Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols

New derivatives of 1,4-dideoxy-1,4-imino-d-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-d-ribitol (13, IC₅₀ ∼2 μM) and its C₁₈-analogues (IC₅₀ <10 μM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC₅₀ ∼8 μM) growth of JURKAT cells.     

Interactive effects of salicylic acid and nitric oxide on soybean plants under NaCl salinity

The effects of salicylic acid (SA), sodium nitroprusside (SNP), a nitric oxide donor, and their combination (SA+SNP) on some physiological parameters of 23-day-old soybean seedlings grown under saline and nonsaline conditions were studied. The changes in the leaf area, shoot fresh and dry weights, contents of chlorophylls and carotenoids, amounts of MDA and hydrogen peroxide showed that the addition of 100 μM SA and/or 100 μM SNP markedly declined the oxidative damage to soybean plants induced by 50 and 100 μM NaCl. Our results proved that combined action of SA and nitric oxide donor significantly activated catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), which contributed to the decay of H₂O₂ in soybean leaves under NaCl toxicity. The protective action of (SA+SNP) against saltinduced oxidative damage was often more efficient than effects of SA and SNP alone. We also observed that the accumulation of proline was apparently accelerated by these substances under salt stress. As well, it was observed that the interaction between SA and nitric oxide had synergistic effects in decreasing of the damages induced by NaCl salinity.     

Photosystem II supercomplex remodeling serves as an entry mechanism for state transitions in Arabidopsis

Within dense plant populations, strong light quality gradients cause unbalanced excitation of the two photosystems resulting in reduced photosynthetic efficiency. Plants redirect such imbalances by structural rearrangements of the photosynthetic apparatus via state transitions and photosystem stoichiometry adjustments. However, less is known about the function of photosystem II (PSII) supercomplexes in this context. Here, we show in Arabidopsis thaliana that PSII supercomplex remodeling precedes and facilitates state transitions. Intriguingly, the remodeling occurs in the short term, paralleling state transitions, but is also present in a state transition-deficient mutant, indicating that PSII supercomplex generation is independently regulated and does not require light-harvesting complex phosphorylation and movement. Instead, PSII supercomplex remodeling involves reversible phosphorylation of PSII core subunits (preferentially of CP43) and requires the luminal PSII subunit Psb27 for general formation and structural stabilization. Arabidopsis knockout mutants lacking Psb27 display highly accelerated state transitions, indicating that release of PSII supercomplexes is required for phosphorylation and subsequent movement of the antenna. Downregulation of PSII supercomplex number by physiological light treatments also results in acceleration of state transitions confirming the genetic analyses. Thus, supercomplex remodeling is a prerequisite and an important kinetic determinant of state transitions.     

ETOILE regulates developmental patterning in the filamentous brown alga Ectocarpus siliculosus

Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell-cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell-cell communication during the establishment of the developmental pattern in this brown alga.     

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.     

Understanding emerging treatment paradigms in rheumatoid arthritis

Treatment strategies for rheumatoid arthritis (RA) will continue to evolve as new drugs are developed, as new data become available, and as our potential to achieve greater and more consistent outcomes becomes more routine. Many patients will find both symptom relief and modest control of their disease with disease-modifying antirheumatic drugs (DMARDs), yet this course of therapy is clearly not effective in all patients. In fact, despite strong evidence that intensive treatment in the early stages of RA can slow or stop disease progression and may prevent disability, many patients continue to be managed in a stepwise manner and are treated with an ongoing monotherapy regimen with DMARDs. There is now a large body of evidence demonstrating the success of treating RA patients with anti-TNF therapy, usually in combination with methotrexate. As a result of the increased use of anti-TNF therapy, treatment paradigms have changed - and our practice is beginning to reflect this change. In the present review, we summarize the salient points of several recently proposed and emerging treatment paradigms with an emphasis on how these strategies may impact future practice.     

Complex recombination patterns arising during geminivirus coinfections preserve and demarcate biologically important intra-genome interaction networks

Genetic recombination is an important process during the evolution of many virus species and occurs particularly frequently amongst begomoviruses in the single stranded DNA virus family, Geminiviridae. As in many other recombining viruses it is apparent that non-random recombination breakpoint distributions observable within begomovirus genomes sampled from nature are the product of variations both in basal recombination rates across genomes and in the over-all viability of different recombinant genomes. Whereas factors influencing basal recombination rates might include local degrees of sequence similarity between recombining genomes, nucleic acid secondary structures and genomic sensitivity to nuclease attack or breakage, the viability of recombinant genomes could be influenced by the degree to which their co-evolved protein-protein and protein-nucleotide and nucleotide-nucleotide interactions are disreputable by recombination. Here we investigate patterns of recombination that occur over 120 day long experimental infections of tomato plants with the begomoviruses Tomato yellow leaf curl virus and Tomato leaf curl Comoros virus. We show that patterns of sequence exchange between these viruses can be extraordinarily complex and present clear evidence that factors such as local degrees of sequence similarity but not genomic secondary structure strongly influence where recombination breakpoints occur. It is also apparent from our experiment that over-all patterns of recombination are strongly influenced by selection against individual recombinants displaying disrupted intra-genomic interactions such as those required for proper protein and nucleic acid folding. Crucially, we find that selection favoring the preservation of co-evolved longer-range protein-protein and protein DNA interactions is so strong that its imprint can even be used to identify the exact sequence tracts involved in these interactions.     

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.     

The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.