Historical (A.D. 1800s) and Modern (A.D. 1984–2009) Montane Forests Across 624,156?ha of the Colorado Front Range

There are concerns that recent fires, following a century of land uses, are burning in dry western forests in an uncharacteristic manner with large patches of higher-severity fire affecting long-term ecosystem dynamics. For example, it is well documented that a mixed-severity fire regime predominated over montane forests of the Colorado Front Range. However, much about the historical fire regime is unknown including the size, frequency, and distribution of higher-severity fires. We addressed these questions utilizing data from the original land surveyors who recorded locations of burned timber along survey lines resulting in a coarse-scale transect of fire occurrence across 624,156?ha. We reconstructed higher-severity burn patches, size distribution, and fire rotation for the 1800s (A.D. 1809–1883) and compared to the characteristics of modern fires over a recent 26-year period (A.D. 1984–2009) taken from remotely sensed data. We found the historical geometric mean higher-severity patch was 170.9?ha and the maximum patch size was 8,331?ha; the higher-severity fire rotation was 248.7?years. In addition, we confirmed that higher-severity fires were historically less common at elevations below 2,200?m. Modern fires had a geometric mean patch size of 90.0?ha (patches >20?ha) and a maximum size of 5,183?ha; the higher-severity fire rotation was 431?years. The distributions of higher-severity patches were only 63.5% similar, as the historical distribution had fewer small patches and more large patches. The mixed-severity fire regime, historically, included a significant portion of higher-severity fire and large burn patches; modern fires appear to be within the range of historical variability.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.