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.     

Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element-companion cell complex

Serpins are unique inhibitors of serine proteases that are located in various plant tissues and organs. An orthologue of the pumpkin (Cucurbita maxima) phloem serpin CmPS-1 was amplified from cucumber (Cucumis sativus) RNA by RT-PCR, cloned, and designated as CsPS-1 (GenBank accession no. AJ866989). Alternative amino acid sequences in the reactive centre loop suggest distinct inhibitory specificity between CmPS-1 and CsPS-1. A difference in the electrophoretic mobility of these serpins was used in heterografts to establish that serpins are phloem-mobile. Immuno light microscopy revealed that the phloem serpins are localized exclusively to sieve elements (SE), while the phloem filament protein CmPP1, used as a reference, is localized to both SEs and companion cells (CCs). Similar to CmPS-1, CsPS-1 accumulates over time in phloem exudates, indicating that serpins differ from other phloem-mobile proteins whose concentrations appear to be stable in phloem exudates. These differences could reflect alternative mechanisms regulating protein turnover and/or inaccessibility of protein degradation. The functionality of the pore/plasmodesma units connecting SEs and CCs was tested with graft-transmitted CmPP1 as a transport marker. The occurrence of CmPP1 in the CCs of the Cucumis graft partner shows that translocated 88 kDa phloem filament protein monomers can symplasmically exit the SE and accumulate in the CC. By contrast, serial sections probed with the serpin antibody demonstrate that the 43 kDa serpin does not enter CCs. Collectively, these data indicate that CCs play a decisive role in homeostasis of exudate proteins; proteins not accessing the CCs accumulate in SEs and display a time-dependent increase in concentration.     

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the β-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining β-cell mass in both type 1 and type 2 diabetic patients.     

Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp(171) (AspIV:20), Asp(262) (AspVI:23), and Glu(288) (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp(171) in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp(262) and Glu(288) from TM-VI and -VII, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral bioavailability can be designed to mimic this interaction and thereby efficiently and selectively block the CXCR4 receptor.     

Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes.     

How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor

Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar surface of the B-chain. Evidence is presented that two contiguous sites in an alpha-helix, Val(B12) and Tyr(B16), contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of Tyr(B16) by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 beta-helix of the alpha-subunit. Because substitution of Val(B12) by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val(B12) by alanine, threonine, and alpha-aminobutyric acid leads to activities of 1(+/-0.1)%, 13(+/-6)%, and 14(+/-5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(+/-10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain alpha-helix, functioning as an essential recognition element, docks against the L1 beta-helix of the insulin receptor.