Effects of fat loss and low energy availability on the serum cardiometabolic profile of physique athletes.

Low energy availability (LEA) is a health concern for athletes, although it may paradoxically lead to improved cardiometabolic health in the general population. We investigated the associations between LEA, body composition, and serum cardiometabolic profile in 23 physique athletes (DIET) and 21 controls (CONT) during a 5-month pre-competition diet (MID), followed by 1 week of increased energy availability (COMP) and a 5-month weight regain period (POST). Quantification of 250 serum metabolome variables was conducted by NMR spectroscopy, body composition by dual-energy x-ray absorptiometry, dietary intake by food diaries, and exercise levels by training logs. Body fat percentage decreased from 19.5 ± 7.0% to 8.3 ± 5.3% (p < 0.001) in DIET through increased exercise levels and decreased energy intake, while CONT maintained those constant. In MID, DIET had increased (FDR < 0.01) HDL cholesterol, HDL particle size and number, and decreased (FDR < 0.05) VLDL lipids, serum triglycerides, and low-grade inflammation (glycoprotein acetyls) compared to baseline and CONT. The changes were associated with reduced android fat mass (-78 ± 13%) and energy intake (-28 ± 10%). In COMP, most of the metabolic changes found in MID persisted, except for altered triglycerides in all lipoprotein classes. After weight regain in POST, serum metabolome, body composition, energy intake, and exercise levels had reverted to baseline levels. In conclusion, fat loss and LEA may have beneficial yet transient effects on the serum cardiometabolic profile of lean individuals. Especially the HDL lipidome and lipoprotein triglycerides offer potential novel biomarkers for detecting LEA in athletes.

Taurine, an osteocyte metabolite, protects against oxidative stress-induced cell death and decreases inhibitors of the Wnt/ß-catenin signaling pathway.

Taurine has been shown to have positive effects on bone mass, which are thought to be due in part to its cytoprotective effects on osteoblasts and here we show that taurine also protects osteocytes against cell death due to reactive oxygen species. Using the IDG-SW3 cell line, the expression of the taurine uptake transporter Taut/Slc6a6 is increased during osteoblast to osteocyte differentiation. Taurine had no effect on genes associated with osteoblast to osteocyte differentiation such as Dmp1, Phex or osteocalcin, even at high doses, but a slight yet significant inhibition of alkaline phosphatase was observed at the highest dose (50 mM). No effect was seen on the osteoclast regulatory genes Rankl and Opg, however the wnt antagonist Sost/sclerostin was potently and dose-dependently downregulated in response to taurine supplementation. Taurine also significantly inhibited Dkk1 mRNA expression, but only at 50 mM. Interestingly, osteocytes were found to also be able to synthesize taurine intracellularly, potentially as a self-protective mechanism, but do not secrete the metabolite. A highly significant increase in the expression of cysteine dioxygenase (Cdo), a key enzyme necessary for the production of taurine, was observed with osteoblast to osteocyte differentiation along with a decrease in methionine, the precursor of taurine. For the first time, we describe the synthesis of taurine by osteocytes, potentially to preserve viability and to regulate bone formation through inhibition of sclerostin.

The application of metabonomics to predict drug-induced liver injury.

The occurrence of drug-induced liver injury (DILI) presents a significant safety issue for patients and represents a major cause of regulatory action. The methods that are in current use for early detection and prediction of DILI in patients are not adequate. The liver is the major site of synthesis of endogenous metabolites, and data suggest that alterations in the profiles of endogenous metabolites ("the metabolome") may precede development of clinically overt DILI. Metabonomics involves the application of analytical technologies such as nuclear magnetic resonance and mass spectrometry to detect changes in the metabolome. In this review, we describe the emerging role of metabonomics in predicting and understanding the mechanisms underlying DILI. Recent human clinical trials of drugs, including acetaminophen (APAP) and ximelagatran, have shown that the metabonomics of biofluids (plasma and urine) collected before and immediately after dosing can identify individual patients who are likely to develop DILI. These studies support the need to include metabonomic investigations in clinical trials of potentially hepatotoxic medications.

Use of pharmaco-metabonomics for early prediction of acetaminophen-induced hepatotoxicity in humans.

Achieving the ability to identify individuals who are susceptible to drug-induced liver injury (DILI) would represent a major advance in personalized medicine. Clayton et al. demonstrated that the pattern of endogenous metabolites in urine could predict susceptibility to acetaminophen-induced liver injury in rats. We designed a clinical study to test this approach in healthy adults who received 4 g of acetaminophen per day for 7 days. Urine metabolite profiles obtained before the start of treatment were not sufficient to distinguish which of the subjects would develop mild liver injury, as indicated by a rise in alanine aminotransferase (ALT) to a level more than twice the baseline value (responders). However, profiles obtained shortly after the start of treatment, but prior to ALT elevation, could distinguish responders from nonresponders. Statistical analyses revealed that predictive metabolites included those derived from the toxic metabolite N-acetyl paraquinone imine (NAPQI), but that the inclusion of endogenous metabolites was required for significant prediction. This "early-intervention pharmaco-metabonomics" approach should now be tested in clinical trials of other potentially hepatotoxic drugs.

The "random-coil" state of proteins: comparison of database statistics and molecular simulations.

This study presents a comparison of two models of the random-coil state, one based on statistical distributions from the structural database and the other based on molecular dynamics simulations. The database model relies on the assumption that the random- or statistical-coil state of a particular residue can be described by its conformational distribution in a sufficiently diverse subset of protein structures. The molecular dynamics model is based on distributions from molecular simulations carried out on "dipeptide" models (single residues with N-terminal acetyl and C-terminal N'-methyl amide blocking groups). A comparison of the two models for the residues Ala, Asn, Asp, Gly, and Val indicates that the database distributions are greatly influenced by long-range interactions and dominated by specific recognizable elements of protein structure. In contrast, the limited structural scope of the dipeptide models presents the extreme case of a peptide under the influence of only short-range interactions. The models were evaluated by a comparison of scalar coupling constants calculated from the conformational distributions and compared with experimentally values determined for unstructured peptides. Although the models gave different distributions, there was similar agreement with experiment. This comparison emphasizes the differences and limitations in each model and highlights the difficulty in presenting an accurate picture of the random-coil state. Proteins 1999;36:407- 418.

Botulinum toxin A versus fixed cast stretching for dynamic calf tightness in cerebral palsy.

OBJECTIVE: To compare botulinum toxin A injections with fixed plaster cast stretching in the management of cerebral palsied children with dynamic (i.e. non-fixed) calf tightness. METHODS: The settings were the Women's and Children's Hospital (WCH) and the Crippled Children's Association of South Australia (CCA), Adelaide, South Australia. Twenty children were selected by two paediatric rehabilitation specialists. A prospective, randomized, single-blind controlled study, was carried out, with 10 children in each arm. The clinicians were blinded as to the allocated interventions. The outcome measures for 6 months post intervention were clinical assessment, modified Ashworth Scale, Gross Motor Function Measure, 2 D-video ratings using a modified Physical Rating Scale and a global scoring scale and a parent satisfaction questionnaire. RESULTS AND CONCLUSION: Botulinum toxin A injections were of similar efficacy to serial fixed plaster casting in improving dynamic calf tightness in ambulant or partially ambulant children with cerebral palsy. The ease of outpatient administration, reduction of muscle tone and safety with botulinum toxin A was confirmed. Parents consistently favoured botulinum toxin A and highlighted the inconvenience of serial casting.

Inhibition of glucose transport in human red blood cells by adenosine antagonists.

Previous studies have suggested that adenosine antagonists can interfere with normal glucose uptake in perfused rat heart. In the present studies, fluorine-19 nuclear magnetic resonance spectroscopy was used to study the effect of the adenosine antagonist, BW-A1433U, on the equilibrium exchange of fluorinated glucose analogs in human erythrocytes. Studies of the equilibrium exchange of both 2-fluoro-2-deoxy-D-glucose and 3-fluoro-3-deoxy-D-glucose with either one-dimensional magnetization transfer or two-dimensional exchange spectroscopy were performed, and significant inhibition was observed in all cases. From concentration-dependent studies, an inhibition constant for the equilibrium exchange measured at 37 degrees C of 24 microM was determined.

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor.

19F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E. coli periplasmic glucose/galactose receptor (GGR). The protein was derived from E. coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and [2,4,6,7-2H4]-5-fluorotryptophan. As a result of the large gamma-isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored. Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism. In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on [2,4,6,7-2H4]-5-fluorotryptophan. Analysis of the observed 19F powder pattern lineshape resulted in anisotropy and asymmetry parameters of delta sigma = -93.5 ppm and eta = 0.24. Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S2 of approximately 1, and by correlation times for internal motion approximately 10(-11)s. Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with tau i set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S2 = 0.89 for fluorotryptophan residues 183, 127, 133, and 195. By contrast, the calculated order parameter for FTrp284, located on the surface of the protein, was 0.77. Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R1F183 > R1F127 approximately R1F133 approximately R1F195 > R1F284. Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data. In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule. These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp284, located in the hinge region of the receptor.

Identification of 2-fluoro-2-deoxy-D-glucose metabolites by 19F(1H) hetero-RELAY.

It has been proposed that in mammalian systems the glucose analog 2-fluoro-2-deoxy-D-glucose (FDG) is phosphorylated and subsequently converted to the corresponding mannose derivative via the action of phosphoglucose isomerase. As is generally true in metabolic studies of fluorinated molecules, the fluorine spectrum alone is suggestive, without providing definitive structural evidence, while the use of 1H NMR techniques generally suffers from a lack of adequate selectivity. A 1H-19F version of the hetero-RELAY experiment has been applied to this problem. Formation of the corresponding C-6 phosphorylated 2-FDG analog with hexokinase, followed by treatment of the resulting phosphorylated products with phosphoglucose isomerase, resulted in the observation of additional 19F resonances consistent with the corresponding 2-fluoro-2-deoxy-D-mannose-6-phosphate metabolite. A more definitive product identification was obtained using the hetero-RELAY experiment, which provides a complete 19F-decoupled proton spectrum for each of the fluorinated species.

The Adelaide preschool language unit: results of follow-up.

OBJECTIVE: To determine educational, social and behavioural functioning of children who had been involved in a preschool language intervention programme between 1982 and 1990. METHODOLOGY: Fifty children who attended the Unit were available for follow-up. The children were divided into three groups: (i) language disorder; (ii) speech disorder; and (iii) mixed speech and language disorder. A psychologist administered educational, cognitive and social behavioural tests. Speech, language and articulation were assessed by a speech pathologist. RESULTS: Cognitively, the 'mixed' speech and language group obtained lower scores than the speech and language disorder children; results on educational tests were also generally lower. All three groups were significantly underachieving in areas of language, reading, spelling and arithmetic relative to their performance IQ. No socialization problems were found. CONCLUSIONS: Severe speech and language disorders in young children, even after periods of intensive intervention, have a significant effect on later educational achievement even when children appear to be 'coping' in their educational settings.

Anomeric dependence of fluorodeoxyglucose transport in human erythrocytes.

The transport of several n-fluoro-n-deoxy-D-glucose derivatives across the human erythrocyte membrane has been studied under equilibrium exchange conditions using one- and two-dimensional nuclear magnetic resonance (NMR) techniques. This approach is based on the intracellular 19F shift, which was found to depend on the anomeric form and on the F/OH substitution position. Since the transport behavior of both glucose anomers can be followed simultaneously, this approach is particularly sensitive to differences in anomeric permeability. For 2-, 3-, 4-, and 6-fluorodeoxyglucose analogs, the alpha anomers permeate more rapidly, and the P alpha/P beta ratio is dependent on the position of fluorination, with values of 1.1, 1.3, 2.5, and 1.6, respectively, obtained at 37 degrees C. These results have been analyzed in terms of a simple alternating conformation model for the glucose transporter. Although mutarotase activity has been reported for red cells, mutarotation behavior for all anomers was found to be completely negligible on the transport and spin-lattice relaxation time scales. Metabolic transformation of the fluorinated glucose analogs, primarily to fluorinated gluconate and sorbitol analogs, is very slow and does not significantly interfere with the transport measurements. A mean ratio of 2.6 was found for the extracellular/intracellular fluorine spin-lattice relaxation rates.

Structure and dynamics of tosylchymotrypsin at pH 7 examined by tritium NMR spectroscopy.

3H-NMR spectroscopy of specifically tritiated and tritiated/deuterated derivatives of tosylchymotrypsin has been used to examine the behavior of the tosyl group in this protein at pH 7. The presence of several tritiated isotopomers complicates analysis of experiments and extensive computer simulations of T1 relaxation, line widths, and various nuclear Overhauser experiments for the collection of tritiated species present in the samples were used to the interpret the observations made. These analyses suggests that the tosyl group of tosylchymotrypsin at pH 7 is largely retained within the substrate specificity pocket observed in the crystal structure. This outcome is in strong contrast to the situation observed at pH 4, where the tosyl group is mobile enough to be found outside the specificity pocket an appreciable fraction of the time, and may be the result of protein association at pH 7.

Cloned cDNA sequence for the human mesothelial protein 'mesosecrin' discloses its identity as a plasminogen activator inhibitor (PAI-1) and a recent evolutionary change in transcript processing.

Mesosecrin, a Mr approximately 46 x 10(3) glycoprotein secreted in abundance by human mesothelial cells in culture, was recently described by this laboratory. We isolated partial cDNA clones for mesosecrin from a human mesothelial cell cDNA library in lambda gt11 using a specific antiserum. Comparison of mesosecrin cDNA sequences with the recently published sequence for plasminogen activator inhibitor-1 (PAI-1) cloned from cDNA libraries of endothelial and other cell types revealed that mesosecrin and PAI-1 are the same protein. Reverse fibrin autography of electrophoretically fractionated medium from mesothelial cell cultures confirmed that mesosecrin is functional as a plasminogen activator inhibitor. The mesosecrin/PAI-1 cDNA clones hybridized to abundant 3.6 and 2.6 kb (kb = 10(3) bases) mRNAs on Northern blots of cultured human mesothelial cell and endothelial cell RNA. These mRNA sizes correspond to those recently published for human endothelial and fibrosarcoma PAI-1 mRNA, which most likely result from alternate polyadenylation sites. Messages 3.6 and 2.6 kb long were also detected in cells cultured from orangutans and African green monkeys, but only an approximately 3.6 kb mRNA was detected in cells of lower primates and several other mammalian species. Thus the extra polyadenylation site in the PAI-1 gene, responsible for the shorter form of the RNA, apparently has been acquired recently during primate evolution. Because they are more easily propagated in culture than endothelial cells, human mesothelial cells offer a new and advantageous system for PAI-1 production and study of its regulation and function.

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.

Mesosecrin: a secreted glycoprotein produced in abundance by human mesothelial, endothelial, and kidney epithelial cells in culture.

Human mesothelial cells, endothelial cells, and type II kidney epithelial cells growing in culture devote approximately 3% of their total protein synthesis to the production of an Mr approximately 46-kD, pI 7.1, secreted glycoprotein (designated Sp46). Fibroblasts make about 1/10th as much Sp46 as these cell types, and their synthesis is dependent upon hydrocortisone. Keratinocytes, urothelial cells, conjunctival epithelial cells, and mammary epithelial cells do not make detectable amounts of Sp46. Mesothelial cells secrete Sp46 onto the substratum, and from there it is subsequently released into the medium. Immunofluorescence analysis using specific antisera discloses that Sp46 is deposited beneath cells as a fine coating on the substratum. In sparse cultures, Sp46 is detected in trails behind motile cells. In contrast, secreted fibronectin coalesces into fibers, most of which remain in contact with and on top of the cells; thus Sp46 does not preferentially bind to fibronectin. About 6 kD of the mass of human Sp46 is N-linked oligosaccharide, which is terminally sialated before secretion. Sp46 has a low glycine content, indicating that it is not a collagenlike protein. Its NH2-terminal sequence over the first 40 amino acids does not resemble any protein for which sequence information is available. Sp46 appears to be a novel extracellular glycoprotein, high-level constitutive expression of which is restricted to mesoderm-derived epithelial and endothelial cells. We therefore propose for it the name "mesosecrin."