Variable domains and a VpreB-like molecule are present in a jawless vertebrate

Immunoglobulins (Igs) and T cell antigen receptors (TCRs) that undergo somatic diversification have not been identified in the two extant orders of jawless vertebrates, which occupy essential positions in terms of understanding the evolution of the emergence of adaptive immunity. Using a single motif-dependent PCR-based approach coupled with a vector that allows selection of cDNAs encoding secretion signal sequences, four different genes encoding Ig V-type domains were identified in the sea lamprey (Petromyzon marinus). One of the predicted proteins encoded by these genes shares structural characteristics with mammalian VpreB molecules, including the absence of a recognizable transmembrane region, a relatively high proportion of charged amino acids in its C-terminal tail and distinctive features of its secretion signal peptide. This is the first indication of a molecule related to the B cell receptor (BCR) complex in a species that diverged prior to the jawed vertebrates in which RAG-mediated adaptive immunity is first encountered.Sequences described in this paper have been deposited in GenBank, with accession numbers AY576797–AY576800.     

Regulatory Sequences of Orthologous petD Chloroplast mRNAs are Highly Specific among Chlamydomonas Species

The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′ UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.     

Trafficking in persulfides: delivering sulfur in biosynthetic pathways

The presence of sulfur in cofactors has been appreciated for over a century, but the trafficking and delivery of sulfur to cofactors and nucleosides is still not fully understood. In the last decade, great strides have been made toward understanding those processes and the enzymes that conduct them, including cysteine desulfurases and rhodanese homology domain proteins. The persulfide group (R-S-SH) predominantly serves as the sulfur donor, and sulfur incorporation pathways share enzymes to a remarkable degree. Mechanisms for the use of persulfide groups are illustrated with the relatively simple case of 4-thiourdine generation, and further possibilities are illuminated by the 2-thiouridine and cofactor biosynthetic systems. The rationale and ramifications of sharing enzymes between sulfur incorporation pathways are discussed, including implications for interpreting genetic or genomic data that indicate a role for a sulfur transfer protein in a particular biological process.     

A metabolomic,geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization

Background

Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective

This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

Methods

An NMR metabolomics approach was used to measure metabolite concentrations in 26 pollen extracts. Pollen count data was analyzed from five cities to model exposure.

Results

A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content: glucose, fructose, sucrose, and myo-inositol. In extracts of 10 mg of pollen/ml, the adenosine was highest for grasses (45 µM) followed by trees (23 µM) and weeds (19 µM). Pollen count data showed that tree pollen was typically 5–10 times the amount of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to be only 1.1, 0.11, and 0.12 µg, respectively. Seasonal models of pollen exposure and respiration suggest that it would be a rare event limited to tree pollen season for concentrations of pollen-derived adenosine to approach physiological levels.

Conclusion

Sugar content and other metabolites may be useful in classifying pollens. Unless other factors create localized exposures that are very different from these models, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.

     

Selected anthropometric variables and aerobic fitness as predictors of cardiovascular disease risk in children

The aim of this study was to assess the suitability of body mass index, waist circumference, waist-to-height ratio and aerobic fitness as predictors of cardiovascular risk factor clustering in children. A cross-sectional study was conducted with 290 school boys and girls from 6 to 10 years old, randomly selected. Blood was collected after a 12-hour fasting period. Blood pressure, waist circumference (WC), height and weight were evaluated according to international standards. Aerobic fitness (AF) was assessed by the 20-metre shuttle-run test. Clustering was considered when three of these factors were present: high systolic or diastolic blood pressure, high low-density lipoprotein (LDL) cholesterol, high triglycerides, high plasma glucose, high insulin concentrations and low high-density lipoprotein (HDL) cholesterol. A ROC curve identified the cut-off points of body mass index (BMI), WC, waist-to-height ratio (WHtR) and AF as predictors of risk factor clustering. BMI, WC and WHR resulted in significant areas under the ROC curves, which was not observed for AF. The anthropometric variables were good predictors of cardiovascular risk factor clustering in both sexes, whereas aerobic fitness should not be used to identify cardiovascular risk factor clustering in these children.     

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.     

Reggies/flotillins regulate cytoskeletal remodeling during neuronal differentiation via CAP/ponsin and Rho GTPases

The reggies/flotillins were discovered as proteins upregulated during axon regeneration. Here, we show that expression of a trans-negative reggie-1/flotillin-2 deletion mutant, R1EA, which interferes with oligomerization of the reggies/flotillins, inhibited insulin-like growth factor (IGF)-induced neurite outgrowth in N2a neuroblastoma cells and impaired in vitro differentiation of primary rat hippocampal neurons. Cells expressing R1EA formed only short and broad membrane protrusions often with abnormally large growth cones. R1EA expression strongly perturbed the balanced activation of the Rho-family GTPases Rac1 and cdc42. Furthermore, focal adhesion kinase (FAK) activity was also enhanced by R1EA expression, while other signaling pathways like ERK1/2, PKC or PKB signaling were unaffected. These severe signaling defects were caused by an impaired recruitment of the reggie/flotillin-associated adaptor molecule CAP/ponsin to focal contacts at the plasma membrane. Thus, the reggies/flotillins are crucial for coordinated assembly of signaling complexes regulating cytoskeletal remodeling.     

Five Arabidopsis Reticulon Isoforms Share Endoplasmic Reticulum Location,Topology, and Membrane-Shaping Properties

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.     

Fine-scale genetic pattern and evidence for sex-biased dispersal in the túngara frog, Physalaemus pustulosus

Túngara frogs (Physalaemus pustulosus) are a model system for sexual selection and communication. Population dynamics and gene flow are of major interest in this species because they influence speciation processes and microevolution, and could consequently provide a deeper understanding of the evolutionary processes involved in mate recognition. Although earlier studies have documented genetic variation across the species' range, attempts to investigate dispersal on a local level have been limited to mark-recapture studies. These behavioural studies indicated high mobility at a scale of several hundred metres. In this study we used seven highly polymorphic microsatellite loci to investigate fine-scaled genetic variation in the túngara frog. We analysed the influence of geographical distance on observed genetic patterns, examined the influence of a river on gene flow, and tested for sex-biased dispersal. Data for 668 individuals from 17 populations ranging in distance from 0.26 to 11.8 km revealed significant levels of genetic differentiation among populations. Genetic differentiation was significantly correlated with geographic distance. A river acted as an efficient barrier to gene flow. Several tests of sex-biased dispersal were conducted. Most of them showed no difference between the sexes, but variance of Assignment Indices exhibited a statistically significant male bias in dispersal.