Sucrose and fructan metabolism of different wheat cultivars at chilling temperatures

Four wheat ( Triticum aestivum L.) varieties cultivated in different climates from subtropics to North Patagonia were used to study sucrose and fructan metabolism in plants when submitted to a cold period. Higher levels of sugars were found in the more cold tolerant cultivars. Sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14) activities showed a 2–3 fold increase when plants were grown at 4°C for 10 days. The more cold-tolerant wheat cultivars also showed the higher levels of enzyme activities. These metabolical changes were not due to anatomical or morphological differences produced during growth at 4°C     

Developmental Aspects of Detoxifying Enzymes in Fish (Salmo Iridaeus)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism.

A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development.

In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney.

No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.     

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.     

Liver steatosis in obese prepubertal children: a possible role of insulin resistance

Objective: To determine whether in obese prepubertal children insulin resistance (IR) is associated with the development of liver steatosis. Methods and Procedures: Cross‐sectional study evaluating the prevalence of liver steatosis in 100 severely obese prepubertal children and comparing IR indexes between children with (group 1) and without steatosis (group 2). Furthermore, IR indexes were compared to values of 50 normal weight children. Fasting blood samples were collected for the evaluation of liver function tests, lipid profile, plasma glucose, and insulin levels. All children underwent an oral glucose tolerance test and anthropometric measurements. Hepatic ultrasound was performed according to international criteria and by one single operator. Analysis was performed by Mann–Whitney U‐test, Pearson correlation, and logistic regression. Results: Liver steatosis was found in 52% obese children and was equally distributed between the two sexes. Obese children were more insulin resistant when compared to controls (homeostasis model assessment of IR (HOMA‐IR): P = 0.0001; whole body insulin sensitivity index (WBISI): P = 0.0005; fasting glucose/fasting insulin ratio (G/I): P = 0.0001), and group 1 presented an even higher degree of IR when compared to group 2 (HOMA‐IR P = 0.0001; WBISI P = 0.0004; G/I P = 0.0001). The area under the curve (AUC) for insulin was significantly higher in group 1 when compared to group 2, while no difference was found in the AUC for glucose. There was no association between IR and adiposity indexes (P >0.05). The role of IR as a predictor for the development of steatosis was analyzed by multiple logistic regression, which documented that IR indexes were significantly related to steatosis independently of BMI‐SDS. Discussion: Liver steatosis is an emerging problem in prepubertal severely obese children, and it appears to be an association between liver steatosis and IR in these subjects.     

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.     

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.     

Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and humero-spinal dysostosis

Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.     

Involvement of dopamine receptors and beta-arrestin in metamphetamine-induced inclusions formation in PC12 cells

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of β-adrenergic receptors is promoted by β-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether β-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) β-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of β-arrestin; (iii) dopamine antagonists reduced both MA-induced β-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the β-arrestin dominant negative mutant, βarrV53D and was increased by the persistently ubiquitinated β-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of β-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.