Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach

Neurotensin (NT) was demonstrated in the central nucleus of the rat amygdala (CNA) using a modification of the avidin-biotin complex immunohistochemical technique. Electron-dense reaction product (particles were 15-25 nm in diameter) was localized in perikarya, dendrites, axons, and axon terminals. It was found also associated with profiles of rough endoplasmic reticulum, mitochondria, microtubules, and small agranular as well as large granular vesicles. In distal dendrites, the reaction product was associated with microtubules, vesicles, and postsynaptic densities. Axon terminals of three types formed synaptic contracts with NT-immunoreactive neurons in the CNA: one was characterized by numerous round or oval agranular vesicles, the second by numerous pleomorphic vesicles, and the third by agranular vesicles that were loosely distributed and pleomorphic. All three types formed symmetric axosomatic and asymmetric axodendritic contacts. NT-immunoreactive axon terminals containing small round agranular vesicles stood out clearly from the intermingling profiles of immunonegative structures. We found numerous glomeruli, each consisting of a central NT-immunoreactive dendrite surrounded by all three types of axon terminals. We observed that some NT-immunoreactive terminals formed symmetric axoaxonal contacts with each other, providing evidence for the presence of local NT-to-NT circuits, whereas many others synapsed with axon terminals devoid of NT immunoreactivity.     

Changes in the lipogenic response to feeding of liver, white adipose tissue and brown adipose tissue during the development of obesity in the gold-thioglucose-injected mouse.

Lipogenic response to feeding was measured in vivo in liver, epididymal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT), during the development of obesity in gold-thioglucose (GTG)-injected mice. The fatty acid synthesis after a meal was higher in all tissues of GTG-treated mice on a total-tissue basis, but the magnitude of this increase varied, depending on the tissue and the time after the initiation of obesity. Lipogenesis in BAT from GTG mice was double that of control mice for the first 2 weeks, but subsequently decreased to near control values. In WAT, lipogenesis after feeding was highest 2-4 weeks after GTG injection, and in liver, lipid synthesis in fed obese mice was greatest at 7-12 weeks after the induction of obesity. The post-prandial insulin concentration was increased after 2 weeks of obesity, and serum glucose concentration was higher in fed obese mice after 4 weeks. These results indicate that increased lipogenesis in GTG-injected mice may be due to an increase in insulin concentration after feeding and that insulin resistance (assessed by lipogenic response to insulin release) is apparent in BAT before WAT and liver.     

A class of linear spectral models and analyses for the study of longitudinal data

Longitudinal data can always be represented by a time series with a deterministic trend and randomly correlated residuals, the latter of which do not usually form a stationary process. The class of linear spectral models is a basis for the exploratory analysis of these data. The theory and techniques of factor analysis provide a means by which one component of the residual series can be separated from an error series, and then partitioned into a sum of randomly scaled metameters that characterize the sample paths of the residuals. These metameters, together with linear modelling techniques, are then used to partition the nonrandom trend into a determined component, which is associated with the sample paths of the residuals, and an independent inherent component. Linear spectral models are assumption-free and represent both random and nonrandom trends with fewer terms than any other mixed-effects linear model. Data on body-weight growth of juvenile mice are used in this paper to illustrate the application of linear spectral models, through a relatively sophisticated exploratory analysis.     

Mobility of secondary structure units of horse-muscle acylphosphatase. Relation to antigenicity

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.     

Modification of Lignins by Growing Cells of the Sulfate-Reducing Anaerobe Desulfovibrio desulfuricans

The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans was grown on medium supplemented with either Kraft lignin or lignosulfonate. Only lignosulfonate contributed to the growth of D. desulfuricans cells, by replacing sulfate, a natural electron acceptor for this microorganism. Kraft lignin added to the culture medium could not substitute for lactate or sulfate, both necessary culture medium components. However, it was found to enhance the viability of D. desulfuricans cells. When changes occurring in lignin during growth of Desulfovibrio cultures were monitored, it was found that both lignin preparations could be partially depolymerized. Spectrophotometric and elemental analysis of biologically treated lignins suggested that both the polyphenolic backbone and lignin functional groups were affected by D. desulfuricans. After treatment, a twofold increase in the sulfur content of Kraft lignin and a minor decrease (14%) in the sulfur content of lignosulfonate were observed. After biological treatment, Kraft lignin and lignosulfonate both bound larger quantities of heavy metals.     

Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme

C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hormonal responses to repetitive brief maximal exercise in humans

The responses of nine men and nine women to brief repetitive maximal exercise have been studied. The exercise involved a 6-s sprint on a non-motorised treadmill repeated 10 times with 30 s recovery between each sprint. The total work done during the ten sprints was 37,693 +/- 3,956 J by the men and 26,555 +/- 4,589 J by the women (M greater than F, P less than 0.01). This difference in performance was not associated with higher blood lactate concentrations in the men (13.96 +/- 1.70 mmol.l-1) than the women (13.09 +/- 3.04 mmol.l-1). An 18-fold increase in plasma adrenaline (AD) occurred with the peak concentration observed after five sprints. The peak AD concentration in the men was larger than that seen in the women (9.2 +/- 7.3 and 3.7 +/- 2.4 nmol.l-1 respectively, P less than 0.05). The maximum noradrenaline (NA) concentration occurred after ten sprints in the men (31.6 +/- 10.9 nmol.l-1) and after five sprints in the women (27.4 +/- 20.8 nmol.l-1). Plasma cardiodilatin (CDN) and atrial natriuretic peptide (ANP) concentrations were elevated in response to the exercise. The peak ANP concentration occurred immediately post-exercise and the response of the women (10.8 +/- 4.5 pmol.l-1) was greater than that of the men (5.1 +/- 2.6 pmol.l-1, P less than 0.05). The peak CDN concentrations were 163 +/- 61 pmol.l-1 for the women and 135 +/- 61 pmol.l-1 for the men. No increases in calcitonin gene related peptide (CGRP) were detected in response to the exercise. These results indicate differences between men and women in performance and hormonal responses. There was no evidence for a role of CGRP in the control of the cardiovascular system after brief intermittent maximal exercise.     

De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. coli.

The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E. coli mutant using an avian liver cDNA expression library. In E. coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins. The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities. Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced. The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins. Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS. The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis. Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.