Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

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.     

Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter

The chloramphenicol acetyltransferase (CAT) gene is widely used in recombinant constructs employed to study promoter and enhancer control of gene expression. However, CAT-based assays require a laborious, multi-step procedure for quantitation of promoter activity. We have applied the recently described firefly luciferase (LUC) reporter gene to the study of the interleukin-2 (IL2) promoter and have further defined the properties of this reporter gene system. We find that IL2-LUC constructs have multiple advantages over IL2-CAT constructs. The LUC assay is highly sensitive and requires 1/10 the cells used in the CAT system. A final quantitative measure of promoter activity can be obtained within 25 h following transfection with IL2-LUC, compared to 108-160 h with IL2-CAT. Light emission significantly (fourfold) above background is detectable 3 h after induction in a direct assay of extracts from transfected cells. We have described the variability of the assay, the minimum number of transfected cells required to detect light, the stability of luciferase in cell extracts, the effect of Triton X-100 on the assay, and a rapid cell lysis procedure. The luciferase system is a simple, rapid, and sensitive method for the study of promoter activity in transfected cells, particularly for weakly expressed genes such as IL2 which give low activity in the CAT assay.     

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)     

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1-2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.