Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.     

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.     

Effective induction of anti-melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2

RNA-based genetic immunization represents an alternative novel strategy for antigen-specific cancer vaccines. In the present paper we investigate the use of synthetic messenger RNA in an experimental melanoma model. We show that gene gun-based immunization using synthetic RNA mediates gene expression in the epidermis and effectively induces antigen-specific cellular and humoral immunity in mice in vivo. Importantly, bombardment of the skin with RNA coding for the melanocytic self-antigen TRP2 linked to the immunogenic protein EGFP was associated with protection against experimentally induced B16 melanoma lung metastases and vitiligo-like fur depigmentation. Our results provide a scientific basis for clinical trials using synthetic mRNA encoding melanocytic antigens linked to immunogenic helper proteins for vaccination of patients with melanoma. Julia Steitz and Cedrik M. Britten contributed equally to this work.     

Fitness consequences for copepods feeding on a red tide dinoflagellate: deciphering the effects of nutritional value, toxicity, and feeding behavior

Phytoplankton exhibit a diversity of morphologies, nutritional values, and potential chemical defenses that could affect the feeding and fitness of zooplankton consumers. However, how phytoplankton traits shape plant–herbivore interactions in the marine plankton is not as well understood as for terrestrial or marine macrophytes and their grazers. The occurrence of blooms of marine dinoflagellates such as Karenia brevis suggests that, for uncertain reasons, grazers are unable to capitalize on, or control, this phytoplankton growth—making these systems appealing for testing mechanisms of grazing deterrence. Using the sympatric copepod Acartia tonsa, we conducted a mixed diet feeding experiment to test whether K. brevis is beneficial, toxic, nutritionally inadequate, or behaviorally rejected as food relative to the palatable and nutritionally adequate phytoplankter Rhodomonas lens. On diets rich in K. brevis, copepods experienced decreased survivorship and decreased egg production per female, but the percentage of eggs that hatched was unaffected. Although copepods showed a 6–17% preference for R. lens over K. brevis on some mixed diets, overall high ingestion rates eliminated the possibility that reduced copepod fitness was caused by copepods avoiding K. brevis, leaving nutritional inadequacy and toxicity as remaining hypotheses. Because egg production was dependent on the amount of R. lens consumed regardless of the amount of K. brevis eaten, there was no evidence that fitness costs were caused by K. brevis toxicity. Copepods limited to K. brevis ate 480% as much as those fed only R. lens, suggesting that copepods attempted to compensate for low food quality with increased quantity ingested. Our results indicate that K. brevis is a poor food for A. tonsa, probably due to nutritional inadequacy rather than toxicity, which could affect bloom dynamics in the Gulf of Mexico where these species co-occur.     

Insect-antifeedant and antibacterial activity of diterpenoids from species of Plectranthus

Bio-assay guided fractionation of an acetone extract of leaf material from Plectranthus saccatus Benth. resulted in the isolation of a beyerane diterpenoid. This compound, characterised by spectroscopic methods as ent-3beta-(3-methyl-2-butenoyl)oxy-15-beyeren-19-oic acid, showed insect antifeedant activity against Spodoptera littoralis. Known quinonoid abietane diterpenoids obtained from new sources included a mixture of the (4R,19R) and (4R,19S) diastereoisomers of coleon A from P. aff. puberulentus J.K. Morton, coleon A lactone from P. puberulentus J.K. Morton, and coleon U and coleon U quinone from P. forsteri 'Marginatus' Benth. These compounds, and the crude acetone extracts from the leaf surfaces of 11 species of Plectranthus, were tested for antifeedant activity against S. littoralis, antibacterial activity against Bacillus subtilis and Pseudomonas syringae and antifungal activity against Cladosporium herbarum. The coleon A mixture showed potent antifeedant activity against S. littoralis, whereas coleon U showed the greatest antimicrobial activity.     

DNA polymerases and human diseases

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.     

The meiotic chromosomal bouquet: SUN collects flowers

In the early stages of meiosis, all the telomeres in the cell attach to the nuclear envelope and gather near the centrosome. This polarized chromosomal array is known as the bouquet, as the clustered telomeres resemble the gathered stems of a floral arrangement. In this issue of Cell, Chikashige et al. (2006) provide intriguing clues about the molecular details underlying this conserved meiotic event.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.