RNA interference and plant parasitic nematodes

RNA interference (RNAi) has recently been demonstrated in plant parasitic nematodes. It is a potentially powerful investigative tool for the genome-wide identification of gene function that should help improve our understanding of plant parasitic nematodes. RNAi should help identify gene and, hence, protein targets for nematode control strategies. Prospects for novel resistance depend on the plant generating an effective form of double-stranded RNA in the absence of an endogenous target gene without detriment to itself. These RNA molecules must then become available to the nematode and be capable of ingestion via its feeding tube. If these requirements can be met, crop resistance could be achieved by a plant delivering a dsRNA that targets a nematode gene and induces a lethal or highly damaging RNAi effect on the parasite.     

Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

Background

Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery.

Methods

30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC^®) for quantification of these cells.

Results

In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months.

Conclusion

We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.     

Caspase-dependent and -independent activation of acid sphingomyelinase signaling

Recent evidence suggests clustering of plasma membrane rafts into ceramide-enriched platforms serves as a transmembrane signaling mechanism for a subset of cell surface receptors and environmental stresses (Grassme, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R., and Gulbins, E. (2001) J. Biol. Chem. 276, 20589-20596; Cremesti, A., Paris, F., Grassme, H., Holler, N., Tschopp, J., Fuks, Z., Gulbins, E., and Kolesnick, R. (2001) J. Biol. Chem. 276, 23954-23961). Translocation of the secretory form of acid sphingomyelinase (ASMase) into microscopic rafts generates therein the ceramide that drives raft coalescence. This process serves to feed forward Fas activation, with approximately 2% of full caspase 8 activation sufficient for maximal ASMase translocation, leading to death-inducing signaling complex formation within ceramide-rich platforms, and apoptosis. Here we report that treatment of Jurkat T cells with UV-C also induces ASMase translocation into rafts within 1 min, catalyzing sphingomyelin hydrolysis to ceramide and raft clustering. In contrast to Fas, UV-induced ASMase translocation and activation were caspase-independent. Nonetheless, ceramide-rich platforms promoted UV-C-induced death signaling, because ASMase inhibition or raft disruption inhibited apoptosis, improving clonogenic cell survival. These studies thus define two distinct mechanisms for biologically relevant ASMase activation within rafts; a Fas-mediated mechanism dependent upon caspase 8 and FADD, and a UV-induced mechanism independent of caspase activation. Consistent with this notion, genetic depletion or pharmacologic inhibition of caspase 8 or FADD, which render Jurkat cells incapable of sphingolipid signaling and apoptosis upon Fas ligation, did not impair these events upon UV-C stimulation.     

Domain-specific effector interactions within the central cavity of human adult hemoglobin in solution and in porous sol-gel matrices: evidence for long-range communication pathways

The water-filled central cavity of human adult hemoglobin (Hb A) is the binding or interaction site for many different allosteric effectors. Oxygen binding titrations reveal that pyrenetetrasulfonate (PyTS), a fluorescent analogue of 2,3-diphosphoglycerate, behaves like an allosteric effector. The ligation state, pH, and concentrations of other effectors (IHP, L35, and chloride) alter PyTS fluorescence for both solution-phase and sol-gel-encapsulated Hb samples. These conditions also alter the resonance Raman spectra and rates of geminate recombination of CO-ligated Hb. Together, these results demonstrate that there are conformational and functional consequences resulting from interactions between specific domains of the central cavity and individual effectors as well as from long-range synergistic effects that are mediated through the central cavity.     

A new S4-ligated zinc-peptide 1:2 complex for the hydrolytic cleavage of DNA

A new sulfur-ligated Zn-peptide 1:2 complex, [Zn(II)(Boc-NH-Cys-Gly-Cys-OMe)2]2- (2), was prepared, characterized, and tested for its DNA-binding and -cleavage properties. Complex 2 was found to cleave DNA hydrolytically. The negative charge in 2 reduces the affinity of the complex for DNA, and enhances its binding specificity.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

eQTL Explorer: integrated mining of combined genetic linkage and expression experiments

The development of computational resources to visualize and explore data from combined genome-wide expression and linkage studies is essential for the development of testable hypotheses. eQTL Explorer stores expression profiles, linkage data and information from external sources in a relational database and enables simultaneous visualization and intuitive interpretation of the combined data via a Java graphical interface. eQTL Explorer provides a new and powerful tool to interrogate these very large and complex datasets.     

Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.     

Characterization of secretory sphingomyelinase activity, lipoprotein sphingolipid content and LDL aggregation in ldlr-/- mice fed on a high-fat diet

The propensity of LDLs (low-density lipoproteins) for aggregation and/or oxidation has been linked to their sphingolipid content, specifically the levels of SM (sphingomyelin) and ceramide. To investigate this association in vivo, ldlr (LDL receptor)-null mice (ldlr-/-) were fed on a modified (atherogenic) diet containing saturated fats and cholesterol. The diet led to significantly elevated SM content in all serum lipoproteins. In contrast, ceramide increased only in the LDL particles. MS-based analyses of the lipid acyl chain composition revealed a marked elevation in C16:0 fatty acid in SM and ceramide, consistent with the prevalence of palmitic acid in the modified diet. The diet also led to increased activity of the S-SMase [secretory SMase (sphingomyelinase)], a protein that is generated by ASMase (acid SMase) and acts on serum LDL. An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity. ASMase-deficient mice (asm-/-/ldlr-/-) lacked S-SMase activity and were protected from diet-induced elevation in LDL ceramide. LDL from asm-/-/ldlr-/- mice fed on the modified diet were less aggregated and oxidized than LDL from asm+/+/ldlr-/- mice. When tested in vitro, the propensity for aggregation was dependent on the SM level: only LDL from animals on modified diet that have high SM content aggregated when treated with recombinant S-SMase. In conclusion, LDL-SM content and S-SMase activity are up-regulated in mice fed on an atherogenic diet. S-SMase mediates diet-induced changes in LDL ceramide content and aggregation. S-SMase effectiveness in inducing aggregation is dependent on diet-induced enrichment of LDL with SM, possibly through increased hepatic synthesis.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.