Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.     

Presynaptic regulation of dopaminergic transmission in the striatum

1. In vitro studies have indicated that several transmitters present in the striatum can regulate presynaptically the release of dopamine (DA) from nerve terminals of the nigrostriatal DA neurons. 2. The receptors involved in these local regulatory processes are located or not located on DA nerve terminals. 3. Recent in vivo investigations have demonstrated that the corticostriatal glutamatergic neurons facilitate presynaptically the release of DA and have allowed the analysis of the respective roles of presynaptic events and nerve activity in the control of DA transmission.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

Nasal allergy to fungi in guinea pigs

Sensitized guinea pigs produced specific IgG and IgE antibodies toward Cladosporium and Alternaria. In presence of fungal extracts, nasal mast cells degranulate. Ultrastructural modifications of the cells during degranulation have been established. The ciliary epithelium and the ciliary beating are not affected by fungal allergens.     

Galanin and pancreastatin inhibit stimulated insulin secretion in the mouse: comparison of effects

The effects of the two intrapancreatic peptides galanin and pancreastatin on basal and stimulated insulin and glucagon secretion in the mouse were compared. It was found that at 2 min after intravenous injection of galanin or pancreastatin (4.0 nmol/kg), basal plasma glucagon and glucose levels were slightly elevated. Galanin was more potent than pancreastatin to elevate basal plasma glucagon levels: they increased from 60 +/- 15 to 145 +/- 19 pg/ml (p less than 0.01) after galanin compared to from 35 +/- 5 to 55 +/- 8 pg/ml (p less than 0.05) after pancreastatin. Plasma insulin levels were lowered by galanin (p less than 0.05), but not by pancreastatin. CCK-8 (6.3 nmol/kg) or terbutaline (3.6 mumol/kg) markedly increased the plasma insulin levels. Galanin (4.0 nmol/kg) completely abolished the insulin response to CCK-8 (p less than 0.001), but pancreastatin (4.0 nmol/kg) was without effect. Galanin inhibited the insulin response to terbutaline by approximately 60% (p less than 0.01), but pancreastatin inhibited the insulin response to terbutaline by approximately 35% only (p less than 0.05). CCK-8 and terbutaline did both elevate plasma glucagon levels by moderate potencies: neither pancreastatin nor galanin could affect these responses. Thus, in the mouse, galanin and pancreastatin both inhibit basal and stimulated insulin secretion, and stimulate basal glucagon secretion. Galanin is thereby more potent than pancreastatin. The study also showed that galanin potently inhibits insulin secretion stimulated by the octapeptide of cholecystokin and by the beta 2-adrenoceptor agonist terbutaline, and that pancreastatin inhibits terbutaline-induced insulin secretion.     

Effects of training at simulated altitude on performance and muscle metabolic capacity in competitive road cyclists

Differences between the effects of training at sea level and at simulated altitude on performance and muscle structural and biochemical properties were investigated in 8 competitive cyclists who trained for 3-4 weeks, 4-5 sessions/week, each session consisting of cycling for 60-90 min continuously and 45-60 min intermittently. Four subjects, the altitude group (AG), trained in a hypobaric chamber (574 torr = 2300 m above sea level), and the other four at sea level (SLG). Before and after training work capacity was tested both at simulated altitude (574 torr) and at sea level, by an incremental cycle ergometer test until exhaustion. Work capacity was expressed as total amount of work performed. Venous blood samples were taken during the tests. Leg muscle biopsies were taken at rest before and after the training period. AG exhibited an increase of 33% in both sea level and altitude performance, while SLG increased 22% at sea level and 14% at altitude. Blood lactate concentration at a given submaximal load at altitude was significantly more reduced by training in AG than SLG. Muscle phosphofructokinase (PFK) activity decreased with training in AG but increased in SLG. All AG subjects showed increases in capillary density. In conclusion, work capacity at altitude was increased more by training at altitude than at sea level. Work capacity at sea level was at least as much improved by altitude as by sea level training. The improved work capacity by training at altitude was paralleled by decreased exercise blood lactate concentration, increased capillarization and decreased glycolytic capacity in leg muscle.     

The efferent innervation of the genital chamber by an identified serotonergic neuron in the female cricket Acheta domestica

Summary The serotonergic innervation of the genital chamber of the female cricket, Acheta domestica, has been investigated applying anti-serotonin (5-HT) immunocyto-chemistry at both light- and electron-microscopic levels as well as using conventional electron microscopy. Whole mount and pre-embedding chopper techniques of immuno-cytochemistry reveal a dense 5-HT-immunoreactive network of varicose fibers in the musculature of the genital chamber. All of these immunoreactive fibers originate from the efferent serotonergic neuron projecting through the nerve 8v to the genital chamber (Hustert and Topel 1986; Elekes et al. 1987). At the electron-microscopic level, 5-HT-immunoreactive nerve terminals, which contain small (50–60 nm) and large ( 100 nm) agranular vesicles as well as granular vesicles (100nm), contact the muscle fibers or the sarcoplasmic processes without establishing specialized neuromuscular connections. In addition to the 5-HT-immunoreactive axons, two types of immunonegative axons can also be found in the musculature. By use of conventional electron microscopy, three ultrastructurally distinct types of axon processes can be observed, one of which resembles 5-HT-immunoreactive axons. While the majority of the varicosities do not synapse on the muscle fibers, terminals containing small (50–60 nm) agranular vesicles occasionally form specialized neuromuscular contacts. It is suggested that the 5-HTergic innervation plays a non-synaptic modulatory role in the regulation circular musculature in the genital chamber of the cricket, while the musculature as a whole may be influenced by both synaptic and modulatory mechanisms.Fellow of the Alexander von Humboldt-Stiftung     

Degradation of diarylethane structures by Pseudomonas fluorescens biovar I

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.