Liposome-based homogeneous luminescence resonance energy transfer

There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor-ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin-streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.     

Sleep Restriction Increases the Risk of Developing Cardiovascular Diseases by Augmenting Proinflammatory Responses through IL-17 and CRP

Background

Sleep restriction, leading to deprivation of sleep, is common in modern 24-h societies and is associated with the development of health problems including cardiovascular diseases. Our objective was to investigate the immunological effects of prolonged sleep restriction and subsequent recovery sleep, by simulating a working week and following recovery weekend in a laboratory environment.

Methods and Findings

After 2 baseline nights of 8 hours time in bed (TIB), 13 healthy young men had only 4 hours TIB per night for 5 nights, followed by 2 recovery nights with 8 hours TIB. 6 control subjects had 8 hours TIB per night throughout the experiment. Heart rate, blood pressure, salivary cortisol and serum C-reactive protein (CRP) were measured after the baseline (BL), sleep restriction (SR) and recovery (REC) period. Peripheral blood mononuclear cells (PBMC) were collected at these time points, counted and stimulated with PHA. Cell proliferation was analyzed by thymidine incorporation and cytokine production by ELISA and RT-PCR. CRP was increased after SR (145% of BL; p<0.05), and continued to increase after REC (231% of BL; p<0.05). Heart rate was increased after REC (108% of BL; p<0.05). The amount of circulating NK-cells decreased (65% of BL; p<0.005) and the amount of B-cells increased (121% of BL; p<0.005) after SR, but these cell numbers recovered almost completely during REC. Proliferation of stimulated PBMC increased after SR (233% of BL; p<0.05), accompanied by increased production of IL-1β (137% of BL; p<0.05), IL-6 (163% of BL; p<0.05) and IL-17 (138% of BL; p<0.05) at mRNA level. After REC, IL-17 was still increased at the protein level (119% of BL; p<0.05).

Conclusions

5 nights of sleep restriction increased lymphocyte activation and the production of proinflammatory cytokines including IL-1β IL-6 and IL-17; they remained elevated after 2 nights of recovery sleep, accompanied by increased heart rate and serum CRP, 2 important risk factors for cardiovascular diseases. Therefore, long-term sleep restriction may lead to persistent changes in the immune system and the increased production of IL-17 together with CRP may increase the risk of developing cardiovascular diseases.     

Nitrogen retention by peatland buffer areas at six forested catchments in southern and central Finland

We studied the nitrogen retention capacity of six peatland buffer areas constructed in forested catchments in southern and central Finland. The buffers (0.1–4.9% of the total catchment area) were either undrained mires or drained peatlands rewetted 4–7 years before the present study. The N retention capacity was studied by adding ammonium nitrate (NH₄NO₃–N) solution into the inflow waters of the buffers once (one area) or twice (five areas) during a period of 4–6 years. Except for the first N addition in one area, the three largest buffer areas (relative size > 1%) retained the added inorganic N almost completely; their retention efficiencies during the year of addition were >93% for both NO₃–N and NH₄–N. Two of the three small buffers (relative size < 0.25%) were also able to reduce inorganic N from the through-flow waters effectively; their retention capacities for inorganic nitrogen varied between 58 and 89%. However, one small buffer area had a retention capacity of only <20%. The factors contributing to efficient N retention were hydrological load during N addition, relative size of the buffer area, and its length, i.e. the distance between the inflow and outflow points. If there was any release of the added N, it mostly occurred within a relatively short-time period (<100 days) after the treatment. The buffer areas appeared to be efficient and long-term sinks for inorganic nitrogen because the release of N during the 2–4 years after N addition was minor.     

Chromosomal assignment of R-spondin genes in the donkey (equus asinus, 2n=62)

R-spondins constitute a recently discovered small family of growth factors, and the evidence of their role in several developmental pathways is growing fast. In this work we describe the chromosomal location of the fourRSPO genes in the donkey. Using horse BACs, we localizedRSPO1 on EAS 5q23,RSPO2 on EAS 12q13,RSPO3 on EAS 24q26, andRSPO4 on EAS 15p13. Moreover,RSPO2, RSPO3, andRSPO4 are the first genes mapped on donkey chromosomes 12, 24, and 15, respectively.     

Life history and sex allocation in the simultaneously hermaphroditic polychaete worm Ophryotrocha diadema: the role of sperm competition

Sex allocation theory predicts that, in hermaphroditic organisms,individuals allocate a fixed amount of resources divided amongmale and female functions to reproduction and that the proportiondevoted to each sex depends on the mating group size. As themating group size increases, hermaphrodites are predicted toallocate proportionally more resources to the male and lessresources to the female function (approaching equal allocationto both sexes) to face increased sperm competition. Up to nowlittle experimental evidence has been provided to support thetheory in hermaphroditic animals. Facultative shift betweenmale and female allocation in response to variation in localgroup size does occur in several taxa but not always in theexpected direction and not with similar patterns. In the protandricand then simultaneously hermaphroditic polychaete worm Ophryotrochadiadema reproductive resources are flexibly allocated in theprotandrous and the hermaphroditic phase. The cost of male reproductionduring adolescence is spread over the whole energy budget ofthe animal as shown by the shortening of lifespan and the loweringof growth rate in individuals with enhanced male expenditureduring the protandrous phase. Moreover, in this species, shortterm sex allocation adjustments differ from those describedin other taxa. Individuals regulate their reproductive outputso that where reproductive competitors are present, the numberof female gametes is strongly reduced but the number of malegametes (although it changes) is not significantly increased.Resources subtracted from the female function are not directlyallocated to sperm production, but to expensive male behaviorsthat are likely to enhance male reproductive success. Theseresults are discussed in the light of the relevance of sexualselection in large populations of hermaphrodites.     

Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site

Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.     

Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation

The human ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho GTPases and regulates cytokinesis. Although the oncogenic form of ECT2 contains an N-terminal truncation, it is not clear how the structural abnormality of ECT2 causes malignant transformation. Here we show that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of ECT2. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant negative Rho GTPases, suggesting the involvement of Rho GTPases in ECT2 transformation. Although deletion of the N-terminal cell cycle regulator-related domain (N) of ECT2 did not activate its transforming activity, removal of the small central domain (S), which contains two nuclear localization signals (NLSs), significantly induced the activity. The ECT2 N domain interacted with the catalytic domain and significantly inhibited the focus formation by oncogenic ECT2. Interestingly, the introduction of the NLS mutations in the S domain of N-terminally truncated ECT2 dramatically induced the transforming activity of this otherwise non-oncogenic derivative. Among the known Rho GTPases expressed in NIH 3T3 cells, RhoA was predominantly activated by oncogenic ECT2 in vivo. Therefore, the mislocalization of structurally altered ECT2 might cause the untimely activation of cytoplasmic Rho GTPases leading to the malignant transformation.     

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.