Forward and reverse genetics of rapid-cycling Brassica oleracea

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today’s B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Peripheral sweat gland function,but not whole-body sweat rate,increases in women following humid heat acclimation

The purpose of this study was to compare both the whole-body and pharmacological-induced sweat rates of men and women following humid heat acclimation.     

Blood lactate and ammonium ion accumulation during graded exercise in humans

The purpose of this study was to determine the pattern of blood lactate and ammonium ion (NH+4) accumulation during graded exercise in humans. Six adult volunteers performed a maximum O2 uptake (VO2 max) test on a bicycle ergometer. Blood samples were collected each minute of the test. Both blood lactate (r = 0.92) and NH4+ (r = 0.70) increased exponentially in relation to increased work. However, closer examination of individual curves revealed that both metabolites remained near resting levels during mild exercise (less than 40% VO2 max) and then demonstrated abrupt upward break points at increased work loads (greater than 50% VO2 max). There was a significant linear relationship (r = 0.96) between the work load at which the lactate break point (LBP) and NH4+ break point (ABP) occurred in each subject. In addition, there was a significant linear relationship (r = 0.82) between the blood concentrations of NH4+ and lactate during exercise. The results suggest a connection between NH4+ production and glycolytic energy metabolism during exercise. Several possible explanations are offered; however, further work at the cellular level is needed before the exact relationship between NH4+ and lactate can be determined.     

Plasma adrenocorticotropin and cortisol responses to brief high-intensity exercise in humans

The purpose of this study is to examine plasma cortisol and adrenocorticotropin (ACTH) levels following a brief high-intensity bout of exercise. Each subject (n = 6) performed a 1-min bout of exercise on a cycle ergometer at 120% of his maximum O2 uptake. Blood samples were collected at rest, immediately following the exercise bout, and at 5, 15, and 30 min postexercise. Mean (+/- SE) plasma ACTH levels increased significantly (P less than 0.05) from 2.2 +/- 0.4 pmol/l at rest to 6.2 +/- 1.7 pmol/l immediately following exercise. Mean (+/- SE) plasma cortisol levels increased significantly from 0.40 +/- 0.04 mumol/l at rest to 0.52 +/- 0.04 mumol/l at 15 min postexercise. These data show that brief high-intensity exercise results in significant increases in plasma cortisol and ACTH levels. Furthermore, the temporal sequence between the two hormones suggests that the increase in plasma cortisol levels following brief high-intensity exercise is the result of ACTH-induced steroidogenesis in the adrenal cortex.     

Exaggerated ANF response to exercise in middle-aged vs. young runners

Hormonal, electrolyte, and renal responses were measured before, during, and after a marathon (42.2 km) in 14 runners: 8 young (Y) (mean age 27.8 yr) and 6 middle aged (MA) (mean aged 46.7 yr). No differences between groups in prerun values for heart rate (HR), plasma osmolality (OSM), antidiuretic hormone (ADH), aldosterone (ALDO), atrial natriuretic factor (ANF), or plasma renin activity (PRA) were found. Renal and urinary measurements were also similar between groups before the marathon. After 10 km of running, both groups had significant increases in HR, ALDO, ANF, and PRA, while OSM, Na+, and ADH remained unchanged from prerun values. The increase in plasma ANF concentrations at this point was significantly greater in the MA subjects compared with the Y (mean increase 104.1 vs. 42.8 pg/ml, respectively; P less than 0.01). Immediate postmarathon values for OSM, ADH, and Na+ were significantly higher than initial values in both groups, while HR, PRA, and ALDO continued to increase above the elevated levels found at 10 km. ANF values immediately postmarathon remained higher than prerun concentrations but were significantly reduced from those obtained at 10 km. In contrast, HR continued to rise until the completion of the run. These data are consistent with recent reports of an exaggerated ANF response in older subjects in response to central blood volume expansion.     

Optimization of Large Scale Preparation of 13-(S)-Hydroperoxy-9Z, 11E-Octadecadienoic Acid Using Soybean Lipoxygenase. Application to the Chemoenzymatic Synthesis of (+)-Coriolic Acid

The optimization of 13-S-hydroperoxy-9Z, 11E-octadecadienoic acid synthesis is described using lipoxygenase-1 from soybeans at high substrate concentration. The optimal values of the tested parameters are as follows: oxygen pressure 2.5 bar, temperature 5°C, pH 11, enzyme concentration 4 mg/ml and substrate concentration 0.1 M. All these values were used in a single reaction, allowing chemoenzymatic synthesis of gram amounts of (+)-coriolic acid (99%, e.e. 97%) with a 54% yield starting from linoleic acid.     

Alterations in plasma membrane lipid organization during lymphocyte differentiation

The fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen-induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.     

Effect of aerobic training on the plasma ACTH response to exercise

The purpose of this study was to reexamine the effect of training on plasma adrenocorticotropin (ACTH) levels during exercise. Ten adult volunteers were split into a control and an experimental group. The experimental group participated in a 12-wk training program that resulted in a significant 11% increase in their mean maximal O2 uptake. The plasma ACTH response to a 150-W work rate was measured in both groups before and after the training program. The experimental group demonstrated a significant reduction in the ACTH response (11 vs. 4 pg/ml) to the work rate, whereas the control group demonstrated an unchanged response (16 vs. 13 pg/ml) over the course of the study. These data suggest that the ACTH response to an absolute submaximal work rate is blunted after training.