Warm-up rates during arousal from torpor in heterothermic mammals: physiological correlates and a comparison with heterothermic insects

Summary This study examines the relationship between warm-up rate, body mass, metabolic rate, thermal conductance and normothermic body temperature in heterothermic mammals during arousal from torpor. Predictions based on the assumption that the energetic cost of arousal has been minimised are tested using data for 35 species. The observation that across-species warm-up rate correlates negatively with body mass is confirmed using a comparative technique which removes confounding effects due to the non-independence of species data due to shared common ancestry. Mean warm-up rate during arousal correlates negatively with basal metabolic rate and positively with the temperature difference through which the animal warms, having controlled for other factors. These results suggest that selection has operated to minimise the overall energetic, cost of warm-up. In contrast, peak warm-up rate during arousal correlates positively with peak metabolic rate during arousal, and negatively with thermal conductance, when body mass has been taken into account. These results suggest that peak warm-up rate is more sensitive to the fundamental processes of heat generation and loss. Although heterothermic marsupials have lower normothermic body temperatures and basal metabolic rates, marsupials and heterothermic eutherian mammals do not differ systematically in warm-up rate. Pre-flight warm-up rates in one group of endothermic insects, the bees, are significantly higher than predictions based on rates of arousal of a mammal of the same body mass.Abbreviations BMR basal metabolic rate - ICM independent comparisons method - MWR mean warm-up rate - PMR peak metabolic rate - PWR peak·warm-up rate - Tb_activity body temperature during activity - Tb_torpor body temperature during torpor - T _arousal increase in body temperature during arousal     

Secondary structure-based profiles: use of structure-conserving scoring tables in searching protein sequence databases for structural similarities

The profile method, for detecting distantly related proteins by sequence comparison, has been extended to incorporate secondary structure information from known X-ray structures. The sequence of a known structure is aligned to sequences of other members of a given folding class. From the known structure, the secondary structure (alpha-helix, beta-strand or "other") is assigned to each position of the aligned sequences. As in the standard profile method, a position-dependent scoring table, termed a profile, is calculated from the aligned sequences. However, rather than using the standard Dayhoff mutation table in calculating the profile, we use distinct amino acid mutation tables for residues in alpha-helices, beta-strands or other secondary structures to calculate the profile. In addition, we also distinguish between internal and external residues. With this new secondary structure-based profile method, we created a profile for eight-stranded, antiparallel beta barrels of the insecticyanin folding class. It is based on the sequences of retinol-binding protein, insecticyanin and beta-lactoglobulin. Scanning the sequence database with this profile, it was possible to detect the sequence of avidin. The structure of streptavidin is known, and it appears to be distantly related to the antiparallel beta barrels. Also detected is the sequence of complement component C8, which we therefore predict to be a member of this folding class.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Tyrosine Hydroxylase Activity in Rat Brain Synaptosomes: Direct Measurement Using High Performance Liquid Chromatography

Abstract: By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three- to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.     

Was globin evolution very rapid in its early stages?: A dubious case against the rate-constancy hypothesis

Summary Goodman et al's (1975) claim of accelerated evolution in the early stages of globin evolution is based on an erroneous assignment of the time of divergence of vertebrate myoglobin and hemoglobin. When this is corrected, there is no basis for their claim. The data are much more consistent with the nearly constant rate expected on the neutral mutation-random drift hypothesis than with the uneven rates expected if most amino acid changes were caused by substitution of favorable mutants through Darwinian selection. In addition, the majority of the codons determined by their maximum parsimony method have turned out to be wrong when compared to the actual nucleotide sequences of rabbit and human hemoglobins determined by direct sequencing.Contribution No. 1314 from the National Institute of Genetics, Mishima, 411 Japan.     

C andH NMR spectroscopy as a tool in the configurational analysis of iridoid glucosides

The ¹³C NMR data of 51 iridoid glucosides or glucoside acetates are tabulated. The collection includes 20 pairs of C-6, C-7 or C-8 epimers. Three parameters in using the data for the configurational assignment of 6-O-substituents are given. The chemical shift for C-9 in a range of substituted compounds is shown to be numerically related to the stereochemistry at C-8. This allows the determination of the configuration at this centre for most types of substitution patterns by calculation of the C-9 shift using increments for each substituent. Such increments are given for 25 substituents in three different solvents. A method for simulation of spectra of unknown iridoid glucosides is presented. By this method, the structures of five novel iridoid glucosides have been elucidated, and that of tecomoside has been revised. The methods used to assign the configurations to C-6 and C-8 epimeric iridoid glucosides by ¹H NMR spectroscopy are discussed and a table with selected data is presented. It is suggested that the structures in the literature for ajugol and myoporoside should be interchanged. Consequently, Horeau's method has failed in these instances. Finally, the differences in the ¹³C NMR spectra of pairs of C-6 and C-8 epimeric iridoid glucosides have been interpreted as originating from cis/trans-interactions.     

The effect of sulfur on the response of cotton to urea under alkali soil conditions in pot experiments

Summary The effect of sulfur (S) placement and S rate on the efficiency of urea (U) relative to ammonium sulfate (AS) and ammonium nitrate (AN) for cotton were examined in a pot experiment using sandy clay loam soil (pH 7.9). The results showed that AS and AN application in the absence of S increased the yield than U partly because U-induced damage to plants. The combined application of the N sources with S increased the yields and that, the placement of S in the seed horizon in contact with N was more effective than mixing throughout the soil especially with U. These effects were observed with three cotton cultivars. The addition of S to a maximum of 1.5 g/pot gave further increases in yields or the N content of leaves for U, AS or AN. Using the least squares method, it was found that the presence of S significantly increased the efficiency of U than AS or AN. Incubation of S and the N sources with S in the soil was carried out to understand the growth conditions of cotton fertilized by U in alkalin soil. In the case of U-soil system, the pH increased. NO₂−N accumulated and considerable loss of N took place. The pH, NO₂−N accumulation and the loss of N decreased with S increments.     

Diterpenoids from Ajuga chamaepitys: Two neo-clerodane derivatives

From the whole plant of Ajuga chamaepitys two new neo-clerodane diterpenoids, ajugapitin and its dihydro derivative, have been isolated. Their