Genetic inheritance of gene expression in human cell lines.

Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits.

Futile cycles revisited: a markov chain model of simultaneous glycolysis and gluconeogenesis.

We have used a random walk model of glycolysis and gluconeogenesis to investigate the bioenergetic implications of considering the cell cytoplasm to be a uniform well-mixed compartment. Radiotracer studies conducted on hepatocytes harvested from fasted rats and incubated with 40 mM glucose and 10 mM lactate demonstrated simultaneous glycolysis and gluconeogenesis, with net glycolysis. Tracer introduced as glycerol was incorporated both into glucose (via gluconeogenesis) and into pyruvate (via glycolysis). The data allow us to place a lower bound on the energetic cost of futile cycles involving adenosine triphosphate (ATP) hydrolysis in the early phosphorylation steps of glycolysis. Applying the Markov Chain model for glucose undergoing metabolism to pyruvate, the expected number of ATP molecules hydrolysed is not less than 15 ATP molecules per glucose molecule. The data suggest that, in hepatocytes under the circumstances of this experiment, either glycolysis is a net consumer of ATP, or glycolysis and gluconeogenesis are compartmentalized to a greater extent than is generally supposed.

AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells.

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet alpha-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.

Effect of creatine supplementation on metabolism and performance in humans during intermittent sprint cycling.

This double blind study investigated the effect of oral creatine supplementation (CrS) on 4 x 20 s of maximal sprinting on an air-braked cycle ergometer. Each sprint was separated by 20 s of recovery. A group of 16 triathletes [mean age 26.6 (SD 5.1) years. mean body mass 77.0 (SD 5.8) kg, mean body fat 12.9 (SD 4.6)%, maximal oxygen uptake 4.86 (SD 0.7) l.min-1] performed an initial 4 x 20 s trial after a muscle biopsy sample had been taken at rest. The subjects were then matched on their total intramuscular creatine content (TCr) before being randomly assigned to groups to take by mouth either a creatine supplement (CRE) or a placebo (CON) before a second 4 x 20 s trial. A muscle biopsy sample was also taken immediately before this second trial. The CrS of 100 g comprised 4 x 5 g for 5 days. The initial mean TCr were 112.5 (SD 8.7) and 112.5 (SD 10.7) mmol.kg-1 dry mass for CRE and CON, respectively. After creatine loading and placebo ingestion respectively, CRE [128.7 (SD 11.8) mmol.kg-1 dry mass] had a greater (P = 0.01) TCr than CON [112.0 (SD 10.0) mmol.kg-1 dry mass]. While the increase in free creatine for CRE was statistically significant (P = 0.034), this was not so for the changes in phosphocreatine content [trial 1: 75.7 (SD 6.9), trial 2: 84.7 (SD 11.0) mmol.kg-1 dry mass, P = 0.091]. There were no significant differences between CRE and CON for citrate synthase activity (P = 0.163). There was a tendency towards improved performance in terms of 1 s peak power (in watts P = 0.07; in watts per kilogram P = 0.05), 5 s peak power (in watts P = 0.08) and fatigue index (P = 0.08) after CrS for sprint 1 of the second trial. However, there was no improvement for mean power (in watts P = 0.15; in watts per kilogram P = 0.1) in sprint 1 or for any performance values in subsequent sprints. Our results suggest that, while CrS elevates the intramuscular stores of free creatine, this does not have an ergogenic effect on 4 x 20 s all-out cycle sprints with intervening 20-s rest periods.

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

The isolated hepatocyte preparation: 30 years on.

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.

A benefit of one's own: older women's entitlement to Social Security retirement.

This article explores differences in Social Security eligibility and benefit levels for older men and women using survey data from the Health and Retirement Study combined with administrative records on actual work histories and Social Security rules. We are able to determine the fully insured status of those persons, how close they are to meeting eligibility criteria when they are not fully insured, and their prospects for benefits. Around three-quarters of older women nearing retirement today will be fully insured for Social Security old-age benefits on the basis of their own accounts, but the rest would need substantial extra employment to rise above the eligibility threshold. Further, two-thirds of older married women who are fully insured have sufficient lifetime earnings to translate into an age-65 primary insurance amount worth at least half their husband's, but the other one-third can expect no additional retirement benefit from contributing to Social Security late in life. Finally, most wives will not be able to improve their benefits by working more under current rules. These results have mixed implications regarding the potential impact of women's rising labor force attachment on eventual retirement benefits. Working more years could increase women's chances of becoming eligible for Social Security benefits, but that effect is likely to be small. Furthermore, even when women do become fully insured according to the rules, not many wives will receive a higher benefit at the margin. The reason is that married women still receive higher Social Security benefits as a spouse than they do on the basis of their own work record. In fact, the net benefit from Social Security due to additional work is negative once one takes into account the Social Security contributions the women paid while employed. Benefits paid to widows are even more likely to be based on the spouse's work history rather than on the woman's. Hence, the rising labor market attachment of women in the future may increase their eligibility for benefits but will produce only modest (and often negative) impacts on their old-age Social Security benefits under current rules.

Hepatic glucose cycling does not contribute to the development of hyperglycemia in Zucker diabetic fatty rats.

Hepatic glucose cycling, whereby glucose is taken up by the liver, partially metabolized, then recycled to glucose, makes a substantial contribution to the development of hyperglycemia in IDDM. This stimulation of glucose cycling appears to be associated with elevated rates of fatty acid oxidation. Whether hepatic glucose cycling also contributes to the development of hyperglycemia in NIDDM is unclear. Using a model of NIDDM, the Zucker diabetic fatty (ZDF) rat, we determined whether glucose cycling was enhanced. Hepatocytes from ZDF rats exhibited higher rates of glucose phosphorylation and glycolysis, but there was no increase in the rate of cycling between glucose and glucose-6-phosphate or between glycolytically derived pyruvate and glucose. Despite the increased rates of glycolysis, the production of CO2 in liver cells from ZDF rats was no different from rates measured in cells from control animals. Instead, there was a large increase in the accumulation of lactate and pyruvate in the ZDF liver cells. The addition of 2-bromopalmitate, an inhibitor of fatty acid oxidation that inhibited glucose cycling in hepatocytes from IDDM rats, had no effect on glucose cycling in cells from ZDF rats. We therefore conclude that, unlike in IDDM, hepatic glucose cycling does not contribute to the development of hyperglycemia in the NIDDM Zucker rat.

Long-term maintenance of low concentrations of fructose for the study of hepatic glucose phosphorylation.

The stimulation of glucose phosphorylation in isolated hepatocytes by low fructose concentrations is transient due to the rapid metabolism of fructose. To prolong this stimulatory effect fructose was enzymically generated in the incubation medium from either sucrose with invertase or inulin with inulinase. A maximal rate of glucose phosphorylation was achieved when fructose was formed at at least 0.01 micromol/min, which maintained a concentration of 70 microM fructose in the medium. In the presence of a fructose concentration of 70 microM, the rate of phosphorylation with 5 mM glucose was doubled and remained constant over a 2.5 h period. Under these conditions the rate of glycolysis was increased more than 3-fold. The stimulation of flux through glucokinase by low concentrations of fructose decreased the proportion of glucose phosphorylated, which was cycled between glucose and glucose 6-phosphate, and increased the proportion that was glycolysed. The method described for maintaining the stimulation of glucose phosphorylation by isolated hepatocytes over prolonged incubation periods is especially suited to the further study of the control of glucokinase activity, in particular how the variation of flux through glucokinase affects the flux through all the pathways that utilize the product, glucose 6-phosphate.

Differential induction of chromosomal instability by DNA strand-breaking agents.

To investigate the role of DNA strand breakage as the molecular lesion responsible for initiating genomic instability, five different strand-breaking agents, bleomycin, neocarzinostatin, hydrogen peroxide, restriction endonucleases, and ionizing radiation, were examined for their capacity to induce delayed chromosomal instability. These studies used GM10115 human-hamster hybrid cells, which contain one copy of human chromosome 4 in a background of 20-24 hamster chromosomes. Chromosomal instability was investigated using fluorescence in situ hybridization to visualize chromosomal rearrangements involving the human chromosome. Rearrangements are detected multiple generations after treatment, in clonal populations derived from single progenitor cells surviving treatment of the specified DNA-damaging agents. Clastogenic and cytotoxic activities of all agents were tested by examining chromosome aberration yields in first-division metaphases and by clonogenic survival assays. Analysis of over 250 individual clones representing over 50,000 metaphases demonstrates that when compared at comparable levels of cell kill, ionizing radiation, bleomycin, and neocarzinostatin are equally effective at eliciting delayed genomic instability. These observations document, for the first time, the persistent destabilization of chromosomes following chemical treatment. In contrast, the analysis of nearly 300 clones and 60,000 metaphases, involving treatment with four different restriction endonucleases and/or hydrogen peroxide, did not show any delayed chromosomal instability. These data indicate that DNA strand breakage per se does not necessarily lead to chromosomal instability but that the complexity or quality of DNA strand breaks are important in initiating this phenotype.

Isolated hepatocytes--past, present and future.

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca(2+)-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase. The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.

MEG-based imaging of focal neuronal current sources.

We describe a new approach to imaging neural current sources from measurements of the magnetoencephalogram (MEG) associated with sensory, motor, or cognitive brain activation. Many previous approaches to this problem have concentrated on the use of weighted minimum norm (WMN) inverse methods. While these methods ensure a unique solution, they do not introduce information specific to the MEG inverse problem, often producing overly smoothed solutions and exhibiting severe sensitivity to noise. We describe a Bayesian formulation of the inverse problem in which a Gibbs prior is constructed to reflect the sparse focal nature of neural current sources associated with evoked response data. We demonstrate the method with simulated and experimental phantom data, comparing its performance with several WMN methods.

Who takes early Social Security benefits? The economic and health characteristics of early beneficiaries.

Using the 1992 and 1994 Waves of the Health and Retirement Survey, we compare individuals who first take Social Security benefits at age 62 with those who don't and find that the income and net assets of these two groups are similar in the years just prior to eligibility. However, there is great diversity within the groups, so that poor health appears to be more closely related to lower economic well-being than is early Social Security acceptance status. Our results suggest that raising the Social Security retirement age is not likely to dramatically lower the economic well-being of the typical person aged 62 since only 3% of men aged 62 are receiving Social Security retirement benefits, are in poor health, and have Social Security retirement benefits as their only source of pension income.

Strength of silicone breast implants.

Rupture and leakage are recognized problems associated with silicone breast implants. Data are scarce about the durability of the silicone shell, and the life span of this device is unknown. The purpose of this study was to investigate the strength of silicone breast implants. Thirty implant shells were subjected to mechanical testing. Twenty-nine of the shells were tested after explanation, and one unused implant served as a control to validate the testing method. Implantation time varied from 4 months to 20 years, and all shells were tested, regardless of condition. Fourteen implant shells were intact, eight were leaking, and seven were ruptured. All ruptured implants had been in place for 10 years or longer. The breaking force of all excised shell specimens ranged from 2.6 to 22.4 N (0.6 to 5.0 lb.). Specimens from the control "high performance" shell required 15.5 to 25.6 N (3.5 to 5.8 lb) of force to fail. The weakest group was from thin-shelled implants between 10 and 16 years of age. More than half these specimens failed with less than 1 lb of force. The average breaking force of ruptured shell material was less than that of intact shells. A comparison of strength data in this study with manufacturers' data suggests that breaking force is dependent on implant type, shell thickness, and implantation time.

Suppression of glycolysis is associated with an increase in glucose cycling in hepatocytes from diabetic rats.

Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes.

Effects of thyroid status on glucose cycling by isolated rat hepatocytes.

The effects of alterations in thyroid status on glucose metabolism have been investigated in rat hepatocytes. Addition of 10 or 40 mmol/L glucose induced increases in respiration rate that were significantly larger in cells from hyperthyroid rats than from hypothyroid animals. The responses of hepatocytes from euthyroid rats were intermediate. In cells from hyperthyroid rats, most of the increase occurred upon addition of 10 mmol/L glucose, with only a further small stimulation resulting when glucose concentration was increased to 40 mmol/L. For a given glucose concentration, glycolytic rates, determined by measuring release of tritium from [6-3H]glucose, were comparable in all thyroid states. Studies with 10 mmol/L [2-3H]glucose showed that cycling between glucose-6-phosphate and glucose was almost twofold higher in euthyroid and hyperthyroid states as compared with the hypothyroid state, although the magnitude of the increase in cycling rate was only approximately 0.2 mumol glucose.min-1.g-1. When 40 mmol/L [2-3H]glucose was added, over 44% of the glucose that was phosphorylated to glucose-6-phosphate was cycled back to glucose, but this cycling was independent of thyroid status. Cycling between fructose-1,6-bisphosphate and fructose-6-phosphate was negligible in all thyroid states. Rates of glycogen synthesis were comparable in hypothyroid and hyperthyroid states and slightly less than in the euthyroid state. Glycolytically formed pyruvate was cycled back to glucose in hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats. During a 60-minute incubation period, cycling to glucose in the presence of 10 mmol/L or 40 mmol/L glucose was up to twofold higher in cells from euthyroid and hyperthyroid rats than in hepatocytes from hypothyroid animals. The measured increases in cycling rates induced by thyroid hormone were small and in theory could have been satisfied by a much smaller increase in respiration rate than was observed.

Abolition of the inhibitory effect of ethanol oxidation on gluconeogenesis from lactate by asparagine or low concentrations of ammonia.

When isolated hepatocytes from fasted rats were incubated with 10 mM lactate, the [lactate]/[pyruvate] ratio measured at the beginning of the incubation was raised above 70:1 but declined to a steady level of about 8:1 within 40 min. The rate of gluconeogenesis from lactate was initially slow but gradually increased over the incubation period becoming maximal by 30 min. The simultaneous addition of lactate and ethanol resulted in an initial [lactate]/[pyruvate] ratio above 250:1 which by 60 min had declined to a new steady-state level of approx. 60:1. The lactate, ethanol combination also brought about a prolongation of the lag phase before glucose synthesis became maximal; however, by 40 min the rate of gluconeogenesis was independent of the presence of ethanol. Thus the inhibitory effect of ethanol on glucose synthesis was manifest only over the early portion of the incubation period. When asparagine, a precursor of malate/aspartate components, was added to the incubation mixture, the lag before maximal rates of glucose formation from lactate in the absence or presence of ethanol was almost abolished. The presence of asparagine also rapidly lowered the [lactate]/[pyruvate] ratio of hepatocytes incubated with lactate plus ethanol establishing a steady-state level of 15:1 within 10-15 min. Asparagine enhanced the rate of lactate-stimulated ethanol oxidation, particularly during the early part of the incubation. In endeavouring to elucidate which of the products of asparagine catabolism (i.e. ammonia and aspartate) were responsible for these effects, we found that a small and constant level of ammonia, formed by the degradation of urea by urease, almost reproduced the effects of asparagine on the [lactate]/[pyruvate] ratio, glucose synthesis and ethanol oxidation. A bolus addition of 10 mM aspartate or 4 mM ammonia to cells metabolising lactate and ethanol were less effective than a steady-state low ammonia concentration, generated from urea/urease. Our studies suggest that asparagine or a low concentration of ammonia, by providing components of the malate/aspartate shuttle, can ameliorate some of the metabolic effects of ethanol on the liver.

Stimulation of gluconeogenesis leads to an increased rate of beta-oxidation in hepatocytes from fasted diabetic but not from fasted normal rats.

We have investigated the effects of imposing an ATP demand, generated by the addition of lactate, on hepatocytes isolated from fasted normal and streptozocin-induced diabetic rats. The stimulation of O2 consumption upon lactate addition was much greater in hepatocytes from diabetic rats, as a result of a lactate-induced stimulation of beta-oxidation that was not observed in control cells. This lactate-induced increment in beta-oxidation was extremely sensitive to inhibition by low levels of a number of inhibitors of energy transduction, implying that the increment was tightly coupled to ATP synthesis. Such sensitivity of the beta-oxidative pathway to the addition of similar low concentrations of these inhibitors was not seen in control cells. Inhibitors of the gluconeogenic pathway were also more effective in decreasing beta-oxidation in cells from diabetic animals than in cells from normal rats. The increment in beta-oxidation was not accompanied by increased rates of glucose synthesis, fatty acid esterification or ureogenesis. We propose that it may be associated with higher rates of glucose cycling in cells from diabetic rats.

The contribution of glucose cycling to the maintenance of steady-state levels of lactate by hepatocytes during glycolysis and gluconeogenesis.

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady-state concentrations of these metabolites became established. The rate of glycolysis, determined with [6-3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one-third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20-80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose. Rates of glycolysis were also measured using 40 mM [6-3H]glucose and [U-14C]glucose added alone, or in combination with a steady-state lactate concentration (3 mM). There was no effect on the rate of glycolysis determine with [6-3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflect the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 mumol C3 equivalents min-1.g-1 respectively. Identical studies employing [U-14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady-state conditions and that the formation of lactate from [U-14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.