NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

Statistical shape modelling of the thoracic spine for the development of pedicle screw insertion guides

Spinal fixation and fusion are surgical procedures undertaken to restore stability in the spine and restrict painful or degenerative motion. Malpositioning of pedicle screws during these procedures can result in major neurological and vascular damage. Patient-specific surgical guides offer clear benefits, reducing malposition rates by up to 25%. However, they suffer from long lead times and the manufacturing process is dependent on third-party specialists. The development of a standard set of surgical guides may eliminate the issues with the manufacturing process. To evaluate the feasibility of this option, a statistical shape model (SSM) was created and used to analyse the morphological variations of the T4–T6 vertebrae in a population of 90 specimens from the Visible Korean Human dataset (50 females and 40 males). The first three principal components, representing 39.7% of the variance within the population, were analysed. The model showed high variability in the transverse process (~ 4 mm) and spinous process (~ 4 mm) and relatively low variation (< 1 mm) in the vertebral lamina. For a Korean population, a standardised set of surgical guides would likely need to align with the lamina where the variance in the population is lower. It is recommended that this standard set of surgical guides should accommodate pedicle screw diameters of 3.5–6 mm and transverse pedicle screw angles of 3.5°–12.4°.

     

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

Effects of glucose, glucose plus branched-chain amino acids, or placebo on bike performance over 100km

Madsen, Klavs, Dave A. MacLean, Bente Kiens, and DirkChristensen. Effects of glucose, glucose plus branched-chain aminoacids, or placebo on bike performance over 100 km. J. Appl. Physiol. 81(6): 2644-2650, 1996.This studywas undertaken to determine the effects of ingesting either glucose(trial G) or glucose plusbranched-chain amino acids (BCAA; trialB), compared with placebo (trialP), during prolonged exercise. Nine well-trained cyclists with a maximal oxygen uptake of 63.1 ± 1.5 mlO₂ ^· min^{1 ·} kg¹performed three laboratory trials consisting of 100 km of cycling separated by 7 days between each trial. During these trials, the subjects were encouraged to complete the 100 km as fast as possible ontheir own bicycles connected to a magnetic brake. No differences inperformance times were observed between the three trials (160.1 ± 4.1, 157.2 ± 4.5, and 159.8 ± 3.7 min, respectively). Intrial B, plasma BCAA levels increased from339 ± 28 µM at rest to 1,026 ± 62 µM after exercise(P < 0.01). Plasma ammoniaconcentrations increased during the entire exercise period for allthree trials and were significantly higher intrial B compared withtrials G andP (P < 0.05). The respiratory exchange ratio was similar in the threetrials during the first 90 min of exercise; thereafter, it tended todrop more in trial P than intrials G andB. These data suggest that neitherglucose nor glucose plus BCAA ingestion during 100 km of cyclingenhance performance in well-trained cyclists.

     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.