HSP70 level in the leaves of Phaseolus vulgaris intoxicated with cadmium.

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Constitutively elevated blood serotonin is associated with bone loss and type 2 diabetes in rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin

Expression analysis of buckwheat (Fagopyrum esculentum Moench) metallothionein-like gene (MT3) under different stress and physiological conditions

The buckwheat metallothionein-like (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. MT3 mRNAs were detected from the early stage of seed development to the end of maturation, reaching the highest level during the mid-maturation stage. High MT3 mRNA level was noticed for both green and senescent leaves. The influence of raising Cu ion concentrations on MT3 gene expression was studied only in leaves, while the effect of Zn ions was analyzed through seed development as well. It was found that Cu and Zn ions had stimulatory effects on expression in leaves. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions, while after this stage, influence of Zn ions was not detected. After H2O2/NaCl treatment, MT3 mRNA level was decreased in green leaves, contrary to senescent leaves where expression levels remained unchanged. H2O2 treatment caused the increase of MT3 mRNA levels in the mid-maturation stage of seed development. NaCl had no effect on expression levels in seeds. According to obtained results, proposed functions in different plant organs regarding oxidative stress and metal homeostasis are discussed

Long-term imipramine treatment affects rat brain and pituitary corticosteroid receptors and heat shock proteins levels in a gender-specific manner

Gender-related differences in the effects of imipramine, on the protein levels of glucocorticoid receptor (GR), and heat shock proteins Hsp90 and Hsp70, as well as on dexamethasone binding to corticosteroid receptors (CRs) in the pituitary, hypothalamus, hippocampus and brain cortex of non-depressed rats were studied. Differences between female and male animals in the GR protein level in the tissues of untreated animals were not noticed. However, imipramine led to opposite changes in the cellular level of GR protein in the brain of female and male rats, as well as to gender- and tissue-specific changes in in vitro dexamethasone binding to GR and mineralocorticoid receptor (MR) in the hippocampus and brain cortex. Gender-related differences in the expression of Hsp90 and Hsp70 were noticed mainly in the hippocampus, only after imipramine treatment. The observed changes in the response of GR to imipramine suggest that this antidepressant may affect both the level of the receptor protein and the mechanisms regulating its binding ability in a gender-related manner.nul

Gender-related differences in the effects of antidepressant imipramine on glucocorticoid receptor binding properties and association with heat shock proteins in the rat liver and kidney

Gender-related differences in susceptibility to stress and stress-related disorders such as depression, and in response to treatment with antidepressants have been observed, but the underlying molecular mechanisms are still unknown. Considering the role of glucocorticoid hormones in the systemic reaction against stress and in pathogenesis of depression, the aim of the present work was to study gender-related differences in glucocorticoid signaling and in response of this system to a typical antidepressant drug, imipramine. Gender-related differences in glucocorticoid receptor functional properties were assessed using hepatic and renal whole cell extracts of female and male rats before and after long-term imipramine treatment. The receptor's hormone-binding parameters, B(max) and K(D), were determined by radioligand binding assay, the glucocorticoid receptor and heat shock proteins (Hsp70 and Hsp90) levels by quantitative immunoblotting, and the interaction of these proteins within glucocorticoid receptor heterocomplex by co-immunoprecipitation. Glucocorticoid receptor binding potency (B(max)/K(D) ratio) was significantly higher in males than females both before and after treatment with imipramine. Gender-specific changes in the glucocorticoid receptor binding parameters in the examined tissues were observed in response to imipramine, and were found to be associated with alterations in the receptor interaction with Hsp70 and Hsp90. The results of the study point to sexual dimorphism in the glucocorticoid signaling and imply that glucocorticoid receptor functional alterations contribute to gender-related differences in vulnerability to stress and stress-related disorders, and in response to antidepressant drugs. (c) 2009 Elsevier B.V. All rights reserved.Ministry of Science of the Republic of Serbia [143003

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms

PCR detection of genetically modified soy bean and maize in food/feed stuffs

Potencijalni štetni efekti koje na zdravlje ljudi i životinja može imati upotreba hrane poreklom od genetički modifikovanih organizama (GMO), kao i eventualni neželjeni efekti koje gajenje genetički modifikovanih biljaka može imati na životnu sredinu, doveli su do potrebe da se u EU strogo ograniči distribucija GMO. Aktima Komisije EU, 49/2000 i 50/2000, propisano je da se prehrambeni artikli proizvedeni od GMO ili koji sadrže GMO, moraju jasno obeležiti. Ovoj obavezi ne podležu oni proizvodi koji sadrže manje od 1% genetički modifikovanog materijala. Prisustvo GMO u hrani za humanu i animalnu upotrebu može biti ustanovljeno bilo detekcijom DNK kojom je urađena modifikacija (PCR metode), bilo detekcijom proteina kodiranog ovom DNK (imunološke metode). U Institutu za molekularnu genetiku i genetičko inženjerstvo standardizovane su kvalitativna i kvantitativna metoda zasnovane na tehnici PCR. Da li uzorak sadrži GMO ili ne određuje se detektovanjem CaMV35S promotora, dok se za detektovanje specifičnih genetičkih modifikacija u RoundUp Ready soji tolerantnoj na herbicid RoundUp i Bt kukuruzu otpornom na insekte, koriste prajmeri dizajnirani iz odgovarajućih specifičnih transgena - gena EPSPS iz A. tumefaciens (za soju) i gena CrylA iz B. thuringiensis (za kukuruz). Eksperimentalno utvrdjena granica osetljivosti metoda je 1pg DNK, što omogućava detektovanje 0,01% GM DNK u testiranom uzorku i odgovara kriterijumima evropskih laboratorija koje su ovlašćene da izdaju sertifikat.Public concern about possible negative health effects due to consumption of GM plants, together with concerns about possible environmental dangers, have led the European Commission to limit strictly the distribution of GM plants in the EU. The EU regulations No 49 and 50/2000 require the food industry to label, as GMO containing, those food products not certified free of transgenic plant material. Foods are considered "clean" and may be left unlabeled, if the GM content does not exceed 1%. The presence of GM in food and feed products can be evaluated by detecting the transgenic DNA (PCR technique), or by detecting proteins derived from this DNA (immunological methods). At the Institute of Molecular Genetics and Genetic Engineering both qualitative and quantitative PCR-based tests are standardized. Commonly used primers are designed from the CaMV35S promoter for the answer yes/no in general GMO screening procedure. Specific modifications, as in herbicide tolerant RoundUp Ready Soybean or insect resistant maize (Bt176 corn), are identified by primers designed from specific genes e.g. the EPSPS - gene from A.tumefaciens (for soybean) and the CrylA gene from B.thuringiensis (for maize). The experimentally determined detection limit is 1pg, which allows detection of O,01% GM DNA in test-samples and meets criteria prescribed for the EU laboratories accredited to issue a certificate

Cloning and computer analysis of the promoter region of the legumin-like storage protein gene from buckwheat, Fagopyrum esculentum Moench

Using the modified 5’-RACE approach, a fragment containing the 955 bp long 5’- regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced and the sequence analyzed by computer prediction of cis-regulatory elements possibly involved in tissue specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research, but also as a useful tool for biotechnological application