Current understanding of <Emphasis Type="Italic">Phaeocystis</Emphasis> ecology and biogeochemistry,and perspectives for future research

The phytoplankton genus Phaeocystis has well-documented, spatially and temporally extensive blooms of gelatinous colonies; these are associated with release of copious amounts of dimethyl sulphide (an important climate-cooling aerosol) and alterations of material flows among trophic levels and export from the upper ocean. A potentially salient property of the importance of Phaeocystis in the marine ecosystem is its physiological capability to transform between solitary cell and gelatinous colonial life cycle stages, a process that changes organism biovolume by 6–9 orders of magnitude, and which appears to be activated or stimulated under certain circumstances by chemical communication. Both life-cycle stages can exhibit rapid, phased ultradian growth. The colony skin apparently confers protection against, or at least reduces losses to, smaller zooplankton grazers and perhaps viruses. There are indications that Phaeocystis utilizes chemistry and/or changes in size as defenses against predation, and its ability to create refuges from biological attack is known to stabilize predator–prey dynamics in model systems. Thus the life cycle form in which it occurs, and particularly associated interactions with viruses, determines whether Phaeocystis production flows through the traditional “great fisheries” food chain, the more regenerative microbial food web, or is exported from the mixed layer of the ocean.Despite this plethora of information regarding the physiological ecology of Phaeocystis, fundamental interactions between life history traits and system ecology are poorly understood. Research summarized here, and described in the various papers in this special issue, derives from a central question: how do physical (light, temperature, particle distributions, hydrodynamics), chemical (nutrient resources, infochemistry, allelopathy), biological (grazers, viruses, bacteria, other phytoplankton), and self-organizational mechanisms (stability, indirect effects) interact with life-cycle transformations of Phaeocystis to mediate ecosystem patterns of trophic structure, biodiversity, and biogeochemical fluxes? Ultimately the goal is to understand and thus predict why Phaeocystis occurs when and where it does, and the bio-feedbacks between this keystone species and the multitrophic level ecosystem.     

The cholesterol-raising factor from coffee beans, cafestol, as an agonist ligand for the farnesoid and pregnane X receptors

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR). Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, sterol 12alpha-hydroxylase, and Na(+)-taurocholate cotransporting polypeptide in the liver of wild-type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild-type mice, but did increase expression of the positive FXR-target genes intestinal bile acid-binding protein and fibroblast growth factor 15 (FGF15) in the intestine. Because FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of cytochrome P450 3A11 and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR, and this may contribute to its impact on cholesterol homeostasis.     

A Genome-Wide Assessment of the Role of Untagged Copy Number Variants in Type 1 Diabetes

Genome-wide association studies (GWAS) for type 1 diabetes (T1D) have successfully identified more than 40 independent T1D associated tagging single nucleotide polymorphisms (SNPs). However, owing to technical limitations of copy number variants (CNVs) genotyping assays, the assessment of the role of CNVs has been limited to the subset of these in high linkage disequilibrium with tag SNPs. The contribution of untagged CNVs, often multi-allelic and difficult to genotype using existing assays, to the heritability of T1D remains an open question. To investigate this issue, we designed a custom comparative genetic hybridization array (aCGH) specifically designed to assay untagged CNV loci identified from a variety of sources. To overcome the technical limitations of the case control design for this class of CNVs, we genotyped the Type 1 Diabetes Genetics Consortium (T1DGC) family resource (representing 3,903 transmissions from parents to affected offspring) and used an association testing strategy that does not necessitate obtaining discrete genotypes. Our design targeted 4,309 CNVs, of which 3,410 passed stringent quality control filters. As a positive control, the scan confirmed the known T1D association at the INS locus by direct typing of the 5′ variable number of tandem repeat (VNTR) locus. Our results clarify the fact that the disease association is indistinguishable from the two main polymorphic allele classes of the INS VNTR, class I-and class III. We also identified novel technical artifacts resulting into spurious associations at the somatically rearranging loci, T cell receptor, TCRA/TCRD and TCRB, and Immunoglobulin heavy chain, IGH, loci on chromosomes 14q11.2, 7q34 and 14q32.33, respectively. However, our data did not identify novel T1D loci. Our results do not support a major role of untagged CNVs in T1D heritability.     

Synthesis,properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH₃-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH₃-analogs and the corresponding analogs containing S instead of BH₃ (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH₃-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2, respectively, than for in vitro transcribed mRNA capped with m₂^7,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2 favorable for anticancer immunization.     

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.     

Oncostatin M Mediates STAT3-Dependent Intestinal Epithelial Restitution via Increased Cell Proliferation,Decreased Apoptosis and Upregulation of SERPIN Family Members

Objective

Oncostatin M (OSM) is produced by activated T cells, monocytes, and dendritic cells and signals through two distinct receptor complexes consisting of gp130 and LIFR (I) or OSMR-β and gp130 (II), respectively. Aim of this study was to analyze the role of OSM in intestinal epithelial cells (IEC) and intestinal inflammation.

Methods

OSM expression and OSM receptor distribution was analyzed by PCR and immunohistochemistry experiments, signal transduction by immunoblotting. Gene expression studies were performed by microarray analysis and RT-PCR. Apoptosis was measured by caspases-3/7 activity. IEC migration and proliferation was studied in wounding and water soluble tetrazolium assays.

Results

The IEC lines Caco-2, DLD-1, SW480, HCT116 and HT-29 express mRNA for the OSM receptor subunits gp130 and OSMR-β, while only HCT116, HT-29 and DLD-1 cells express LIFR mRNA. OSM binding to its receptor complex activates STAT1, STAT3, ERK-1/2, SAPK/JNK-1/2, and Akt. Microarray analysis revealed 79 genes that were significantly up-regulated (adj.-p≤0.05) by OSM in IEC. Most up-regulated genes belong to the functional categories “immunity and defense” (p = 2.1×10⁻⁷), “apoptosis” (p = 3.7×10⁻⁴) and “JAK/STAT cascade” (p = 3.4×10⁻⁶). Members of the SERPIN gene family were among the most strongly up-regulated genes. OSM significantly increased STAT3- and MEK1-dependent IEC cell proliferation (p<0.05) and wound healing (p = 3.9×10⁻⁵). OSM protein expression was increased in colonic biopsies of patients with active inflammatory bowel disease (IBD).

Conclusions

OSM promotes STAT3-dependent intestinal epithelial cell proliferation and wound healing in vitro. Considering the increased OSM expression in colonic biopsy specimens of patients with active IBD, OSM upregulation may modulate a barrier-protective host response in intestinal inflammation. Further in vivo studies are warranted to elucidate the exact role of OSM in intestinal inflammation and the potential of OSM as a drug target in IBD.     

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.     

The effect of TSPP-mediated photodynamic therapy and Parecoxib in experimental tumours

Aims

The study investigated the effects of the combined treatment Parecoxib (Pcox) and 5,10,15,20-tetra-sulphonato-phenyl-porphyrin(TSPP)-mediated photodynamic therapy on Walker 256 carcinosarcoma.

Main methods

Five groups of male Wistar rats were used: the control group, treated with TSPP, group 2, irradiated 24 h thereafter, group 3, treated with Pcox and irradiated 24 h thereafter, groups 4 and 5 treated with combined therapies, TSPP and Pcox before irradiation, and Pcox 24 h after TSPP and irradiation respectively. Tumour inflammation, growth and non-growth factors, apoptosis/necrosis rate and oxidative/nitrosative stress markers were investigated.

Key findings

Malondialdehyde levels and cyclooxygenase (COX)-2 expression increased significantly in the group treated with Pcox after TSPP-PDT when compared with TSPP + IR group (p < 0.05, p < 0.001 respectively), in correlation with a decrease in glutathione levels (p < 0.05). The quantification of apoptosis, based on the TUNEL-assay, and necrosis rate revealed an increase of apoptotic/necrotic index in the same group (p < 0.05). On the other hand, Pcox administered before irradiation showed a significant increase in both vascular endothelial growth factor (VEGF) and COX-2 levels (p < 0.05) and in nitric oxide production (p < 0.01), when compared with the control group.

Significance

The administration of Pcox after TSPP-mediated PDT showed promising antitumoural effects, leading to an increase in oxidative and nitrosative stress as well as apoptosis/necrosis rate in tumour tissue. These results show that combined regimens that involve selective COX-2 inhibitors administration after irradiation may improve the therapeutic effectiveness of PDT.     

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.     

Characterization of Bacillus thuringiensis isolates from Argentina that are potentially useful in insect pest control

In order to find novel strains of Bacillus thuringiensis that are toxic to some of the major pests that impact economically important crops in Argentina, we initiated a search for B. thuringiensis isolates native to Argentina. We succeeded in assembling a collection of 41 isolates, some of which show a high potential to be used in biological control programs against lepidopteran and coleopteran pests. About 90% of the strains showed toxicity against Spodoptera frugiperda and Anticarsia gemmatalis, two important lepidopteran pests in Argentina. It is noteworthy that only one of these strains contained a cry1-type gene, while another isolate showed a dual toxicity against the lepidopteran and coleopteran insects assayed. Genetic characterization of the strains suggests that the collection likely harbors novel Cry proteins that may be of potential use in biological insect pest control.