Designing REDD+ to be just : considerations for a legally binding instrument

The international climate regime is in the process of negotiating a legally binding instrument concerning Reducing Emissions from Deforestation and Degradation (REDD+). The paper starts by exploring the complex web of decisions and advices that currently regulate REDD+ initiatives within the international climate regime. This is followed by an analysis of justice issues raised by non-state actors in the REDD+ international negotiations. The paper concludes by building on this analysis to identify some relevant considerations when seeking to design a just and legally binding REDD+ instrument. These considerations include: the impact of market- versus fund-based investment channels, the importance of defining a clear objective; the inclusion and role of international principles such as sovereignty, preventative action, common but differentiated responsibility, sustainable development, and Free, Prior, and Informed Consent; the appropriate design of REDD+ safeguards and the inclusion of grievance mechanisms within the instrument which provide guidance on resolving disputes associated with REDD+ investment.

Integrin-functionalized artificial membranes as test platforms for monitoring small integrin ligand binding by surface plasmon-enhanced fluorescence spectroscopy

The design and synthesis of molecularly or supramolecularly defined interfacial architectures have seen in recent years a remarkable growth of interest and scientific research activities for various reasons. On the one hand, it is generally believed that the construction of an interactive interface between the living world of cells, tissue, or whole organisms and the (inorganic or organic) materials world of technical devices such as implants or medical parts requires proper construction and structural (and functional) control of this organism–machine interface. It is still the very beginning of generating a better understanding of what is needed to make an organism tolerate implants, to guarantee bidirectional communication between microelectronic devices and living tissue, or to simply construct interactive biocompatibility of surfaces in general. This exhaustive book lucidly describes the design, synthesis, assembly and characterization, and bio-(medical) applications of interfacial layers on solid substrates with molecularly or supramolecularly controlled architectures. Experts in the field share their contributions that have been developed in recent years.

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro

Transcriptional gene silencing (TCS) is often associated with an increased level of cytosine methylation in the affected promoters. The effect of methylation of the cauliflower mosaic virus (CaMV) 35S promoter sequence on its binding to factors present in the nuclei was analyzed by electrophoretic mobility shift assays using extracts of petunia flowers. Specific DNA-protein interactions were detected in the region of the CaMV 35S promoter that contains the as-1 element and the region between -345 and -208. The binding of protein factor(s) to the as-1 element was influenced by cytosine methylation, whereas the binding to the region between -345 and -208 was unaffected. The results suggest that cytosine methylation of the as-1 element potentially affects the activity of the CaMV 35S promoter. © Georg Thieme Verlag KG Stuttgart.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism

Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo. Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosageand RNA-dependent manner. In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro. Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632. Furthermore, overexpression of RBP29 suppresses the temperaturesensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles. These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA.

UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen and human uroepithelial cells

Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

DNA binding study of isophorone in rats and mice

Following isophorone exposure, in a 2-year study with F344 rats and B6C3F1 mice performed under the National Toxicology Program (NTP), an elevated incidence of tumors was observed in male rats (kidney tumors) and male mice (liver tumors). Female rats and mice showed no elevation of tumor rates by isophorone (NTP 1986).