The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose.

The biological interconversion of galactose and glucose takes place only by way of the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P uridylyltransferase, and UDP-galactose 4-epimerase. The only biological importance of these enzymes appears to be to provide for the interconversion of galactosyl and glucosyl groups. Galactose mutarotase also participates by producing the galactokinase substrate alpha-D-galactose from its beta-anomer.

Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants.

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L.

Brightness of uniform stabilized fields.

Brightness of uniform fields during normal and stabilized viewing was determined as a function of adapting luminance, field size, and luminance gradient of the edges of the adapting field. In one set of experiments, it was found that, over a range of adapting luminances from 6 to 9600 td, a uniformly-illuminated 7.5 deg hemifield appeared about 1 log unit brighter in normal viewing than when it was retinally-stabilized.

Prevalence of self-reported erectile dysfunction in people with long-term IDDM.

Objective: The purpose of this report is to examine the prevalence of erectile dysfunction and relationships to other characteristics in men with younger-onset diabetes.

Research Design And Methods: In a population-based cohort study in southern Wisconsin, prevalence of erectile dysfunction was measured based on self reports in men who were 21 years of age or older, were < 30 years of age at diagnosis of diabetes, had 10 or more years of diabetes, and were taking insulin (n = 365).

Results: Of the study group, 20% reported a history of erectile dysfunction.

Observer weighting of concurrent binaural information.

Two experiments examined how listeners weight binaural information of individual components in a multicomponent complex when they are instructed to use this information in various ways. On each experimental trial of the first experiment, a two-component complex consisting of a 553- and 753-Hz pure tone was presented to listeners over headphones. Each component had an interaural difference of time (IDT) that was randomly chosen from a single distribution.

Crystals and structures of cytochrome c oxidases--the end of an arduous road.

Crystal structures of cytochrome c oxidases, one of which is the largest membrane-bound protein complex crystallized to date in a form suitable for X-ray diffraction, have recently been solved. The information from these accomplishments confirms many of the structural properties known from earlier spectroscopic and analytical studies, and provides a basis for understanding the complex mechanisms of electron transfer and proton pumping.

Mechanisms of phosphoryl and acyl transfer.

Acyl and phosphoryl transfer are important biochemical reactions. We have been using isotope effects caused by O-18, N-15, C-13, and deuterium substitution to examine the mechanisms and transition-state structures for enzymatic and nonenzymatic transfers of phosphoryl and acyl groups. Phosphoryl transfers from phosphate monoesters are highly dissociative, although not truly stepwise in protic solvents or in enzymatic reactions.

Three-dimensional structure of galactose-1-phosphate uridylyltransferase from Escherichia coli at 1.8 A resolution.

Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDP-galactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here.

Characterization of a 5.4 kb cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases.

Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70-80 kb. Although titin has been studied by several laboratories, barely more than half of the cDNA sequence (approximately 45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule).

Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates. One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) [Ballinger, M. D.

Pulsed electron paramagnetic resonance studies of the lysine 2,3-aminomutase substrate radical: evidence for participation of pyridoxal 5'-phosphate in a radical rearrangement.

The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance between the unpaired electron in the alpha-radical of beta-lysine, a steady-state intermediate in the reaction, and deuterium at the C4' position of the cofactor, PLP. [4'-2H]PLP was synthesized and exchanged into the enzyme.

Stimulus intensity and fundamental frequency effects on duplex perception.

Duplex perception occurs when part of the acoustic signal is used for both a speech and a nonspeech percept. This phenomenon has been interpreted as evidence of a distinct system for speech perception that precedes other specialized systems of general auditory processing (such as auditory grouping, and perception of pitch, loudness, and timbre). This interpretation was investigated by using an intensity-dependent form of duplex perception with the acoustic pair /da/ and /ga/.

Ten-year incidence of gross proteinuria in people with diabetes.

There are few population-based epidemiological data describing the long-term incidence of gross proteinuria in people with diabetes. We performed a population-based study in southern Wisconsin of individuals with diabetes diagnosed at either < 30 years of age and taking insulin (younger-onset, n = 666) or > or = 30 years of age either taking (older-onset taking insulin, n = 376) or not taking insulin (older-onset not taking insulin, n = 418). The presence of gross proteinuria (> or = 0.

Influence of culture conditions on biofilm formation by Escherichia coli O157:H7.

Biofilms of Escherichia coli O157:H7 were developed on stainless steel chips in trypticase soy broth (TSB), 1/5 dilution of TSB, 0.1% Bacto peptone (BP) and a minimal salts medium (MSM) supplemented with 0.04% of one of the following carbon sources: glucose, glycerol, lactose, mannose, succinic acid, sodium pyruvate or lactic acid.

Expression of deletion-containing dystrophins in mdx muscle: implications for gene therapy and dystrophin function.

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane.

Longitudinal effects of aging on lung function at rest and exercise in healthy active fit elderly adults.

We retested 18 healthy, active, and highly fit [maximal O2 consumption (VO2max) 201 +/- 12% of predicted] older adults over a 6-yr period (mean age 67-->73 yr) to determine the longitudinal effects of aging on lung function at rest and during exercise. In the 6-yr period, total lung capacity (TLC), functional residual capacity, and diffusion capacity did not change; vital capacity, forced expiratory volume in 1 s, and maximal volitional flow rates decreased; and residual volume and closing capacity/TLC increased 11-13%, all of which were greater than predicted from cross-sectional data. At maximum exercise over the 6-yr period, VO2max fell 11.

Galactose-1-phosphate uridylyltransferase from Escherichia coli, a zinc and iron metalloenzyme.

Galactose-1-P uridylyltransferase purified from Escherichia coli cells grown in enriched medium contains approximately 1.2 mol of tightly bound zinc/mol of subunits as well as variable amounts of iron, up to 0.7 mol/mol of subunits, and no detectable Ca, Cd, Cu, Mo, Ni, Co, Mn, As, Pb, or Se.

The total costs of illness: a metric for health care reform.

Systems thinking is a fundamental element of quality management and should be a fundamental element of health care reform. An implication of systems thinking is that one aim of health care should be to minimize the total costs of illness, not simply the direct medical expenditures. If we are to continue to improve health care over time, we should measure its impact on the total costs of illness to the patient, family, employer, and society.

Subcutaneous fat necrosis, hypercalcemia, and prostaglandin E.

We present two patients with subcutaneous fat necroses (SCFN) in whom endocrinologic studies revealed an association with elevated prostaglandin E (PGE) levels. A boy born after prolonged labor complicated by meconium aspiration developed erythematous, indurated plaques over the back, arms, buttocks, and cheeks at 4 days of age. A biopsy specimen of involved skin showed panniculitis with foci of necrotic adipocytes containing radially arranged, needle-shaped clefts and a granulomatous infiltrate in the septae.

Respiratory sinus arrhythmia in humans: an obligatory role for vagal feedback from the lungs.

Respiratory sinus arrhythmia (RSA) is used as a noninvasive measure of vagal cardiac input, but its causative mechanisms in humans remain undetermined. We compared the RSA of five lung-denervated double-lung transplant patients with intact hearts to six normal (N) control subjects, five heart-denervated patients, and two liver transplant patients at matched tidal volumes (VT's) and breathing frequencies. In N and liver transplant subjects, RSA was significant during eupnea and increased two- to threefold with increasing VT and inspiratory effort.

Effects of REM sleep on the ventilatory response to airway occlusion in the dog.

We determined the effects of sleep state on the ventilatory response following transient airway occlusion and on the response to vagal blockade in the unanesthetized sleeping dog. Three tracheotomized dogs underwent repeated occlusions (159 trials) during rapid eye movement (REM) and nonrapid eye movement (NREM) sleep. In all sleep states we found significant but variable transient hyperventilation following release of occlusion.

Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase.

A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid.

A simplified procedure for synthesizing nucleoside 1-thiotriphosphates: dATP alpha S, dGTP alpha S, UTP alpha S, and dTTP alpha S.

A procedure for synthesizing a nucleoside 1-thiotriphosphate in a single reaction vessel beginning with the nucleoside, PSCl3, and PPi is described. Reaction of the dried nucleoside with PSCl3 in anhydrous triethylphosphate is followed by addition of the tetrabutylammonium salt of PPi. Addition of excess triethylamine precipitates the nucleotides.