High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.     

Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.     

Single crystals of V amylose complexed with isopropanol and acetone

Single crystals of amylose complexed with isopropanol or acetone were prepared by adding these precipitants to a metastable aqueous solution of amylose. With both precipitants, similar micrometre sized platelet crystals were obtained. They gave indistinguishable electron diffraction diagrams which could be indexed in an orthorhombic unit cell, with a = 28.26 A, b = 29.30 A, c = 8.01 A and in a space group P2(1)2(1)2(1) or P2(1)2(1)2. Within the unit cell, the amylose chains are organized in antiparallel pairs of parallel 6(5) amylose helices occupying 70% of the cell content, the remaining 30% consisting of isopropanol/acetone and water, with an estimate of 10 isopropanol/acetone molecules for 52 water molecules per unit cell. If the crystals are suspended in pure isopropanol at various temperatures or in pure methanol at room temperature, they undergo a de-solvation process that ultimately converts them into VH amylose. De-solvation with isopropanol left the crystals intact whereas with methanol, they became cracked during the shrinkage. An explanation is proposed for such difference.     

Plasma catecholamines in the aorta and the phrenicoabdominal vein in exercising dogs

Plasma epinephrine and norepinephrine concentrations were measured in the aorta and phrenicoabdominal vein in five dogs at rest and during short-duration mild- and moderate-intensity exercise and during prolonged mild-intensity exercise. Plasma epinephrine and norepinephrine concentrations increased with exercise in both the aorta and the phrenicoabdominal vein. Plasma epinephrine concentration was much higher in the phrenicoabdominal vein than in the aorta (24-43 times). Plasma epinephrine concentrations in the aorta and phrenicoabdominal vein were significantly correlated (r = 0.88). This confirms that peripheral epinephrine concentration is a reliable index of the activity of the adrenal medulla during exercise. The epinephrine-to-norepinephrine ratio in the phrenicoabdominal vein was stable (4:1) throughout the experimental protocol, suggesting that the proportion of the two amines released by the adrenal medulla did not vary through this range of adrenal activity in dogs.     

Exogenous 13C glucose oxidation during exercise: North American vs Western European studies

The purpose of this study was to test the hypothesis that the well-documented changes in background ¹³C enrichment of expired CO₂ observed in response to exercise and carbohydrate ingestion, in subjects living on a North American diet, are not present in subjects living on a Western European diet. The experimental protocol used by Pirnay et al. in 1977 and by Krzentowski et al. in 1984 in subjects living on a Western European diet (4 h of exercise on a treadmill at 50% VO_2max with ingestion of 100 g of glucose in 400 ml of water) was duplicated as closely as possible in six subjects living on a North American diet. The actual amounts of exogenous glucose oxidized, computed with a high artificial ¹³C enrichment of glucose (+189.7 ¹³C PDB-1) which allows one to neglect the 1–2 changes in ¹³C background, were [mean (SEM)] 54.7 (5.4) and 84.2 (3.4) g over 2 h and 4 h of exercise, respectively. These values compare well with data computed by Pirnay et al. [56.6 (13.1) and 94.9 (4.2) g] and by Krzentowski et al. [55.0 (6.2) and 88.0 (4.5) g] using a natural enrichment of glucose (–11.21 and –10.63 ¹³C PDB-1, respectively) assuming no change in ¹³C background in their Western European subjects. Under the same assumption and using a natural enrichment of glucose (–11.30 ¹³C PDB-1) the oxidation of exogenous glucose was overestimated by 30–40% in our North American subjects. This result indicates that because of a lower input of ¹³C in their diet, the difference between the isotopic composition of carbohydrate and fat stores are smaller, and changes in ¹³C background are small or absent in response to moderate workload in Western European subjects, when compared to their North American counterparts.     

Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.     

HGH20k species and variability of GH responses to long-duration exercise in male cyclists fed different food supplements.

Exercise studies dealing with hGH have always considered this hormone as a unique molecular entity. We postulated that the well-known variability in blood total hGH response could possibly be explained, at least in part, by concomitant changes in blood hGH20k levels, variant form possibly expressing some of the hGH anti-insulinic properties. Six male trained cyclists were imposed a 2-hr long ergocycle exercise. Food supplements were given prior to and/or during exertion to exacerbate a possible contribution from hGH20k to total hGH variability by modification of substrate availability. Both blood total hGH and hGH20k levels increased with exercise, the largest increases being observed in absence of supplementation. Large variability of responses were observed in both blood total hGH and hGH20k levels, the latter variant contributing minimally to total blood hGH response (4.3 +/- 0.8%), and being closely associated with the main species (r = 0.90; p less than 0.001). It was concluded that variations associated with hGH20k increases observed in response to prolonged exercise cannot explain the large intra-and inter-individual variability measured in blood total hGH response.