Overexpression of the lemon basil alpha-zingiberene synthase gene increases both mono- and sesquiterpene contents in tomato fruit

alpha-Zingiberene synthase (ZIS), a sesquiterpene synthase gene that was isolated from lemon basil (Ocimum basilicum L.), encodes an enzyme that catalyzes the formation of alpha-zingiberene, and other sesquiterpenes, from farnesyl diphosphate. Transgenic tomato fruits overexpressing ZIS under the control of the fruit ripening-specific tomato polygalacturonase promoter (PG) accumulated high levels of alpha-zingiberene (224-1000 ng g(-1) fresh weight) and other sesquiterpenes, such as alpha-bergamotene, 7-epi-sesquithujene, beta-bisabolene and beta-curcumene, whereas no sesquiterpenes were detected in non-transformed control fruits. The ZIS-transgenic fruits also produced monoterpenes, such as alpha-thujene, alpha-pinene, beta-phellandrene and gamma-terpinene (1-22 ng g(-1) fresh weight), which were either not detected or were found only in minute concentrations in control fruits. Recombinant ZIS overexpressed in Escherichia coli catalyzed the formation of these monoterpenes from geranyl diphosphate. As the ZIS protein apparently lacks a transit peptide, and is localized in the cytosol, the production of monoterpenes in the transgenic tomatoes suggests that a pool of geranyl diphosphate is available in the cytosol. The phenotype of the ZIS-transgenic tomatoes was the same as that for wild-type tomatoes, with regard to plant vigor and shape, but transgenic plants exhibited a small decrease in lycopene content. This study thus showed that the synthesis of both mono- and sesquiterpenes can be enhanced by the ectopic expression of a single transgene in tomato fruit, and it further demonstrated the interconnection between the pools of terpenoid precursors in the plastids and the cytosol.     

Pheromone trail following in the ant Lasius niger: high accuracy and variability but no effect of task state

The use of pheromone trails in ant colony organization is an important model for understanding collective decision‐making and complex adaptive systems. The ant Lasius niger L. (Hymenoptera: Fomicidae) is one of the main model organisms used for such studies. Key to understanding pheromone trail use by ants is knowing how well trails are followed. The results of a previous study suggest that L. niger trail following is poor, with between 60% and 70% accuracy at a T bifurcation. It is hypothesized that the true trail following accuracy is higher, and that the low accuracy reported previously is the result of a methodological error. Specifically, it is hypothesized that ‘task state’ (i.e. what the ants ‘thought they were doing’) affected pheromone following accuracy. In the present study, the task state of the ants is set experimentally to one of three states: scouting (completely naive), recruited (having information that food has been found, but not where it is) and shuttling (having a strong memory of the location of a food source). Trail following accuracy is tested for each group. Trail following is found to be more accurate than previously reported: 83%, 82% and 74% correct decisions for scouts, recruits and shuttlers, respectively. However, the difference between the three groups is not significant. Importantly, very high inter‐trial variation is reported both in the present study and in experiments from other research groups. This variation is unexplainable by trail strengths or colony‐level differences, and is highlighted as an important factor when experimentally measuring trail following.     

Aminopeptidase P from human leukocytes.

Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.     

Synthetic probes for the alpha-factor receptor

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

How well do older persons tolerate moderate altitude?

We studied the physiologic and clinical responses to moderate altitude in 97 older men and women (aged 59 to 83 years) over 5 days in Vail, Colorado, at an elevation of 2,500 m (8,200 ft). The incidence of acute mountain sickness was 16%, which is slightly lower than that reported for younger persons. The occurrence of symptoms of acute mountain sickness did not parallel arterial oxygen saturation or spirometric or blood pressure measurements. Chronic diseases were present in percentages typical for ambulatory elderly persons: 19 (20%) had coronary artery disease, 33 (34%) had hypertension, and 9 (9%) had lung disease. Despite this, no adverse signs or symptoms occurred in our subjects during their stay at this altitude. Our findings suggest that persons with preexisting, generally asymptomatic, cardiovascular or pulmonary disease can safely visit moderate altitudes.