The Influence of Tissue Plasminogen Activator I/D Polymorphism on the tPA Response to Exercise.

The purpose was to determine if the Alu-insertion (I)/deletion (D) polymorphism of the tissue plasminogen activator (tPA) gene influences the tPA response to maximal exercise. Fifty male subjects (age = 23.6 ± 4.7 yrs) completed a maximal treadmill exercise test. Blood samples were drawn before and immediately after exercise for determination of plasma tPA antigen and activity. Isolated DNA was amplified via polymerase chain reaction, electrophoresed, and visually amplified to determine tPA genotype. Subjects were classified as possessing the D allele (D) (n = 28) or being homozygous for the I allele (I) (n = 22). Differences in tPA antigen and activity were assessed using a two-factor (genotype and time) repeated measures analysis of variance. There were significant main effects for time for tPA antigen and activity (p < 0.05), but no main effect for genotype. Furthermore, there was no genotype x time interaction due to a similar increase in tPA antigen in the D group (pre-exercise = 5.83 + 2.39 ng/ml, post-exercise = 21.88 + 7.38 ng/ml) and the I group (pre-exercise = 5.61 + 2.82 ng/ml, post-exercise = 19.05 + 7.67 ng/ml) and a similar increase in tPA activity in the D group (pre-exercise = 0.39 ± 0.19 IU/ml, post-exercise = 9.73 ± 4.22 IU/ml) and I group (pre-exercise = 0.45 ± 0.29 IU/ml, post-exercise = 9.76 ± 5.50 IU/ml). The I/D polymorphism of the tPA gene does not influence the tPA antigen nor tPA activity responses to maximal exercise in healthy, young, sedentary males.

Cyclooxygenase-2 gene transcription in a macrophage model of inflammation.

Infections involving LPS-bearing, Gram-negative bacteria can lead to acute inflammation and septic shock. Cyclooxygenase-2 (COX-2), the target of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors, is importantly involved in these responses. We examined the dynamics of COX-2 gene expression in RAW264.7 murine macrophages treated with LPS as a model for COX-2 gene expression during inflammation. We established, using Northern blotting, nuclear run-on assays, and RT-PCR, that COX-2 transcriptional activation continues for at least 12 h after LPS treatment and involves at least three phases. Previous studies with murine macrophages identified an NF-kappaB site, a C/EBP site, and a cAMP response element-1 (CRE-1) as cis-acting elements in the COX-2 promoter. We identified three additional functional elements including a second CRE (CRE-2), an AP-1 site, and an E-box that overlaps CRE-1. The E-box mediates transcriptional repression whereas the other cis-elements are activating. Using electrophoretic mobility supershift and chromatin immunoprecipitation assays, we cataloged binding to each functional cis element and found them occupied to varying extents and by different transcription factors during the 12 h following LPS treatment. This suggests that the cis elements and their cognate transcription factors participate in a sequential, coordinated regulation of COX-2 gene expression during an inflammatory response. In support of this concept, we found, using inhibitors of Jun kinase and NF-kappaB p50 nuclear localization, that COX-2 gene transcription was completely dependent on phospho-c-Jun plus p50 at 6 h after LPS treatment but was only partially dependent on the combination of these factors at later treatment times.

Partnering between monomers of cyclooxygenase-2 homodimers.

Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin H2 in the committed step of prostanoid biosynthesis. These enzymes are pharmacological targets of nonsteroidal antiinflammatory drugs and cyclooxygenase (COX) 2 inhibitors. Although PGHSs function as homodimers and each monomer has its own COX and peroxidase active sites, the question of whether there is cross-talk between monomers has remained unresolved. Here we describe two heterodimers in which a native subunit of human PGHS-2 has been coupled to a subunit having a defect within the COX active site at some distance from the dimer interface. Native/G533A PGHS-2, a heterodimer with a COX-inactive subunit, had the same specific COX activity as the native homodimer. Native/R120Q PGHS-2, a heterodimer in which both subunits can oxygenate arachidonic acid but in which the R120Q subunit cannot bind the COX inhibitor flurbiprofen, was inhibited by flurbiprofen to about the same extent as native PGHS-2. These results imply that native PGHS-2 exhibits half-of-sites reactivity. Isothermal titration calorimetry established that only one monomer of the native PGHS-2 homodimer binds flurbiprofen tightly. In short, binding of ligand to the COX site of one monomer alters its companion monomer so that it is unable to bind substrate or inhibitor. We conclude that PGHS monomers comprising a dimer, although identical in the resting enzyme, differ from one another during catalysis. The nonfunctioning subunit may provide structural support enabling its partner monomer to catalyze the COX reaction. This subunit complementarity may prove to be characteristic of other dimeric enzymes having tightly associated monomers.

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Bt: mode of action and use.

The insecticidal toxins from Bacillus thuringiensis (Bt) represent a class of biopesticides that are attractive alternatives to broad-spectrum "hard" chemistries. The U.S. Food Quality Protection Act and the European Economic Council directives aimed at reducing the use of carbamate and organophosphate insecticides were expected to increase the use of narrowly targeted, "soft" compounds like Bt. Here we summarize the unique mode of action of Bt, which contributes to pest selectivity. We also review the patterns of Bt use in general agriculture and in specific niche markets. Despite continued predictions of dramatic growth for biopesticides due to US Food Quality Protection Act-induced cancellations of older insecticides, Bt use has remained relatively constant, even in niche markets where Bt has traditionally been relatively high.