Effects of 2 wk of GH administration on 24-h indirect calorimetry in young, healthy, lean men

The present study was designed as a randomized, double-blind placebo (Plc)-controlled study to determine the effect of 2 wk of growth hormone administration (GH-adm.) on energy expenditure (EE) and substrate oxidation in healthy humans. Sixteen young healthy men were divided into two groups. The study consisted of two 24-h measurements (indirect calorimetry), separated by 2 wk of either Plc or GH injections (6 IU/day). At baseline, no significant differences were observed between the two groups in any of the measured anthropometric, hormonal, or metabolic parameters, neither did the parameters change over time in the Plc group. GH-adm. resulted in a 4.4% increase in 24-h EE (P < 0.05) and an increase in fat oxidation by 29% (P < 0.05). However, a decrease in the respiratory quotient was only observed in the postabsorptive phase after an overnight fast (0.84 +/- 0.1 to 0.79 +/- 0.1, P < 0.05). Furthermore, lean body mass (LBM) was increased by GH-adm. only [62.8 +/- 2.5 kg (baseline) vs. 64.7 +/- 2.4 kg (after), P < 0.001]. In conclusion, GH-adm. increases 24-h EE, which may be partly explained by increased LBM. Furthermore, GH-adm. stimulates fat combustion, especially in the postabsorptive state.     

Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor

Both beta- and gammaherpesviruses encode G protein-coupled receptors (GPCRs) with unique pharmacological phenotypes and important biological functions. An example is ORF74, the gamma2-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR, which is highly constitutively active and considered the key oncogene in Kaposi's sarcoma pathogenesis. In contrast, the current annotation of the Epstein-Barr virus (EBV) genome does not reveal any GPCR homolog encoded by this human oncogenic gamma1-herpesvirus. However, by employing bioinformatics, we recognized that the previously established EBV open reading frame BILF1 indeed encodes a GPCR. Additionally, BILF1 is a member of a new family of related GPCRs exclusively encoded by gamma1-herpesviruses. Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelial cell line revealed that BILF1 is expressed as an approximately 50-kDa glycosylated protein. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74. Using chimeric G proteins, we found that human and rhesus EBV-encoded BILF1 are highly potent constitutively active receptors, activating Galphai. Furthermore, BILF1 is able to inhibit forskolin-triggered CREB activation via stimulation of endogenous G proteins in a pertussis toxin-sensitive manner, verifying that BILF1 signals constitutively through Galphai. We suggest that EBV may use BILF1 to regulate Galphai-activated pathways during viral lytic replication, thereby affecting disease progression.     

Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR.     

Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NOx biosensor. The apparent half-saturation constant [Km(app)] value of the NH3 oxidation of N. briensis was 3 microM NH3 for cultures grown both in continuous and batch cultures as determined by a NOx biosensor. Cells grown on the wall of the vessel had a lower Km(app) value of 1.8 microM NH3. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 microM N h(-1), and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH4+, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH4+ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH4+ availability.     

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.     

Chlorosome Proteins Studied by MALDI-TOF-MS: Topology of CsmA in Chlorobium tepidum

Chlorosomes, the light-harvesting apparatus of green bacteria, are a unique antenna system, in which pigments are organized in aggregates rather than associated with proteins. Isolated chlorosomes from the green sulphur bacterium Chlorobium tepidum contain 10 surface-exposed proteins. Treatment of chlorosomes from Chlorobium tepidum with protease caused changes in the spectral properties of bacteriochlorophyll c and digestion of chlorosome proteins. Using SDS-PAGE analysis, immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) we have investigated the topology of the 59-residue CsmA protein. Our results show that at the N-terminus, the only amino acid available for protease degradation is the methionine. At the C-terminus, amino acids can be removed by protease treatment to produce a residual protein containing at least the sequence between residues 2 and 38. These results indicate that the N-terminal portion of the CsmA protein, which is predicted to be mainly hydrophobic, is buried in the chlorosome envelope.     

Human transformations of the Wadden Sea ecosystem through time: a synthesis

Todays Wadden Sea is a heavily human-altered ecosystem. Shaped by natural forces since its origin 7,500 years ago, humans gradually gained dominance in influencing ecosystem structure and functioning. Here, we reconstruct the timeline of human impacts and the history of ecological changes in the Wadden Sea. We then discuss the ecosystem and societal consequences of observed changes, and conclude with management implications. Human influences have intensified and multiplied over time. Large-scale habitat transformation over the last 1,000 years has eliminated diverse terrestrial, freshwater, brackish and marine habitats. Intensive exploitation of everything from oysters to whales has depleted most large predators and habitat-building species since medieval times. In the twentieth century, pollution, eutrophication, species invasions and, presumably, climate change have had marked impacts on the Wadden Sea flora and fauna. Yet habitat loss and overexploitation were the two main causes for the extinction or severe depletion of 144 species (~20% of total macrobiota). The loss of biodiversity, large predators, special habitats, filter and storage capacity, and degradation in water quality have led to a simplification and homogenisation of the food web structure and ecosystem functioning that has affected the Wadden Sea ecosystem and coastal societies alike. Recent conservation efforts have reversed some negative trends by enabling some birds and mammals to recover and by creating new economic options for society. The Wadden Sea history provides a unique long-term perspective on ecological change, new objectives for conservation, restoration and management, and an ecological baseline that allows us to envision a rich, productive and diverse Wadden Sea ecosystem and coastal society.     

Symbiotic and saprophytic survival of three unmarked Rhizobium leguminosarum biovar trifolii strains introduced into the field

The symbiotic and saprophytic persistence of three unmarked Rhizobium leguminosarum biovar trifolii (Rlt) strains introduced into a field site in Iceland were followed. This site was free of clover cultivation and initially devoid of clover-nodulating rhizobia as tested by nodulation studies. Nodule occupancy by strains was identified based on their distinct ERIC-polymerase chain reaction (PCR) DNA fingerprint patterns. The survival and persistence of the individual strains in soil were monitored by the quantitative real-time PCR (qRT-PCR) assay, targeting the host-specific nodE gene. The most dominant strain in the nodule population, Rlt 20-15, showed relatively less saprophytic survival ability and maintained high numbers only in the presence of the appropriate host plant. Conversely, the minor nodule occupant, Rlt 32-28, persisted in soil at a relatively higher abundance both in the presence of its host legumes and in the presence of a non-host grass. The qRT-PCR assay was successfully applied to quantify rhizobial strains directly in soil without culturing or nodulation. However, the assay demonstrated less sensitivity compared with the plant infection most-probable-number (MPN) method for estimating the population size of rhizobia in soil. The quantitative detection limit of our qRT-PCR assays was 1 x 10(3) cells per gram of soil, as opposed to the MPN test which has a detection limit of 10 cells per gram of soil.     

Periphyton-macroinvertebrate interactions in light and fish manipulated enclosures in a clear and a turbid shallow lake

In a clear and a turbid freshwater lake the biomasses of phytoplankton, periphytic algae and periphytonassociated macrograzers were followed in enclosures with and without fish (Rutilus rutilus) and four light levels (100%, 55%, 7% and < 1% of incoming light), respectively. Fish and light affected the biomass of primary producers and the benthic grazers in both lakes. The biomass of primary producers was generally higher in the turbid than the clear lake, and in both lakes fish positively affected the biomass, while shading reduced it. Total biomass of benthic grazing invertebrates was higher in the clear than in the turbid lake and the lakes were dominated by snails and chironomids + ostracods, respectively. While light had no effect on the biomass of grazers in the clear lake, snail breeding was delayed in the most shaded enclosures and presence of fish reduced the number of snails and the total biomass of grazers. In the turbid lake ostracod abundance was not influenced by light, but was higher in fish-free enclosures. Density of chironomids correlated positively with periphyton biomass in summer, while fish had no effect. Generally, light-mediated regulation of primary producers was stronger in the turbid than in the clear lake, but the regulation did not nambiguously influence the primary consumers. However, regulation by fish of the benthic grazer community was stronger in the clear than in the turbid lake, and in both lakes strong top-down effects on periphyton were seen. The results indicate that if present-day climate in Denmark in the future is found in coastal areas at higher latitudes, the effect of lower light during winter in such areas will be highest in clear lakes, with typically lower fish biomass and higher invertebrate grazer density.     

A SNF2-like protein facilitates dynamic control of DNA methylation

DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in Arabidopsis. Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for de novo or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA-directed de novo methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only de novo methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation.