Polybrominated diphenyl ethers enhance the production of proinflammatory cytokines by the placenta.

Polybrominated diphenyl ether(s) (PBDE) are ubiquitous environmental contaminants that bind and cross the placenta but their effects on pregnancy outcome are unclear. It is possible that environmental contaminants increase the risk of inflammation-mediated pregnancy complications such as preterm birth by promoting a proinflammatory environment at the maternal-fetal interface. We hypothesized that PBDE would reduce IL-10 production and enhance the production of proinflammatory cytokines associated with preterm labor/birth by placental explants. Second-trimester placental explants were cultured in either vehicle (control) or 2 µM PBDE mixture of congers 47, 99 and 100 for 72 h. Cultures were then stimulated with 10(6) CFU/ml heat-killed Escherichia coli for a final 24 h incubation and conditioned medium was harvested for quantification of cytokines and PGE(2). COX-2 content and viability of the treated tissues were then quantified by tissue ELISA and MTT reduction activity, respectively. PBDE pre-treatment reduced E. coli-stimulated IL-10 production and significantly increased E. coli-stimulated IL-1ß secretion. PBDE exposure also increased basal and bacteria-stimulated COX-2 expression. Basal, but not bacteria-stimulated PGE(2), was also enhanced by PBDE exposure. No effect of PBDE on viability of the explants cultures was detected. In summary, pre-exposure of placental explants to congers 47, 99, and 100 enhanced the placental proinflammatory response to infection. This may increase the risk of infection-mediated preterm birth by lowering the threshold for bacteria to stimulate a proinflammatory response(s).

Actin is oxidized during myocardial ischemia.

Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p <.05) in actin content of carbonyl groups, which was associated with significant depression (p <.05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins.

Association of increased ubiquitinated proteins with cardiac apoptosis.

Intracellular proteases play an important role in the regulation of apoptosis. A study was performed to determine whether inhibition of the cardiac ATP-dependent ubiquitin 26S protease complex affects cardiomyocyte apoptosis. Isolated rat hearts were perfused for up to 80 min with Krebs-Henseleit buffer +/- the 26S-proteasome inhibitor, MG132 (Z-leu-leu-leucinal). TUNEL-staining of hearts perfused with 25 microM MG132 for 50 min revealed a significant increase (p < 0.05) in the apoptotic index from 1.1% to 15.5% when compared with control hearts perfused with buffer only. Histology of adjacent myocardial sections revealed no signs of necrotic or late apoptotic (nuclear condensation) changes, indicating that the TUNEL-positive nuclei were in the early stages of apoptosis. This early stage of apoptosis was associated with a significant (p < 0.05) reduction in cardiac function. There was a 63% decrease in the rate pressure product in hearts perfused with 25 microM MG132 as compared with a 35% decrease in control hearts over the 80-min perfusion period. Soluble ubiquitin-conjugated proteins, as detected by probing with a specific antibody to ubiquitin, were increased in MG132-treated hearts. In hearts perfused with 50 microM MG132, a greater accumulation of ubiquinated proteins was observed accompanied by a more rapid and greater reduction in hemodynamic function. These observations indicate that prolonged inhibition of the ubiquitin-26S-proteasome results in cardiomyocyte apoptosis accompanied by increased ubiquinated proteins, thus suggesting that accumulation of these abnormal proteins may act as a signal to activate the cell death program.

Promotion of copper excretion from the isolated rat heart attenuates postischemic cardiac oxidative injury.

This study examined the role of Cu as a mediator of cardiac postischemic oxidative injury. Isolated rat hearts were subjected to 20 min of normothermic global ischemia, followed by 30 min of reperfusion; after 20 min of preischemic loading with Krebs-Henseleit buffer +/- 20 or 30 microM zinc-bis-histidinate (Zn-His2), 0.5 mM deferoxamine (DEF) or 42 microM neocuproine (NEO). Postischemic developed systolic pressure and rate-pressure product were highest and postischemic end-diastolic pressure was lowest in hearts treated with 20 or 30 microM Zn-His2 and 0.5 mM DEF. Cu efflux was significantly increased by 225 and 290% (end of preischemic loading), and 325 and 375% (immediate postischemic period) of control basal rates in hearts treated with 30 microM Zn-His2 and 0.5 mM DEF, respectively. NEO did not effect any of these parameters. By the end of ischemia, protein carbonyls were lowest in Zn-His2-treated hearts and highest in DEF-treated hearts when compared with control hearts. The results of this study suggest that removal of redox-active Cu before ischemia has beneficial effects, indicating a mediatory role in postischemic cardiac oxidative injury.

Biological activities of des-His1[Glu9]glucagon amide, a glucagon antagonist.

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His1[Glu9]glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10(6) higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (less than 0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27.

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.

Synthetic peptide antagonists of glucagon.

Several glucagon analogs were synthesized in an effort to find derivatives that would bind with high affinity to the glucagon receptor of rat liver membranes but would not activate membrane-bound adenylate cyclase and, therefore, would serve as antagonists of the hormone. Measurements on a series of glucagon/secretin hybrids indicated that replacement of Asp9 in glucagon by Glu9, found in secretin, was the important sequence difference in the N terminus of the two hormones. Further deletion of His1 and introduction of a C-terminal amide resulted in des-His1-[Glu9]glucagon amide, which had a 40% binding affinity relative to that of native glucagon but caused no detectable adenylate cyclase activation in the rat liver membrane. This antagonist completely inhibited the effect of a concentration of glucagon that alone gave a full agonist response. It had an inhibition index of 12. The pA2 was 7.2. An attempt was made to relate conformation with receptor binding. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18-silica columns.