GDP-fucose synthetase from Escherichia coli: structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site.

BACKGROUND: . In all species examined, GDP-fucose is synthesized from GDP-mannose in a three-step reaction catalyzed by two enzymes, GDP-mannose 4,6 dehydratase and a dual function 3, 5-epimerase-4-reductase named GDP-fucose synthetase. In this latter aspect fucose biosynthesis differs from that of other deoxy and dideoxy sugars, in which the epimerase and reductase activities are present as separate enzymes. Defects in GDP-fucose biosynthesis have been shown to affect nodulation in bacteria, stem development in plants, and are associated with the immune defect leukocyte adhesion deficiency type II in humans. RESULTS: . We have determined the structure of GDP-fucose synthetase from Escherichia coli at 2.2 A resolution. The structure of GDP-fucose synthetase is closely related to that of UDP-galactose 4-epimerase and more distantly to other members of the short-chain dehydrogenase/reductase family. We have also determined the structures of the binary complexes of GDP-fucose synthetase with its substrate NADPH and its product NADP+. The nicotinamide cofactors bind in the syn and anti conformations, respectively. CONCLUSIONS: . GDP-fucose synthetase binds its substrate, NADPH, in the proper orientation (syn) for transferring the 4-pro-S hydride of the nicotinamide. We have observed a single binding site in GDP-fucose synthetase for the second substrate, GDP-4-keto,6-deoxy-mannose. This implies that both the epimerization and reduction reactions occur at the same site in the enzyme. As is the case for all members of the short-chain family of dehydrogenase/reductases, GDP-fucose synthetase retains the Ser-Tyr-Lys catalytic triad. We propose that this catalytic triad functions in a mechanistically equivalent manner in both the epimerization and reduction reactions. Additionally, the X-ray structure has allowed us to identify other residues that are potentially required for substrate binding and catalysis.

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose.

BACKGROUND: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). RESULTS: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. CONCLUSIONS: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Sleep patterns and gastric acid secretion in duodenal ucler disease.

Five normal volunteers and five patients with duodenal ulcer (DU) disease were studied for five consecutive nights. All subjects underwent placement of a nasogastric tube, continuous collection of gastric juice, and continuous electroencephalographic monitoring of sleep. Gastric juice was collected in 20-minute samples by remote suction (Gomco). Blood samples were drawn every 20 minutes on the third night via an indwelling venous needle. Results showed no significant correlations between the sleep variables and the gastric acid secretion measures or between the sleep variables and serum gastrin levels. Acid secretion decreased from hour 1 to hour 2 in controls and in patients with inactive DU disease, while it increased in patients with active DU disease. Each subject had at least one night of recording in which continuous gastric secretion was less than 0.1 mEq per 20-minute sample. It appears unlikely that the gastric discomfort of DU patients can be attributed to acid hypersecretion triggered by rapid eye movement sleep.

Effect of sleep state and position on the incidence of obstructive and central apnea in infants.

Sixty-four infants with a history of apnea were studied to determine the effects of sleeping position and sleep state (rapid eye movement [REM]) v (non-rapid eye movement [NREM]) on the occurrence of central and obstructive apneas. All-night polysomnographic studies were conducted on each infant, and the spontaneous occurrence of central and obstructive apneic events was determined in the prone, supine, and side positions. Sleeping position did not significantly affect the rate or duration of central or obstructive apneas. Furthermore, neither central nor obstructive apneic episodes were significantly altered by sleep state. These data suggest that, in spite of an ostensible predisposition to upper airway obstruction in the supine position and during rapid eye movement sleep, neither sleeping position nor sleep state appears to affect the rate of duration of apneic events.

Sleep disturbances after open heart surgery.

This study was designed to document quantitatively the sleep disturbances that occur after open heart surgery and to investigate a group of patients who underwent a thoracic surgical procedure not involving cardiopulmonary bypass. Nine patients were studied, six after open heart surgery and three after partial or complete pneumonectomy. In each patient, sleep patterns were recorded with use of all night polygraphy before and after operation and for up to 5 weeks on follow-up studies. After open heart surgery, patients manifested considerable suppression of both rapid eye movement and slow wave sleep patterns. In the three patients subjected to thoracotomy these sleep indexes returned to preoperative levels much earlier. Evidence of stage 2 sleep was present in one of the three patients with thoracotomy on the first postoperative night, and in two of the three both rapid eye movement and slow wave sleep returned to preoperative levels by the time of hospital discharge. It is concluded that patients undergoining open heart surgery experience both acute and chronic disruptions of sleep that last well beyond the hospital period of convalescence. These sleep disturbances have considerable relevance for postoperative management.

Postprandial sleepiness: objective documentation via polysomnography.

Fifteen normal volunteers were evaluated to assess the effect of a meal on sleep onset latency. The meal was administered in a counterbalanced design on 1 of 2 successive days. Subjects napped 20 min subsequent to the meal (or at the corresponding time on the no-meal day) and 1 h after the initiation of the first nap. Ten subjects completed the Stanford Sleepiness Scale (SSS) on arriving at the laboratory, and just prior to nap 1 and nap 2. Sleep onset latency after the meal was not significantly different from that obtained under the no-meal condition, but was significantly less on nap 1 as compared with nap 2 irrespective of day or meal. SSS did not reveal subjective differences in sleepiness between the initial estimate and the postmeal estimate. Only five subjects showed a decrease in sleep onset latency postprandially (1-11 min). Although group differences in postprandial sleepiness were not documented, the phenomenon was clearly exhibited by certain individuals. Thus, postprandial sleepiness is not an invariable consequence of meal ingestion; rather, it appears to be affected by numerous variables such as hunger, volume of the meal, and meal constituents.

Progesterone levels and sleep-related breathing during menstrual cycles of normal women.

The respiratory stimulant effect of progesterone has been known for many years and has led to the hypothesis that this hormone protects young, premenopausal women from disordered breathing and apnea during sleep. Therefore, sleep, breathing, and gonadal hormone parameters were evaluated for 11 normal, menstruating women during times of high and low progesterone levels. No sleep or breathing parameter changed significantly with varying levels of progesterone. Although normal women show a significant change in progesterone levels across the course of the menstrual cycle, the levels achieved did not produce significant changes in breathing parameters.

Nocturnal levels of growth hormone in hyperactive children of small stature.

Growth disturbances have been demonstrated in hyperactive children treated with stimulant medication. This study examines sleep related growth hormone secretion in patients with hyperactive syndrome and relative short stature. Five such patients were compared with 9 age matched controls. There were no differences in sleep patterns, and growth hormone levels for the hyperactive children were within normal limits. This study suggests that hyperactive children with short stature have normal sleep patterns and normal sleep related growth hormone secretion.

Hypersomnolent and nonhypersomnolent patients with upper airway obstruction during sleep.

When the syndrome consisting of sleep-induced apnea and hypersomnolence is due to upper airway obstruction, the hypersomnolence is believed to be the direct result of deprivation of sleep related to such obstructions. The purpose of this report is to describe a group of four asymptomatic subjects with upper airway obstruction during sleep. These subjects were matched with a group of patients with the syndrome of sleep-induced apnea and hypersomnolence. There were no significant differences between symptomatic and asymptomatic groups in terms of the absolute number of upper airway obstructions (252 vs 231), their mean duration (20.8 vs 25.9 seconds), the mean arterial carbon dioxide tension during sleep (39 vs 39 mm Hg), or the electroencephalographic patterns during sleep. The only variables that emerged as significantly different between the two groups were the weights (128 vs 90 kg; P less than 0.05), the low arterial oxygen pressure (PaO2) on waking (54 vs 80 mm Hg; P less than 0.002), and the lower PaO2 during sleep (47 vs 70 mm Hg; P less than 0.01) in the symptomatic patients. From these data, we conclude that the hypersomnolence in patients with sleep-induced apnea due to upper airway obstruction cannot be explained by deprivation of sleep, and other factors need to be carefully examined in future studies.

Factors involved in visual capture.

Visual capture was explored using three types of vision-"touch" conflicts. The results indicated that the amount of visual capture differed for the three tasks. In one task (slant judgments) strong, but incomplete, visual capture occurred, in another (a length judgment task) an approximate compromise between the two modalities was found, and in the third task (texture judgments) a trend toward touch capture occurred. In two additional experiments using the slant task, the effects of brief training with one of the competing modalities and the effects of manipulating certain aspects of the stimulus display were explored. The bried training did not alter the resolution of the conflict but varying visual clarity and felt texture of the rod whose slant was being judged did affect visual capture.

Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions.

We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. In developing the system the entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface. A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E. coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin. To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies. We have unambiguously mapped three different antibody epitopes using this method. Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions. This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation.

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.

Computer-assisted chromosome mapping by protoplast fusion in Staphylococcus aureus.

Protoplasts of genetically marked derivatives of Staphylococcus aureus NCTC 8325 were fused with polyethylene glycol and regenerated without selection. Recombinants possessing one specific resistance marker from each parent were selected from the regenerated population and scored for seven or eight unselected markers. The results of these 9- and 10-factor crosses were entered directly into a programmed microcomputer from prescored replica plates. The data then were condensed into an array of phenotypes, together with the frequency with which each occurred. Further analyses by computer included the calculation of coinheritance frequencies for all possible pairs of markers; after entering a proposed order for the markers being analyzed, the minimum number of crossover events required to generate each phenotypic class was calculated. The linkage relationships of markers, based on the protoplast fusion data, were entirely consistent with the linkage relationships of markers already known to exist within each of the three linkage groups previously defined by transformation. The fusion data defined an arrangement of the three linkage groups into a circular chromosome map and predicted the approximate location of four previously unmapped markers (tet-3490, fus-149, purC193::Tn551, and omega [Chr::Tn551]42) on this map.

Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA.

Transformation provided definitive evidence for linkage between tyrB282::Tn551 ermB321 and omega (Chr::Tn551)34, and thus between the separate large linkage groups containing these markers, in Staphylococcus aureus NCTC 8325. Transformation also defined the chromosomal loci for the purC193::Tn551 and omega (Chr::Tn551)42 markers and the linkage of a tetracycline resistance marker (tet-3490) with a fusidic acid resistance marker (fus-149). The use of DNA isolated from protoplasts under conditions that reduced hydrodynamic shear greatly facilitated the demonstration of most of these linkages. These results provide direct evidence confirming several of the linkages predicted by a microcomputer-assisted protoplast fusion analysis in a previous study (M. L. Stahl and P. A. Pattee, J. Bacteriol. 154:395-405, 1983); those markers whose predicted linkages were not confirmed by transformation are probably separated by chromosomal distances that exceed the limits of detection by transformation, even with protoplast DNA.

Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation.

The entire subtilisin structural gene from Bacillus subtilis I168 has been cloned, and its nucleotide sequence has been determined. When expressed on a high-copy-number shuttle vector, a fivefold increase in serine protease activity was observed. The DNA sequence of the gene is 80% homologous to the Bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin BPN'. The chloramphenicol resistance determinant of a plasmid integrated at the subtilisin locus was mapped by PBS1 transduction and was found to be linked to glyB (83%) and argC (60%), but not with metC or purB . The chromosomal locus containing the wild-type subtilisin allele was replaced with an in vitro-derived allele of the gene (delta apr-684) that contained a 684-base-pair deletion. The technique used for introducing the deletion is a variation of the gene replacement methods used in Saccharomyces cerevisiae and Escherichia coli. When used in B. subtilis, deletion mutants could be directly screened among the transformants. Physiological characterization of the delta apr-684 mutation revealed no discernable effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity. A model is presented that outlines the pathway for plasmid integration and deletion formation in B. subtilis.

Immunofluorescent evidence of Proteus mirabilis swarm cell formation on sterilized rat feces.

Swarming Proteus spp. were detected with the use of proteometry (a most-probable-number technique) in the fecal material of selected animal species and in raw sewage from a local sewage treatment plant. Proteus spp. were not detected in any of several soil and freshwater samples examined. Since rat feces harbored high numbers of Proteus mirabilis compared with other habitats examined, we chose to examine it for the possibility of supporting swarming. Immunofluorescent studies with a strain-specific conjugate revealed the morphogenesis of short forms into elongated swarm cells upon the surface of sterilized rat feces that had been inoculated with short forms of P. mirabilis. the same phenomenon was not observed consistently when nonsterile rat feces were inoculated and examined with immunofluorescence.

Extracorporeal circulation causes release of neutrophil gelatinase-associated lipocalin (NGAL).

Extracorporeal circulation (ECC) used during cardiac surgery causes activation of several inflammatory systems. These events are not fully understood but are responsible for complications during the immediate postoperative period. Neutrophil gelatinase-associated lipocalin (NGAL), a member of the expanding lipocalin family, has recently been described as an inflammatory protein. In this study, the release of NGAL into the circulation in 41 patients undergoing heart surgery with ECC was evaluated. A 4- to 5-fold elevation of the concentration of NGAL in plasma was observed during the immediate postoperative course with a rapid elimination during the first postoperative day. Four patients undergoing lung surgery (without ECC) were also studied. The plasma concentration of NGAL only increased with a factor of 1.1-2.2 over the operation. We conclude that NGAL is released into the circulation during heart surgery, probably as a result of the inflammatory activation of leukocytes initiated by the extracorporeal circulation.

Deficiency of vitamin B6 in women taking contraceptive formulations.

The specific activities (S.A.) of the glutamic oxaloacetic transaminase from erythrocytes (EGOT) of 75 women taking 16 diversified contraceptive formulations were determined by the principle (CAS) of unsaturation and saturation of receptors of the Coenzyme-Apoenzyme-System with the coenzyme, pyridoxal 5'-phosphate. 52 women were not taking pyridoxine; 20 were taking 1-5 mg; 3 were taking 25-100 mg. The mean basal S.A. of the 52 women without pyridoxine was lower (p less than 0.01) than for the 20 on 1-5 mg. The mean basal S.A. for the women on 25-100 mg was higher (p less than 0.01) than for the women without pyridoxine. The mean % deficiency for the women on 25-100 mg was negligible and lower (p less than 0.01) than 18+/-8 for the 52 women, and 14+/-6 for the 20 women. These data indicate that 1-5 mg of pyridoxine is inadequate for women on contraceptives. The requisite daily dosage is projected at 50-100 mg. Other data indicate that 5-12 weeks of supplementation with pyridoxine can be required to reach a stable "ceiling" of S.A. of EGOT. This period indicates some regulatory mechanism by diminished levels of the apoenzyme upon gene expression to bring about normal levels of the transaminase.

Sleep and dreaming disturbances in closed head injury patients.

Single night sleep recordings in closed head injury patients 6 to 59 months after injury revealed less stage 1 and a greater number of awakenings compared to age matched controls. Neither the time spent in REM sleep nor the Wechsler Memory Quotient were related to complaints of decreased or absent dreaming following injury. The proportion of REM and number of awakenings, however, showed a moderate relationship to certain behavioural problems.

Organochlorine pesticide and polychlorinated biphenyl residues in Canada geese (Branta canadensis) from Chicago, Illinois.

Breast muscle samples, with or without overlying adipose tissue and skin, were obtained from Canada geese collected in northeastern illinois while undergoing feather molt. Specimens were evaluated for contaminant concentrations to determine if they would be acceptable as human food provided through government-subsidized programs. Samples were baked, allowing fat to drip free, and assayed for persistent organochlorine pesticides and polychlorinated biphenyls. Residues of heptachlor epoxide, dieldrin, DDE and PCBs (as Arochlor 1248) were detected. The specimens contained relatively low concentrations of contaminants, such that US Department of Agriculture residue limits for meat were exceeded in only 1 sample. Baking of breast muscle without the overlying skin and adipose tissue resulted in reductions in concentrations of detectable compounds. Fewer samples baked with the skin attached had detectable concentrations of heptachlor epoxide, dieldrin and PCB then samples cooked without skin; however, the converse was true for DDE. Periodic monitoring for environmental contaminants such as PCBs, exclusion of geese from localities where samples have contaminants such as PCBs, exclusion of geese from localities where samples have contaminants at concentrations that exceed recommended dietary limits, the use of processing and/or cooking methods which remove large amounts of lipid, and advisories that provide information on known health risks are recommended if wild resident Canada geese from the Chicago area are provided as food for underprivileged humans.