Flavonoids,benzoic acids and cinnamic acids isolated from shoots and roots of Italian rye grass (Lolium multiflorum Lam.) with and without endophyte association and arbuscular mycorrhizal fungus

Phenolic compounds present in Lolium multiflorum Lam. were isolated and characterized. Significant differences in their distribution were found, in shoots and roots of plants with (Lm+) and without (Lm−) endophyte association, grown with (Mic+) and without (Mic−) mycorrhizal fungi Glomus intraradices, indicating a systemic effect of these microorganisms on the phenolics metabolism of L. multiflorum.     

Extreme variation in floral characters and its consequences for pollinator attraction among populations of an Andean cactus

Background and aims

A South American cactus species, Echinopsis ancistrophora (Cactaceae), with dramatic among-population variation in floral traits is presented.

Methods

Eleven populations of E. ancistrophora were studied in their habitats in northern Argentina, and comparisons were made of relevant floral traits such as depth, stigma position, nectar volume and sugar concentration, and anthesis time. Diurnal and nocturnal pollinator assemblages were evaluated for populations with different floral trait combinations.

Key Results

Remarkable geographical variations in floral traits were recorded among the 11 populations throughout the distribution range of E. ancistrophora, with flower lengths ranging from 4·5 to 24·1 cm. Other floral traits associated with pollinator attraction also varied in a population-specific manner, in concert with floral depth. Populations with the shortest flowers showed morning anthesis and those with the longest flowers opened at dusk, whereas those with flowers of intermediate length opened at unusual times (2300–0600 h). Nectar production varied non-linearly with floral length; it was absent to low (population means up to 15 µL) in short- to intermediate-length flowers, but was high (population means up to 170 µL) in the longest tubed flowers. Evidence from light-trapping of moths, pollen carriage on their bodies and moth scale deposition on stigmas suggests that sphingid pollination is prevalent only in the four populations with the longest flowers, in which floral morphological traits and nectar volumes match the classic expectations for the hawkmoth pollination syndrome. All other populations, with flowers 4·5–15 cm long, were pollinated exclusively by solitary bees.

Conclusions

The results suggest incipient differentiation at the population level and local adaptation to either bee or hawkmoth (potentially plus bee) pollination.Key words: Pollination, floral biology, Echinopsis ancistrophora, cactus, Cactaceae, hawkmoth, bee     

RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24^−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24^−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24^−/− mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Structure and electrostatic property of cytoplasmic domain of ZntB transporter

ZntB is the distant homolog of CorA Mg²⁺ transporter within the metal ion transporter superfamily. It was early reported that the ZntB from Salmonella typhimurium facilitated efflux of Zn²⁺ and Cd²⁺, but not Mg²⁺. Here, we report the 1.90 Å crystal structure of the intracellular domain of ZntB from Vibrio parahemolyticus. The domain forms a funnel-shaped homopentamer that is similar to the full-length CorA from Thermatoga maritima, but differs from two previously reported dimeric structures of truncated CorA intracellular domains. However, no Zn²⁺ or Cd²⁺ binding sites were identified in the high-resolution structure. Instead, 25 well-defined Cl⁻ ions were observed and some of these binding sites are highly conserved within the ZntB family. Continuum electrostatics calculations suggest that the central pore of the funnel is highly attractive for cations, especially divalents. The presence of the bound Cl⁻ ions increases the stability of cations along the pore suggesting they could be important in enhancing cation transport.     

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10⁻⁸ M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10⁻⁸ M) and GnRH (0.1 and 1 × 10⁻⁹ M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10⁻⁸ M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10⁻⁸ M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10⁻⁸ M), caused no action when used alone, its co-incubation with GnRH (1 × 10⁻⁹ M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.     

A versatile chiral selector for determination of enantiomeric composition of fluorescent and nonfluorescent chiral molecules using steady-state fluorescence spectroscopy

A fluorescent chiral molecular micelle (FCMM), poly (sodium N-undecanoyl-L-phenylalaninate) (poly-L-SUF), was developed as a chiral selector for enantiomeric recognition and determination of enantiomeric composition of four fluorescent and four nonfluorescent chiral molecules by use of steady-state fluorescence spectroscopy. The influence of FCMM concentration, buffer pH and complexation medium on FCMM-analyte host-guest complexation, and the emission spectral properties of the resulting complexes were investigated. The chiral interactions of the analytes,1,1'-binaphthyl-2,2'-diamine, 1-(9-anthryl)-2,2,2-trifluoroethanol, propranolol, naproxen, chloromethyl menthyl ether (CME), citramalic acid, tartaric acid, and limonene (LIM), in the presence of poly-L-SUF were based on diastereomeric complex formation. The figures of merit obtained from the partial-least-squares regression modeling of the calibration samples suggested good prediction ability for the validation of six of the eight chiral analytes. Better host-guest complexation of the more hydrophobic molecules, CME and LIM, were obtained in methanol/water mixtures, resulting in better predictability of the regression models. Prediction ability of the models was evaluated by use of the root-mean-square percent relative error (RMS%RE) and was found to range from 1.77 to 15.80% (buffer), 1.26 to 7.95% (25:75 methanol/water), and 1.21 to 4.28% (75:25 methanol/water).     

The Flowering Unit in the Synflorescences of Amaranthaceae

The structure of the synflorescence and the flowering units in Amaranthaceae are characterized. The synflorescence is polytelic. In the flowering unit we recognize the main florescence and the enrichment zone. The florescences may consist of: (1) Fully developed partial florescences bearing three or more flowers; (2) Partial florescences reduced to one or a few fertile flowers having prophylls with more or less modified axillary productions; or (3) No partial florescences but solitary flowers having prophylls with no axillary productions. We described the flowering unit in species with florescences bearing a solitary flower and the flowering unit in species with florescences bearing partial florescences. Hypothesized developmental processes are described, with a view to finding relationships among different models characterized in the family as well as defining characters for cladistic studies, which may be useful to depict all the variations observed.     

Cytometric analysis for drug-induced steatosis in HepG2 cells

Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2′,7′-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited the typical forward and side scatter features of live cells. The assay was qualified by analyzing a number of selected model drugs with a well documented induction of steatosis in vivo using different mechanisms as positive controls and several non-steatosic compounds as negative controls. For the cytometric screening assay, the concentrations tested were up to the corresponding IC₁₀ value determined by the MTT assay. Among the parameters analyzed, increased BODIPY fluorescence was the most sensitive and selective marker of drug-induced steatosis. However, a more consistent predictive approach was the combination of two endpoints: lipid accumulation and ROS generation. The assay correctly identified 100% of steatosis-positive and steatosis-negative compounds, and a high steatosis risk was predicted for amiodarone, doxycycline, tetracycline and valproate treatments at therapeutic doses. The results suggest that this cell-based assay may be a useful approach to identify the potential of drug candidates to induce steatosis.     

Loss of ��-Dystroglycan Laminin Binding in Epithelium-derived Cancers Is Caused by Silencing of LARGE

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, β-dystroglycan is expressed, but α-dystroglycan is not detected. Here we report that α-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of α-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with the underlying basement membrane to maintain tissue architecture and function. During cancer progression, primitive cancer cells escape from this control by modifying the binding affinities of their cell membrane receptors. Several receptors have been described as important for this process. Of these, the integrins are the best studied (1). The receptor dystroglycan has been reported to be required for the development and maintenance of epithelial tissues (2, 3). A direct requirement for dystroglycan in epithelia is further demonstrated by the profound effect that loss of dystroglycan expression has on cell polarity and laminin binding in cultured mammary epithelial cells (4, 5). However, dystroglycan is not only important in the establishment and maintenance of epithelial structure. Associations have also been made between the loss of α-dystroglycan immunoreactivity and cancer progression in tumors of epithelial origin, including breast, colon, cervix, and prostate cancers (4, 6–9). The dystroglycan loss of function could thus serve as an effective means by which cancerous cells modify their adhesion to the extracellular matrix (ECM).²Dystroglycan is a ubiquitously expressed cell membrane protein that plays a key function in cellular integrity, linking the intracellular cytoskeleton to the extracellular matrix. The dystroglycan gene encodes a preprotein that is cleaved into two peptides (10). The C-terminal component, known as β-dystroglycan, is embedded within the cell membrane, whereas the N-terminal component, α-dystroglycan, is present within the extracellular periphery but remains associated with β-dystroglycan through non-covalent bonds. β-Dystroglycan binds to actin (11), dystrophin (11), utrophin (11), and Grb2 (12) through its C-terminal intracellular domain. α-Dystroglycan, on the other hand, binds to ECM proteins that contain laminin globular domains including laminins (13, 14), agrin (15), and perlecan (16), as well as to the transmembrane protein neurexin (17). α-Dystroglycan is extensively decorated by three different types of glycan modifications: mucin type O-glycosylation, O-mannosylation, and N-glycosylation. The state of α-dystroglycan glycosylation has been shown to be critical for the ability of the protein to bind to laminin globular domain-containing proteins of the ECM (18).Previous studies of epithelium-derived cancers (4, 9) demonstrated that the loss of immunoreactivity of α-dystroglycan antibodies correlates with tumor grade and poor prognosis. This reduced detection of α-dystroglycan, however, is based on a loss of α-dystroglycan reactivity to antibodies (known as IIH6 and VIA4-1) that recognize the laminin-binding glyco-epitope of α-dystroglycan, i.e. the protein is only functional when it is glycosylated in such a way (henceforth, referred to as functional glycosylation). However, in most of the cancer samples that have been studied to date, β-dystroglycan is expressed at normal levels at the cell membrane. Thus, the aforementioned cancer-associated loss of α-dystroglycan expression may reflect a failure in the post-translational processing of dystroglycan rather than in the synthesis of α-dystroglycan itself.A similar defect in dystroglycan has been reported in a group of congenital muscular dystrophies (19). This spectrum of human developmental syndromes involves the brain, eye, and skeletal muscle and shows a dramatic gradient of phenotypic severity that ranges from the most devastating in Walker-Warburg syndrome to the least severe in limb-girdle muscular dystrophy. Six distinct known and putative glycosyltransferases have been shown to underlie these syndromes: protein O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2 (POMT2), protein O-mannose β-1,2-acetylglucosaminyltransferase 1 (POMGnT1), like acetylglucosaminyltransferase (LARGE), Fukutin, and Fukutin-related protein (FKRP) (20–25). Indeed, all muscular dystrophy patients with mutations in any of these genes fail to express the functionally glycosylated α-dystroglycan epitope that is recognized by the IIH6 and VIA4-1 antibodies.To investigate the molecular mechanism responsible for the loss of α-dystroglycan in epithelium-derived cancers and its role in metastatic progression, we examined the expression and glycosylation status of α-dystroglycan in a group of breast, cervical, and lung cancer cell lines. Here we report that although α-dystroglycan is expressed in the metastatic cell lines MDA-MB-231, HeLa, H1299, and H2030, it is not functionally glycosylated. In screening these cell lines for expression of the six known α-dystroglycan-modifying proteins, we observed that only one, LARGE, was extensively down-regulated. We also report that the ectopic restoration of LARGE expression in these cell lines led not only to the production of a functional dystroglycan but also to the reversion of certain characteristics associated with invasiveness, namely cell attachment to ECM proteins and cell migration.