Ghrelin in plants: what is the function of an appetite hormone in plants?

In the present work, we provide compelling evidence for the expression of a ghrelin-like peptide hormone that has only been associated with animals, in various plant tissues. Ghrelin, the appetite stimulating hormone, has been identified from a number of different species including humans, rat, pig, mouse, gerbil, eel, goldfish, bullfrog and chicken. The study here was conducted using an immunohistochemistry assay to screen whether plants have any ghrelin immunoreactivity. In this respect, Prunus x domestica L. and Marus alba were examined. Immunohistochemistry results showed that there is a strong human ghrelin immunoreactivity substance in the parenchyma cells of these plants. This was entirely unexpected since this hormone was considered to be present solely in animals. Thus, this study is the first to report the presence of a peptide with ghrelin-like activity in plants, a finding that has only been observed in the animal kingdom. RIA analysis confirmed that these plants contain significant amounts of this substance. Furthermore, reverse-phase HPLC analyses of plant extracts showed an elution characteristic of the peptide identical to that of human ghrelin. In general, fruit from both plants had higher levels of the peptide than the vegetative parts.     

Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates

A bioanalytical assay for pravastatin and two isomeric metabolites, 3′α-isopravastatin and 6′-epipravastatin, was developed and validated. Mouse plasma and tissue homogenates from liver, kidney, brain and heart were pre-treated using protein precipitation with acetonitrile containing deuterated internal standards of the analytes. The extract was diluted with water and injected into the chromatographic system. This system consisted of a polar embedded octadecyl silica column using isocratic elution with formic acid in a water–acetonitrile mixture. The eluate was transferred to an electrospray interface using negative ionization and the analytes were detected and quantified with the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was successfully validated in a 3.4–7100 ng/ml concentration range for pravastatin, 1.3–2200 ng/ml for 3′α-isopravastatin and 0.5–215 ng/ml for 6′-epipravastatin using only plasma for calibration. For plasma samples, subjected to full validation, within and between day precisions were 1–7% (9–18% at the LLQ level) and accuracies were between 91% and 103%. For tissue homogenates, subjected to partial validation, within and between day precisions were 2–12% (6–19% at the LLQ level) and accuracies were between 87% and 113% (81 and 113% at the LLQ level). Drug and metabolites were shown to be chemically stable under most relevant analytical conditions. Finally, the assay was successfully applied for a pilot study in mice. After intravenous administration of the drug, all isomeric compounds were found in plasma; however, in liver and kidney homogenate only the parent drug showed levels exceeding the LLQ.     

Proteomic response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to the alkaloid extract of <Emphasis Type="Italic">Papaver polychaetum</Emphasis>

The cellular response of Escherichia coli exposed to alkaloids extracted from a biennial endemic plant, Papaver polychaetum, was explored using proteome analysis. Following determination of the minimum inhibitory concentration of the berberine-containing plant extract as 1,250 μg/mL, E. coli cells were grown in the presence of 750 μg/mL extract. The response of the bacteria to the extract, with berberine found as the major alkaloid, was analyzed on two-dimensional gels. The differentially expressed proteins in the presence of 750 μg/mL extract were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These proteins included those that play vital roles for maintenance such as protein synthesis (elongation factor-Ts), transport (oligopeptide-binding protein A, uncharacterized amino-acid ABC transporter ATP binding protein YECC), energy metabolism (alpha-subunit of ATP synthase, pyridine nucleotide transhydrogenase STHA) and regulation. These results provide clues for understanding the mechanism of the alkaloid extract-induced stress and cytotoxicity on E. coli. The altered proteins can serve as potential targets for development of innovative therapeutic agents.     

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and {alpha}5{beta}1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.     

The evaluation of pre-grown mycelial pellets in decolorization of textile dyes during repeated batch process

This study was undertaken for the possibility of application of pre-grown pellets for biotechnological treatment of dyes and textile industry waste waters. Mycelial pellets of five different white rot fungi were tested for their dye decolorization activity. The pellets of Funalia trogii, Phanerochaete chrysosporium and Trametes versicolor were determined as the most effective ones. The decolorization ability of viable pellets was compared with the decolorization (adsorption) ability of dead pellets during repeated batch studies. Astrazon Black dye was decolorized effectively, about 90%, by viable pellets of all fungi during the first use. Viable F. trogii pellets were found as the most effective pellets. Upon pellet treatment not only a high decolorization but also reduced toxicity (antimicrobial activity) of the Astrazon Black dye was recorded. This type of decolorization activity with commercial or crude laccase was partially observed. Growing cells of F. trogii in batch system showed lower efficiency in color removal of mixed dyes compared to the pre-grown pellets in repeated batch system. The results in this study showed that mycelial pellets could effectively be used as an alternative to traditional physicochemical processes.     

A novel mutation in a large family causes a unique phenotype of Mucolipidosis IV

Mucolipidosis type IV is a rare autosomal recessive lysosomal storage disorder reported among Ashkenazi Jews and to a lesser extent in other ethnic groups. Several mutations have been reported in MCOLN1 which is the only known gene associated with the disorder. Here we report the first Saudi patient with Mucolipidosis type IV from a consanguineous family with two branches having a total of five patients carrying a novel transition mutation, c.1307A > G (p.Y436C) in exon 11. The clinical course of the patient was nonspecific and a lysosomal storage disorder was not highly suspected due to lack of coarse facial features, organomegaly and skeletal findings of dysostosis multiplex. The detailed bioinformatics analysis on the deleterious effects of the mutation is discussed. Emphasis is made on the importance of brain magnetic resonance imaging (MRI) findings and serum gastrin level as key clues to the diagnosis of this often subtle neurodevelopmental disorder.     

Pyranose 2-oxidase (P2O): Production from Trametes versicolor in Stirred Tank Reactor andits Partial Characterization

Abstract

Optimization of pyranose-2-oxidase (P2O) production conditions from Trametes versicolor was carried out in shaking cultures containing glucose, malt, and yeast extracts; the optimum concentration values were found to be 1.5% glucose, 1.0% yeast extract, and 1.0% malt extract, pH 5.0, temperature, 26°C, and agitation rate 150 rpm. For the first time, P2O production was also carried out in a stirred tank reactor (STR) with 2.2 L working volume in the optimized medium composition, and biomass, P2O activity, protein, nitrogen and glucose concentrations were also monitored besides pH and dissolved oxygen (DO). In the STR, P2O activity peaked on day 9. Partial enzyme characterization occurred and optimum pH and temperature were detected as 7.0 and 37°C, respectively. K _m value was found to be 1.009 mM.     

A new set up for multi-analyte sensing: at-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO?-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO?-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO?-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.