Differential effects of proinflammatory cytokines on cell death and ER stress in insulin-secreting INS1E cells and the involvement of nitric oxide

Proinflammatory cytokines produced by immune cells destroy pancreatic beta cells in type 1 diabetes. The aim of this study was to investigate the cytokine network and its effects in insulin-secreting cells. INS1E cells were exposed to different combinations of proinflammatory cytokines. Cytokine toxicity was estimated by MTT assay and caspase activation measurements. The NFκB-iNOS pathway was analyzed by a SEAP reporter gene assay, Western-blotting and nitrite measurements. Gene expression analyses of ER stress markers, Chop and Bip, were performed by real-time RT-PCR. Cytokines tested in this study, namely IL-1β, TNFα and IFNγ, had deleterious effects on beta cell viability. The most potent toxicity exhibited IL-1β and its combinations with other cytokines. The toxic effects of IL-1β towards cell viability, caspase activation and iNOS activity were dependent on nitric oxide and abolished by an iNOS blocker. IL-1β was the strongest inducer of the NFκB activation. An iNOS blocker inhibited IL-1β-mediated NFκB activation in the first, initial phase of cytokine action, but did not affect significantly NFκB activation after prolonged incubation. Interestingly iNOS protein expression was induced predominantly by IL-1β and decreased in the presence of an iNOS blocker in the case of a short time exposure. The changes in the expression of ER stress markers were also almost exclusively dependent on the IL-1β presence and counteracted by iNOS blockade. Thus cytokine-induced beta cell death is primarily IL-1β mediated with a NO-independent enhancement by TNFα and IFNγ. The deleterious effects on cell viability and function are crucially dependent on IL-1β-induced nitric oxide formation.     

Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway

Leptin (Lep) is a key factor for the energy homeostasis in mammals, but the available data of its role in teleosts are not conclusive. There are large sequence differences among mammalian and teleost Lep, both at the gene and protein level. Therefore, in order to characterize Lep function in fish, the use of species-specific Lep is crucial. In this study, the cDNA sequence of salmon leptin a1 (lepa1) was used to establish a production protocol for recombinant salmon LepA1 (rsLepA1) in Escherichia coli, that enabled a final yield of 1.7 mg pure protein L^?1 culture. The effects of 20-day administration of rsLepA1 on growth and brain neuroendocrine peptide gene expression [npy, cart, agrp (-1 and -2), pomc (-a1, -a2, -a2s, and -b)] were studied in juvenile, immature Atlantic salmon (96.5 ± 2.1 g) fed a commercial diet to satiation. Intraperitoneal osmotic pumps were used to deliver rsLepA1 at four different concentrations (calculated pumping rates were 0, 0.1, 1.0 and 10 ng g^?1 h^?1). In the highest dosage group (10 ng g^?1 h^?1), the growth rate was significantly reduced, and pomc-a1 gene expression was higher than in controls. The results support the lipostatic hypothesis and suggest that sLepA1 reduces growth in Atlantic salmon by affecting food intake through the central pro-opiomelanocortin pathway.     

Changes in clinical profile, treatment, and mortality in patients hospitalised for acute myocardial infarction between 1985 and 2008

Objectives

To quantify the impact of the implementation of treatment modalities into clinical practice since 1985, on outcome of patients with ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI).

Methods

All consecutive patients admitted for STEMI or NSTEMI at the Thoraxcenter between 1985 and 2008 were included. Baseline characteristics, pharmacological and invasive treatment modalities, and survival status were collected. The study population was categorised in three groups of patients: those hospitalised between 1985–1990, 1990–2000, and 2000–2008.

Results

We identified 14,434 patients hospitalised for myocardial infarction (MI). Both STEMI and NSTEMI patients were increasingly treated with the current guideline based therapy. In STEMI, at 30 days following admission, cumulative mortality rate decreased from 17% in 1985–1990 to 13% in 1990–2000, and to 6% in 2000–2008. Adjusted 30-day and three-year mortality in the last period was 80% and 68% lower than in 1985, respectively. In NSTEMI, at 30 days following admission, cumulative mortality rate decreased from 6% in 1985–1990 to 4% in 1990–2000, and to 2% in 2000–2008. Adjusted 30-day and three-year mortality in the last period was 78% and 49% lower than in 1985, respectively. For patients admitted between 2000 and 2008, 3 year survival of STEMI and NSTEMI patients was 87% and 88%, respectively.

Conclusions

Our results indicate substantial improvements in acute- and long-term survival in patients hospitalised for MI, related to improved acute- as well as long-term treatment. Early medical evaluation in suspected MI and intensive early hospital treatment both remain warranted in the future.     

Effects of sedimentation from water-based drill cuttings and natural sediment on benthic macrofaunal community structure and ecosystem processes

In this study, we aim at investigating the role of physical disturbance in effects of water-based drill cuttings on benthic ecosystems. Today, most of the cuttings discharged from oil and gas installations contain water-based drilling muds, rather than oil-based or synthetic muds. Drill cuttings with water-based muds are assumed to cause only marginal effects on the benthos, mainly resulting from sedimentation. However, this statement has not been experimentally tested, which is the purpose of the present work. Natural sediment particles and water-based drill cuttings were added to benthic communities in layer thicknesses of 3-24 mm in a mesocosm set-up. During the following 6 months, changes in benthic community structure and fluxes of oxygen and nutrients across the sediment water interface were studied. There was a significant reduction in number of taxa, abundance, biomass and diversity of macrofauna with increasing thickness of drill cuttings, which was not observed for the natural sediment particles. The drill cuttings also influenced oxygen consumption and oxygen penetration depth in the sediment, and it was concluded that an organic compound in the drill cuttings initiated a typical eutrophication response. Fluxes of phosphate and silicate were, however, similarly affected by the two types of particles, and maximum fluxes occurred in sediments treated with thin layers (3-6 mm) of particles. As the response of water-based drill cuttings in the present study was a result of factors other than physical disturbance, we recommend a reconsideration of the assumption that water-based drill cuttings only cause sedimentation (burial) effects.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

A strongly bound high-spin iron(II) coordinates cysteine and homocysteine in cysteine dioxygenase

The first experimental evidence of a tight binding iron(II)-CDO complex is presented. These data enabled the relationship between iron bound and activity to be explicitly proven. Cysteine dioxygenase (CDO) from Rattus norvegicus has been expressed and purified with ~0.17 Fe/polypeptide chain. Following addition of exogenous iron, iron determination using the ferrozine assay supported a very tight stoichiometric binding of iron with an extremely slow rate of dissociation, k(off) ~ 1.7 × 10(-6) s(-1). Dioxygenase activity was directly proportional to the concentration of iron. A rate of cysteine binding to iron(III)-CDO was also measured. M?ssbauer spectra show that in its resting state CDO binds the iron as high-spin iron(II). This iron(II) active site binds cysteine with a dissociation constant of ~10 mM but is also able to bind homocysteine, which has previously been shown to inhibit the enzyme.     

Analysis of functional germline polymorphisms for prediction of response to anthracycline-based neoadjuvant chemotherapy in breast cancer

To elucidate the role of predictive factors on individual's drug response, based on genetic variation, we examined the association between eight germline polymorphisms in genes involved in protection against oxidative stress, apoptosis, oncogenic transformation, proliferation, immune response and DNA repair (TP53, NQO1, IL6, TLR4 and XRCC1) and the pathological response to anthracycline-based neoadjuvant chemotherapy in 70 patients with breast cancer. The DNA was genotyped for eight polymorphisms in five genes (TP53, NQO1, IL6, TLR4 and XRCC1) by 5'-exonuclease (TaqMan?) technology. Fisher's exact test was used to evaluate the association between genotype, clinicopathological parameters and pathological response. A good pathological response, defined as a pathological complete response or residual isolated invasive tumor cells, was found significantly more frequently for estrogen (ER) and progesterone receptor (PR) negative breast carcinomas compared to ER and PR positive and ER or PR positive carcinomas, respectively (43.5 vs. 37.5 and 10.3?%, p?=?0.006), and was significantly associated with high tumor grade (G3) (p?=?0.002). A non-significant trend towards a good pathological response was shown in patients carrying the Arg/Arg or Arg/Pro TP53 codon 72 gene variant compared to those harboring the Pro/Pro variant (17.6 or 37.9?% vs. 0; p?=?0.071). No association was found between NQO1 Pro187Ser, IL6 -174G>C, TLR4 Asp299Gly and Thr399Ile, and XRCC1 Arg194Trp, Arg399Gln and Arg280His and pathological response. The present study shows hormone receptor status and tumor grade as predictors for pathological response to neoadjuvant anthracycline-based chemotherapy. Among various functional germline polymorphisms, a potential predictive value was only found for the TP53 Arg72Pro gene variant.