Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Association of polymorphisms in the Angiotensin-converting enzyme gene with Alzheimer disease in an Israeli Arab community

Several lines of evidence support for a role of angiotensin converting enzyme (ACE) in Alzheimer disease (AD). Most genetic studies have focused on an Alu insertion/deletion (I/D) polymorphism in the ACE gene (DCP1) and have yielded conflicting results. We evaluated the association between 15 single-nucleotide polymorphisms (SNPs) in DCP1, including the I/D variant, and AD in a sample of 92 patients with AD and 166 nondemented controls from an inbred Israeli Arab community. Although there was no evidence for association between AD and I/D, we observed significant association with SNPs rs4343 (P = .00001) and rs4351 (P = .01). Haplotype analysis revealed remarkably significant evidence of association with the SNP combination rs4343 and rs4351 (global P = 7.5 x 10(-7)). Individuals possessing the haplotype "GA" (frequency 0.21 in cases and 0.01 in controls) derived from these SNPs had a 45-fold increased risk of developing AD (95% CI 6.0-343.2) compared with those possessing any of the other three haplotypes. Longer range haplotypes including I/D were even more significant (lowest global P = 1.1 x 10(-12)), but the only consistently associated alleles were in rs4343 and rs4351. These results suggest that a variant in close proximity to rs4343 and rs4351 modulates susceptibility to AD in this community.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

Elevated α-Linolenic Acid Content in Extra-plastidial Membranes of Tomato Accelerates Wound-Induced Jasmonate Generation and Improves Tolerance to the Herbivorous Insects Heliothis peltigera and Spodoptera littoralis

Journal of Plant Growth Regulation - In tomato, desaturation of linoleic acid (18:2) to α-linolenic acid (18:3) is mediated in the plastidial membranes by the ω-3 fatty acid desaturases 7...     

NCI Workshop Report: Clinical and Computational Requirements for Correlating Imaging Phenotypes with Genomics Signatures

The National Cancer Institute (NCI) Cancer Imaging Program organized two related workshops on June 26–27, 2013, entitled “Correlating Imaging Phenotypes with Genomics Signatures Research” and “Scalable Computational Resources as Required for Imaging-Genomics Decision Support Systems.” The first workshop focused on clinical and scientific requirements, exploring our knowledge of phenotypic characteristics of cancer biological properties to determine whether the field is sufficiently advanced to correlate with imaging phenotypes that underpin genomics and clinical outcomes, and exploring new scientific methods to extract phenotypic features from medical images and relate them to genomics analyses. The second workshop focused on computational methods that explore informatics and computational requirements to extract phenotypic features from medical images and relate them to genomics analyses and improve the accessibility and speed of dissemination of existing NIH resources. These workshops linked clinical and scientific requirements of currently known phenotypic and genotypic cancer biology characteristics with imaging phenotypes that underpin genomics and clinical outcomes. The group generated a set of recommendations to NCI leadership and the research community that encourage and support development of the emerging radiogenomics research field to address short-and longer-term goals in cancer research.     

Functional trade‐offs increase species diversity in experimental plant communities

Functional trade‐offs have long been recognised as important mechanisms of species coexistence, but direct experimental evidence for such mechanisms is extremely rare. Here, we test the effect of one classical trade‐off – a negative correlation between seed size and seed number – by establishing microcosm plant communities with positive, negative and no correlation between seed size and seed number and analysing the effect of the seed size/number correlation on species richness. Consistent with theory, a negative correlation between seed size and seed number led to a higher number of species in the communities and a corresponding wider range of seed size (a measure of functional richness) by promoting coexistence of large‐ and small‐seeded species. Our study provides the first direct evidence that a seed size/number trade‐off may contribute to species coexistence, and at a wider context, demonstrates the potential role of functional trade‐offs in maintaining species diversity.     

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.     

Silicon promotes cytokinin biosynthesis and delays senescence in Arabidopsis and Sorghum

Silicate minerals are dominant soil components. Thus, plant roots are constantly exposed to silicic acid. High silicon intake, enabled by root silicon transporters, correlates with increased tolerance to many biotic and abiotic stresses. However, the underlying protection mechanisms are largely unknown. Here, we tested the hypothesis that silicon interacts with the plant hormones, and specifically, that silicic acid intake increases cytokinin biosynthesis. The reaction of sorghum (Sorghum bicolor) and Arabidopsis plants, modified to absorb high versus low amounts of silicon, to dark‐induced senescence was monitored, by quantifying expression levels of genes along the senescence pathway and measuring tissue cytokinin levels. In both species, detached leaves with high silicon content senesced more slowly than leaves that were not exposed to silicic acid. Expression levels of genes along the senescence pathway suggested increased cytokinin biosynthesis with silicon exposure. Mass spectrometry measurements of cytokinin suggested a positive correlation between silicon exposure and active cytokinin concentrations. Our results indicate a similar reaction to silicon treatment in distantly related plants, proposing a general function of silicon as a stress reliever, acting via increased cytokinin biosynthesis.     

The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo.

We have analyzed in cell culture the sequence elements that control the level of ribosomal frameshifting in the human T-cell leukemia virus type II (HTLV-2) gag-pro junction. The slippery sequence of HTLV-2 is sufficient to dictate a basal level of frameshifting. This level is enhanced by its upstream sequence context and by the downstream stem-loop structure which is located at an optimal distance of 7 bases. Frameshifting in human immunodeficiency virus gag-pol is similar to that of HTLV-2 gag-pro. However, experiments using hybrid cassettes of HTLV-2 and human immunodeficiency virus type 1 frameshift elements show that while the slippery sequence of HTLV-2 is less efficient, the stem-loop structure is a more efficient enhancer.     

Dynamic combinatorial libraries of artificial repeat proteins

Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (～14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.