Functional genomics the old-fashioned way: chemical mutagenesis in mice

Genetic studies using mutants have led to a greater understanding of the mechanisms underlying the physiology, biochemistry and development of organisms. The increasing availability of complete genome sequences has stimulated genome-wide mutagenesis approaches in model organisms. In an ideal model system, it would be possible to choose from a series of mutations in any given gene to study its function, regulation and interaction with other genes; flies and worms with their rich mutant resources provide such models. Because the mouse is a powerful vertebrate model for human disease, it would be advantageous to have an equally comprehensive mutant collection. Recently, much to the joy of the mouse community, two papers, describe screens to generate such a collection. In an ongoing screen, the groups of Brown and Balling have generated over 40,000 F1 mutant mice by treating males with the super mutagen N-ethyl-N-nitrosourea. 300-500 mice are being screened each week using various objective tests and paradigms for morphological, developmental, clinical and behavioral abnormalities. In combination, these analyses have produced an unbiased set of about 700 new dominant, semidominant and recessive mutations.     

Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance

Insulin resistance contributes to a number of metabolic disorders, including type II diabetes, hypertension, and atherosclerosis. Cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, and hormones, such as growth hormone, are known to cause insulin resistance, but the mechanisms by which they inhibit the cellular response to insulin have not been elucidated. One mechanism by which these agents could cause insulin resistance is by inducing the expression of cellular proteins that inhibit insulin receptor (IR) signaling. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling pathways, the expression of which is regulated by certain cytokines. SOCS proteins are therefore attractive candidates as mediators of cytokine-induced insulin resistance. We have found that SOCS-1 and SOCS-6 interact with the IR when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing the human IR. In SOCS-1-expressing cells, insulin treatment increases the extent of interaction with the IR, whereas in SOCS-6-expressing cells the association with the IR appears to require insulin treatment. SOCS-1 and SOCS-6 do not inhibit insulin-dependent IR autophosphorylation, but both proteins inhibit insulin-dependent activation of ERK1/2 and protein kinase B in vivo and IR-directed phosphorylation of IRS-1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signaling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.     

River network saturation concept: factors influencing the balance of biogeochemical supply and demand of river networks

River networks modify material transfer from land to ocean. Understanding the factors regulating this function for different gaseous, dissolved, and particulate constituents is critical to quantify the local and global effects of climate and land use change. We propose the River Network Saturation (RNS) concept as a generalization of how river network regulation of material fluxes declines with increasing flows due to imbalances between supply and demand at network scales. River networks have a tendency to become saturated (supply???demand) under higher flow conditions because supplies increase faster than sink processes. However, the flow thresholds under which saturation occurs depends on a variety of factors, including the inherent process rate for a given constituent and the abundance of lentic waters such as lakes, ponds, reservoirs, and fluvial wetlands within the river network. As supply increases, saturation at network scales is initially limited by previously unmet demand in downstream aquatic ecosystems. The RNS concept describes a general tendency of river network function that can be used to compare the fate of different constituents among river networks. New approaches using nested in situ high-frequency sensors and spatially extensive synoptic techniques offer the potential to test the RNS concept in different settings. Better understanding of when and where river networks saturate for different constituents will allow for the extrapolation of aquatic function to broader spatial scales and therefore provide information on the influence of river function on continental element cycles and help identify policy priorities.     

Freeze–thaw processes and intense rainfall: the one-two punch for high sediment and nutrient loads from mid-Atlantic watersheds

Large runoff, sediment, and nutrient exports from watersheds could occur due to individual extreme climate events or a combination of multiple hydrologic and meteorological conditions. Using high-frequency hydrologic, sediment, and turbidity data we show that freeze–thaw episodes followed by intense winter (February) rainstorms can export very high concentrations and loads of suspended sediment and particulate organic carbon (POC) and nitrogen (PN) from mid-Atlantic watersheds in the US. Peak suspended sediment (> 5000 mg L^?1), POC (> 250 mg L^?1) and PN (> 15 mg L^?1) concentrations at our 12 and 79 ha forested watersheds for the February rainfall-runoff events were highest on record and the fluxes were comparable to those measured for tropical storms. Similar responses were observed for turbidity values (> 400 FNU) at larger USGS-monitored watersheds. Much of the sediments and particulate nutrients likely originated from erosion of stream bank sediments and/or channel storage. Currently, there is considerable uncertainty about the contribution of these sources to nonpoint source pollution, particularly, in watersheds with large legacy sediment deposits. Future climate projections indicate increased intensification of storm events and increased variability of winter temperatures. Freeze–thaw cycles coupled with winter rain events could increase erosion and transport of streambank sediments with detrimental consequences for water quality and health of downstream aquatic ecosystems.     

Purification,characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from <Emphasis Type="Italic">Adenia hondala</Emphasis>

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC₅₀ of 4.8 μg/ml. AHL can be explored for its clinical potential.     

Evolution of particulate organic matter (POM) along a headwater drainage: role of sources,particle size class,and storm magnitude

Large storm events can not only increase the runoff mass exports of particulate organic matter (POM) from watersheds, but can also alter the sources, size distribution, and composition of POM. We investigated the quantity, particle size distribution, carbon (C) and nitrogen (N) content, and sources of POM for five locations longitudinally along a forested Piedmont stream. POM was sampled for multiple storm events of varying magnitude and intensity over a two-year period. POM was separated into coarse (CPOM), medium (MPOM), and fine (FPOM) size classes, and sources were estimated using stable isotopes of ¹³C and ¹⁵N with a Bayesian mixing model. CPOM largely resembled less-degraded vascular plant material characteristic of forest floor litter, which was estimated to contribute to ~40% of CPOM in upstream locations. FPOM was derived from a more variable mixture of sources with stream beds and stream banks playing a greater role at larger drainage locations (up to ~50 and ~30%, respectively). Contributions from both forest floor litter and humus to CPOM increased with increasing event runoff, and litter contributions increased during events with higher rainfall intensities. Higher C and N content was noted in coarse sediments and finer POM fractions appeared to be more degraded based on C:N and isotope ratios. Climate-change projections predict intensification of large storm events in the Northeastern US. Results of this study suggest that large storms will increase the fluvial exports of coarse, labile, C- and N-rich POM with subsequent impacts on receiving aquatic ecosystems.     

A potent mitogenic lectin from the mycelia of a phytopathogenic fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>, with complex sugar specificity and cytotoxic effect on human ovarian cancer cells

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.     

Interaction and energy transfer studies between bovine serum albumin and CdTe quantum dots conjugates: CdTe QDs as energy acceptor probes

In this paper, a systematic investigation of the interaction of bovine serum albumin (BSA) with water‐soluble CdTe quantum dots (QDs) of two different sizes capped with carboxylic thiols is presented based on steady‐state and time‐resolved fluorescence measurements. Efficient Förster resonance energy transfer (FRET) was observed to occur from BSA donor to CdTe acceptor as noted from reduction in the fluorescence of BSA and enhanced fluorescence from CdTe QDs. FRET parameters such as Förster distance, spectral overlap integral, FRET rate constant and efficiency were determined. The quenching of BSA fluorescence in aqueous solution observed in the presence of CdTe QDs infers that fluorescence resonance energy transfer is primarily responsible for the quenching phenomenon. Bimolecular quenching constant (k_q) determined at different temperatures and the time‐resolved fluorescence data provide additional evidence for this. The binding stoichiometry and various thermodynamic parameters are evaluated by using the van ‘t Hoff equation. The analysis of the results suggests that the interaction between BSA and CdTe QDs is entropy driven and hydrophobic forces play a key role in the interaction. Binding of QDs significantly shortened the fluorescence lifetime of BSA which is one of the hallmarks of FRET. The effect of size of the QDs on the FRET parameters are discussed in the light of FRET parameters obtained.     

Oxalate depletion from leafy vegetables using alginate entrapped banana oxalate oxidase

Summary The feasibility of depleting the high oxalate content of spinach (Spinacia oleracea), amaranthus (Amaranthus gangeticus), and paruppu keerai (Portulaca oleracea) using alginate entrapped banana oxalate oxidase is demonstrated for the first time. The relevance of this work to low-oxalate diet preparation for hyperoxaluric patients is discussed.