Recent improvements to the SMART domain-based sequence annotation resource

SMART (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de) is a web-based resource used for the annotation of protein domains and the analysis of domain architectures, with particular emphasis on mobile eukaryotic domains. Extensive annotation for each domain family is available, providing information relating to function, subcellular localization, phyletic distribution and tertiary structure. The January 2002 release has added more than 200 hand-curated domain models. This brings the total to over 600 domain families that are widely represented among nuclear, signalling and extracellular proteins. Annotation now includes links to the Online Mendelian Inheritance in Man (OMIM) database in cases where a human disease is associated with one or more mutations in a particular domain. We have implemented new analysis methods and updated others. New advanced queries provide direct access to the SMART relational database using SQL. This database now contains information on intrinsic sequence features such as transmembrane regions, coiled-coils, signal peptides and internal repeats. SMART output can now be easily included in users’ documents. A SMART mirror has been created at http://smart.ox.ac.uk.     

Fitness costs of jasmonic acid-induced defense in tomato, Lycopersicon esculentum

The resource allocation hypothesis is based on the assumption that defenses are costly, but relatively few studies have quantified the reproductive price of induced defenses, which represent the best means of measuring such costs in isolation from the genotypic costs that confound research involving constitutive defenses. Jasmonic acid (JA) is a plant signal molecule involved in the defensive responses of plants. It induces many of the same chemicals that are associated with herbivore damage, and thus offers a means of inducing plants without the removal of leaf area, which incurs its own costs. In tomato plants, JA induced resistance to Manduca sexta and increased levels of two defensive enzymes, polyphenol oxidase and peroxidase. We measured the impact of JA-induced defenses in tomato, Lycopersicon esculentum (Solanaceae), on several variables associated with reproductive success: fruit number, fruit weight, ripening time, time of fruit-set, number of seeds per fruit, total seeds per plant, the relationship between fruit weight and seed number, and germination success. Plants were grown in a pest-free greenhouse and treated biweekly with solvent or with JA at either of two concentrations: 10 mM or 1 mM. The high concentration of JA led to fewer but larger fruits, longer ripening time, delayed fruit-set, fewer seeds per plant, and fewer seeds per unit of fruit weight. The reproductive impact of induction was reduced at the lower dose, but still significant; 1 mM JA resulted in delayed fruit-set and fewer seeds per unit of fruit weight, compared to control plants. Our research indicates that JA-induced defenses impose significant costs on tomato plants.     

Chi-dependent DNA strand cleavage by RecBC enzyme

Chi sites enhance in their vicinity homologous recombination by the E. coli RecBC pathway. We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E. coli recC gene, coding for a RecBC enzyme subunit. Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA. These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.     

Enhanced peripheral chemoreflex function in conscious rabbits with pacing-induced heart failure.

The present study aimed to determine whether peripheral and/or central chemoreflex function is altered in chronic heart failure (CHF) and whether altered chemoreflex function contributes to sympathetic activation in CHF. A rabbit model of pacing-induced CHF was employed. The development of CHF (3-4 wk of pacing) was characterized by an enlarged heart, an attenuated contractility, and an elevated central venous pressure. Renal sympathetic nerve activity (RSNA) and minute volume (MV) of ventilation in response to stimulation of peripheral chemoreceptors by isocapnic/hypoxic gases were measured in the conscious state. It was found that the baseline RSNA at normoxia was higher in CHF rabbits than in sham rabbits (35. 00 +/- 4.03 vs. 20.75 +/- 2.87% of maximum, P < 0.05). Moreover, the magnitudes of changes in RSNA and MV in response to stimulation of the peripheral chemoreceptors and the slopes of RSNA-arterial PO2 and MV-arterial PO2 curves were greater in CHF than in sham rabbits. Inhibition of the peripheral chemoreceptors by inhalation of 100% O2 decreased RSNA in CHF but not in sham rabbits. The central chemoreflex function, as evaluated by the responses of RSNA and MV to hyperoxic/hypercapnic gases, was not different between sham and CHF rabbits. These data suggest that an enhancement of the peripheral chemoreflex occurs in the rabbit model of pacing-induced CHF and that the enhanced peripheral chemoreflex function contributes to the sympathetic activation in the CHF state.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

Opportunities at the interface of chemistry and biology

The combination of the tools and principles of chemistry, together with the tools of modern molecular biology, allow us to create complex synthetic and natural molecules, and processes with novel biological, chemical and physical properties. This article illustrates the tremendous opportunity that lies at this interface of chemistry and biology by describing a number of examples, ranging from efforts to expand the genetic code of living organisms to the use of combinatorial methods to generate biologically active synthetic molecules.     

U2 small nuclear RNA localization and expression during bovine preimplantation development.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.     

Site-specific PEGylation of proteins containing unnatural amino acids

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.