Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Pollen size-number trade-off and pollen-pistil relationships in Pedicularis (Orobanchaceae)

Current patterns of floral design in Pedicularis must have undergone an evolutionary process of interacting among components of floral traits, and then formed internal relationships among these traits. To detect such correlations, which may provide insight to understand flower evolution, 40 Pedicularis species representing all corolla types of the genus were studied. Results show that, interspecifically, pollen size correlates negatively with pollen number, but positively with pistil length. This suggests that plants evolve an optimal pollen size, which balances the advantages of large pollen size for gametophytic competition against the fecundity disadvantages of fewer pollen grains. In contrast to sex allocation theory, this study does not find a trade-off, but an interspecific positive correlation between pollen and ovule number. This is consistent with the hypothesis that genetic variation for resource acquisition may in part be responsible for the lack of negative correlation between male and female function.This work was supported by the State Key Basic Research and Development Plan, China (Grant No. G2000046804) to YHG. The authors would like to thank Peter K. Endress and two anonymous reviewers for providing critical comments and helpful suggestions, Qing-Feng Wang, Jing-Yuan Wang and Jin-Ming Chen for their helpful suggestions. Shi-Guo Sun, Jing Xia, and Qian Yu are thanked for their assistance in both the field work and laboratory phases of the project.     

Synthesis of a novel class of glycocluster with a cyclic α-(1→6)-octaglucoside as a scaffold and their binding abilities to concanavalin A

The synthesis of small glycoclusters with high affinity toward lectins is one of the important subjects in glycotechnology. Although cyclic α-(1→6)-d-octaglucoside (CI8) is an attractive scaffold on which to put glycosyl pendants, the compound has only secondary hydroxyl groups, which are relatively unreactive for substitution reactions. The oxidation of the vicinal diols of CI8 and reductive amination of the resultant dialdehydes with 2-aminoethyl mannoside gave mannose-CI8 conjugates with a variety of average mannose incorporation numbers (2-7). The average numbers were deduced from MALDI-TOF mass and ¹H NMR spectroscopy. The binding ability of mannose-CI8 conjugates to concanavalin A increased with the increasing numbers of average mannose incorporation, reaching a plateau at tetravalence, as estimated from a latex bead-based agglutination lectin assay. Toxicity tests demonstrated the biocompatibility of mannose-CI8 conjugates.     

Genetic diversity and population structure of Swertia tetraptera (Gentianaceae), an endemic species of Qinghai-Tibetan Plateau

Swertia tetraptera Maxim is an annual alpine herb endemic to the Qinghai-Tibetan Plateau (QTP). Its populations are locally scattered as isolated patches throughout this region. Genetic variation within and among thirty-four populations of this species was assessed using ISSR fingerprinting with 10 primers. High levels of genetic diversity exist within species (P = 98.9%, I = 0.3475; He = 0.2227), while the within-population diversity is low (P = 32.7%, I = 0.177; He = 0.12). High levels of genetic differentiation were detected among populations based on various statistics, including Nei’s genetic diversity analysis (G_ST = 0.4608), Bayesian analysis (θ^B = 0.476) and AMOVA (F_ST = 0.57). That is, populations shared low levels of genetic identity (I = 0.2622–0.0966). This genetic structure was probably due to severe genetic drift, breeding system and limited gene flow. The observed genetic structure of the populations implies that different populations across the distribution range of the species should be sampled to maintain high genetic diversity when a conservation strategy is implemented.     

Predicting Metabolic Pathways of Small Molecules and Enzymes Based on Interaction Information of Chemicals and Proteins

Metabolic pathway analysis, one of the most important fields in biochemistry, is pivotal to understanding the maintenance and modulation of the functions of an organism. Good comprehension of metabolic pathways is critical to understanding the mechanisms of some fundamental biological processes. Given a small molecule or an enzyme, how may one identify the metabolic pathways in which it may participate? Answering such a question is a first important step in understanding a metabolic pathway system. By utilizing the information provided by chemical-chemical interactions, chemical-protein interactions, and protein-protein interactions, a novel method was proposed by which to allocate small molecules and enzymes to 11 major classes of metabolic pathways. A benchmark dataset consisting of 3,348 small molecules and 654 enzymes of yeast was constructed to test the method. It was observed that the first order prediction accuracy evaluated by the jackknife test was 79.56% in identifying the small molecules and enzymes in a benchmark dataset. Our method may become a useful vehicle in predicting the metabolic pathways of small molecules and enzymes, providing a basis for some further analysis of the pathway systems.     

The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Rates of small-scale species mobility in alvar limestone grassland

Abstract. Small-scale species frequency and cumulative species frequency were studied in four plots in limestone grassland of the Veronica spicata-Avenula pratensis association on Stora Alvaret on the Baltic island of Öland, Sweden. Species mobility was expressed as increase in cumulative species frequency in 20 subplots of 100 cm². Observed cumulative frequencies from 1985–1989 in all four plots, and from 1985–1995 in one plot were compared with values following from two null models, a ‘minimal mobility’ model and a random mobility model. In ca. 50 % of the cases the observed cumulative frequency was not significantly different from the random expectation. However, in many such cases the mean annual frequency was either very high or very low. Three ways of calculating the mobility rate are presented though only one is used: (observed cumulative frequency -lowest annual frequency) / expected cumulative frequency. Values × 100 range from 0 to 100. There were slight differences between the four plots which were interpreted in terms of differences in grazing intensity and soil depth. It is stressed that the idea of the Carousel model has never been meant to suggest that all species would show random mobility, which we now quantify, but that species differ in their mobility rate and that the mean rate is much higher than generally realized.