Rostrocaudal variation of fiber type composition in rat intercostal muscles

Summary We used the histochemical stain for ATPase to compare the fiber-type composition of rat internal and external intercostal muscles from thoracic (T) segments 2–5, 8, and 11. At each level, type II fibers were more numerous than type I fibers, type II B fibers were more numerous than II A fibers, and type I fibers were more numerous in external than in internal intercostals. However, fiber type composition varied from segment to segment. For example, the proportion of type II A fibers increased in a rostrocaudal gradient in internal but not external intercostals, and type I fibers were more prevalent at rostral and caudal than at intermediate levels in both internal and external intercostals. These results provide a basis for interpreting previous physiological and molecular studies which have compared intercostal muscles from different segmental levels.     

Mechanistic studies of cAMP-dependent protein kinase action

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of helix-based vasoactive intestinal peptide analogues: identification of residues required for receptor interaction

Several VIP analogues have been designed on the basis of the hypothesis that the region from residue 6 to residue 28 forms a pi-helical structure when bound to membrane receptors. An empirical approach for the design and construction of analogues based upon distribution frequency and structural homology with several sequence-related peptides is presented. Five peptides were designed, synthesized, and analyzed. One analogue, model 5, containing the native hydrophobic and an altered hydrophilic surface, was an effective VIP agonist in both binding to rat lung membrane receptors (KD1 = 11 +/- 8 pM, KD2 = 6.4 +/- 0.2 nM; VIP KD1 = 21 +/- 13 pM, KD2 = 1.8 +/- 0.6 nM) and stimulation of amylase release from guinea pig pancreatic acini (ED50 = 90 pM; VIP ED50 = 27 pM). The four other analogues were considerably less potent than VIP, yet retained full intrinsic activity. Our results showed that the hydrophobic surface of this helical domain (residues 6-28) contains amino acids important for interaction with receptors, whereas amino acid residues on the hydrophilic surface do not seem to participate strongly in receptor binding or signal transduction. Furthermore, on the basis of high-affinity binding, the stimulation of amylase release in pancreatic acini appears to be coupled to the higher affinity receptors. These results suggest that an approach based on the construction of putative pi-helical structures can be applied to the design of biologically active analogues of VIP. Thus, we have identified several residues within the VIP sequence that are critical for receptor binding using this approach.     

dsg, a gene required for Myxococcus development, is necessary for cell viability.

Previous work identified the dsg gene as necessary for cell-cell interaction in Myxococcus xanthus. Point mutations of this gene, such as dsg-439, are viable, but insertions of Tn5 within the dsg gene (dsg::Tn5) are lethal. Partial diploids, dsg::Tn5/dsg+ or dsg::Tn5/dsg-429 or dsg::Tn5/dsg-439, are also viable, showing that the lethal effect of the haploid insertions is due to loss of function. Thus the evidence implies that the dsg gene is essential for viability as well as development, but its essential quality differs between growth and development because dsg-429 and dsg-439 mutants grow normally, but are unable to develop.     

Experimental determination of nitrogen kinetic isotope fractionation: Some principles; illustration for the denitrification and nitrification processes

Summary A few principles relative to the presentation and use of nitrogen stable isotopic data are briefly reviewed. Some classical relationships between the isotope composition of a substrate undergoing a single-step unidirectional reaction, are introduced. They are illustrated through controlled experiments on denitrification in a soil, and through nitrification by pure cultures ofNitrosomonas europaea. In the latter case, the isotope fractionation is calculated from the isotopic composition of the residual substrate, then of the product and the result is shown to be statistically the same for the two procedures. The isotopic enrichment factor for denitrification is −29.4±2.4‰ at 20°C, and −24.6±0.9‰ at 30°C; for nitrification this factor is −34.7±2.5‰ under the experimental conditions employed.     

Aggregate formation and organo-mineral association affect characteristics of soil organic matter across soil horizons and parent materials in temperate broadleaf forest

Precise assessment of soil organic carbon (OC) storage requires understanding of soil type and depth specific differences in organic matter (OM) stabilization. Therefore, we aimed to analyze OC dynamics down the soil profile and to clarify the effect of depth on the importance of aggregate formation and mineral adsorption for OC storage in mature beech forest soils developed from different parent materials. Aggregate size and density fractions were separated from samples of top and subsoil horizons, which were quantified and analyzed by infrared spectroscopy. We also determined the microbial biomass C (C_mic) and the amount of C decomposed within incubation experiments (CO₂-C) for the bulk soil samples. OC stabilized via aggregate formation and mineral association significantly increased with soil depth. However, this stabilized pool seemed to fuel the labile OM stronger in the subsoil than in the topsoil because the CO₂-C/SOC and CO₂-C/C_mic ratios increased with depth. Measured differences in the magnitude of the detected stabilization and destabilization patterns were attributed to parent material and soil horizon, indicating pronounced spatial and vertical heterogeneity in the contribution of soils under temperate broadleaf forest to terrestrial C sequestration. Considering such site and depth specific differences will improve assessment and modelling of soil OC storage for areas covered with the same forest type but with high pedogenetic diversity.