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Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

This paper is concerned with the challenge of enabling the use of confidential or private data for research and policy analysis, while protecting confidentiality and privacy by reducing the risk of disclosure of sensitive information. Traditional solutions to the problem of reducing disclosure risk include releasing de-identified data and modifying data before release. In this paper we discuss the alternative approach of using a remote analysis server which does not enable any data release, but instead is designed to deliver useful results of user-specified statistical analyses with a low risk of disclosure. The techniques described in this paper enable a user to conduct a wide range of methods in exploratory data analysis, regression and survival analysis, while at the same time reducing the risk that the user can read or infer any individual record attribute value. We illustrate our methods with examples from biostatistics using publicly available data. We have implemented our techniques into a software demonstrator called Privacy-Preserving Analytics (PPA), via a web-based interface to the R software. We believe that PPA may provide an effective balance between the competing goals of providing useful information and reducing disclosure risk in some situations.

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1alpha protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.

Background: The authors reported previously that assessment of the number of positive biopsy cores, maximum tumor length in a core, Gleason score, and prostate volume in an extended biopsy enhanced the accuracy of predicting low-volume/low-grade prostate cancer. On the basis of those findings, they developed a nomogram to predict the probability of low-volume/low-grade prostate cancer specifically for men with a single positive biopsy core., Methods: The study cohort comprised 258 men who underwent radical prostatectomy without neoadjuvant therapy. Prostate cancer was diagnosed in only 1 core of an extended biopsy scheme. Low-volume/low-grade cancer was defined as pathologic organ-confined disease and a tumor volume<0.5 cc with no Gleason grade 4 or 5 cancer. Patient age, prostate-specific antigen (PSA) level, prostate volume, PSA density (PSAD), and tumor length in a biopsy core were examined as variables. A fitted multiple logistic regression model was used to establish the nomogram., Results: One hundred thirty-three patients (51.6%) had low-volume/low-grade cancer. To establish the nomogram, age, PSAD, and tumor length were adopted as variables. The fitted model suggested that older age, higher PSAD values, and greater tumor length would reduce the probability of low-volume/low-grade cancer. The nomogram predicted low-volume/low-grade cancer with good discrimination (an area under the receiver operating characteristic curve of 0.727). Calibration of this nomogram showed good predicted probability., Conclusions: The authors established a nomogram with which to predict low-volume/low-grade cancer in men with 1 positive biopsy core in an extended biopsy scheme, and they recommend this nomogram for use in selecting men for active surveillance., (Copyright (c) 2007 American Cancer Society.)

The skeleton is potential endogenous source of lead during pregnancy and lactation. We have undertaken a longitudinal investigation into the mobilization of lead from the human maternal skeleton to determine whether lead is mobilized from the maternal skeleton during pregnancy and lactation, and if so, when and how much is released. Subjects in the study were migrants to Australia (n=15) whose skeletal lead isotopic composition (endogenous lead) was different to that prevailing in the Australian environment (exogenous lead). This migrant cohort was compared with 6 multi-generational Australian controls. Biological and environmental samples were taken pre-pregnancy where possible, throughout pregnancy and postpartum for at least 6 months. Newly-born infants of the migrant and Australian mothers were monitored for 6 months. Blood lead concentrations for the migrant mothers ranged from 1.5 to 20 microg/dl (geometric mean 2.8) and for Australian mothers ranged from 1.9 to 4.3 microg/dl (geometric mean 2.9). There was minimal change in lead isotopic composition of the Australian pregnant controls although there were increases of approximately 40% in blood lead concentration in 3 of 6 cases during the postpartum period and from 0 to 12% in the other 3. In the migrant pregnant subjects, the geometric mean skeletal lead contribution to blood lead using the isotopic composition was approximately 33% (range 10-88%) for 14 subjects using a revised estimate for exogenous lead. Skeletal contribution to blood lead during the postpartum period was significantly greater than during pregnancy (P<0.001). The skeletal contributions to blood lead are higher and the changes are more consistent in those subjects who conceived within 100 days of arrival in Australia compared with those who conceived longer than 100 days. In the migrant subjects, changes in blood lead concentration during pregnancy and postpartum varied from subject to subject with an overall 20% increase; the increases during the postpartum period were greater than during pregnancy (P<0.001). It was estimated that the amount of maternal skeletal lead mobilized during pregnancy and transferred to the infant via cord blood averaged approximately 79%. The increased skeletal contribution to blood lead is attributed to a low daily calcium intake of approximately 500 mgCa/day, a condition which was present in both migrant and Australian subjects. An ongoing clinical trial is providing a new cohort with calcium supplements. A summary of other aspects of the study is included and covers: additional flux released from the skeleton during pregnancy and postpartum; XRF bone lead results; urinary excretion of lead during pregnancy and postpartum; dietary contribution to blood lead in female adults and children; comparison of rates of exchange of lead in blood of newly-born infants and mothers; relationships of lead in breast milk to lead in blood, urine and diet of the infant and mother; changes in blood lead after cessation of breastfeeding; urinary lead isotopes during pregnancy and postpartum indicate no preferential partitioning of endogenous lead into plasma; a comparison of some aspects of the nonhuman primate and human pregnancy studies.

As an adjunct to a study of lead mobilization during pregnancy and lactation, we have obtained estimates of the daily lead intake and excretion/intake for 15 newly born infants monitored for at least 6 months postpartum. The longitudinal data presented reflect the far lower levels of environmental contribution to lead in blood in the 1990's than that in the earlier studies from the 1970's and early 1980's, the last period for which such dietary information is available in newly born infants. Infants were breast-fed or formula-fed or both and, in the second quarter, were usually fed solid foods (beikost). Lead concentrations were as follows: lead in breast milk, ranged from 0.09 to 3.1 microg/kg with a geometric mean of 0.55 microg/kg, lead in infant formula ranged from 0.07 to 11.4 microg/kg with a geometric mean of 1.6 microg/kg, and lead in beikost ranged from 1.1 to 27 microg/kg with a geometric mean of 2.9 microg/kg. Daily lead intakes ranged from 0.04 to 0.83 microg/kg body weight/day with a geometric mean of 0.23 microg Pb/kg body weight/day, and excretion/intake ranged from 0.7 to 22 with a geometric mean of 2.6. There was no significant difference at the 5% level in lead concentration in daily intakes and excretion/intake for the first quarter versus the second quarter for this small number of subjects. Assuming that there was no contribution from environmental samples such as house dust and ambient air, the contribution of diet to blood has been estimated from lead isotopic measurements with the following ranges: for breast milk only as the dietary source, 40 to 65%; for breast milk and infant formula as the dietary sources, 15 to 70%; and for infant formula and beikost, 20 to 80%. The geometric mean value of the dietary contribution to blood over the 6-month period of approximately 35% is consistent with earlier estimates of uptake of lead in blood in newly born infants when environmental lead concentrations were much higher. Other sources such as air, soil, and dust are considered to contribute minimally to blood lead in these infants because of the low 206Pb/204Pb ratios in environmental media. Thus, we consider that the increased excretion over intake, along with other evidence, reflects mobilization of infant tissues arising especially from rapid bone turnover at this stage of life; the tissue lead has been identified isotopically in urine.

We have compared lead isotopic ratios and lead concentrations in 53 spot urine and 59 24-h urine samples from 13 subjects covering the interval from pre-pregnancy through 180 days postpartum to estimate the amount of lead excreted in urine and renal clearance relative to blood. The total amount of lead excreted in 24-h urine samples ranges from 0.8 to 5.9 microg Pb with an arithmetic mean of 2.2+/-1.1 microg (geometric mean 1.90 microg). This compares with amounts of 0.9-10 microg of extra lead per day estimated to be released into blood from the skeleton during pregnancy and postpartum. There were no differences in excretion rates during the trimesters of pregnancy and between pregnancy and postpartum time periods. The renal clearance relative to blood ranged from 0.8 to 10 g/h (arithmetic mean 3.2+/-1.9; geometric mean 2.7). Renal clearance relative to blood was somewhat higher in trimesters 2 and 3 compared with postpartum 150-180 days (P = 0.004, 0.006, respectively). Reassessment of earlier published blood and dietary data for Australian pregnant controls indicates there is no increased gastrointestinal absorption of lead during pregnancy and postpartum. This differs from calcium, which shows increased absorption during late pregnancy. In light of the inconvenience of sampling and potential contamination at the low levels of lead found in most of these subjects, we do not consider the 24 h urines to provide sufficient useful information.

We have compared lead isotopic ratios and lead concentrations in 51 matched blood and spot urine samples from 13 subjects covering the interval from before pregnancy through 180 days postpartum to evaluate whether mobilization of lead from the maternal skeleton is preferentially partitioned into plasma; we have used urine as an isotopic proxy for plasma. There was no statistically significant difference in the lead 206/lead 204 and lead 207/lead 206 ratios over pregnancy. The urine data for the postpartum period are in the opposite relationships to that predicted for a preferential partitioning hypothesis. These data provide no support for the hypothesis that lead released from the skeleton is preferentially partitioned into plasma.

Immunoneutralization of endogenous gonadal steroids has recently been shown to modify pineal beta-adrenoceptor function in intact Merino ewes. In the current study, interactions between gonadal steroids and these receptors have been further investigated. beta-Adrenoceptor density and binding affinity both showed time-related changes in ewes; the significance of these changes requires further study. Two observations, firstly modification of beta-adrenoceptor function by androstenedione and 17 beta-estradiol in ovariectomized, but not in intact ewes, and secondly that steroid-mediated effects on receptor density and binding affinity in the pineal of ovariectomized Merino ewes could be demonstrated during anestrus, but not during the breeding season for intact ewes, indicate that gonadal steroids may regulate pineal beta-adrenoceptor variables in Merino ewes. It is suggested that gonadal steroids may regulate ovine pineal function in ewes, and that the seasonal differences in sensitivity of luteinizing hormone release to steroid feedback may be mediated in part via effects on the pineal gland.

This study deals with fetal growth retardation in heat-exposed sheep, and provides information on mechanisms of acclimation to heat. Radioactive microspheres were used to measure regional capillary blood flows in conscious sheep 80-100 days pregnant, at first in a thermoneutral environment, next after 2.5-6 h exposure to a hot environment of 40 degrees C, 25 mmHg water vapour pressure, and then 7 days and 15-20 days later, after spending 9 h daily in the heat. Maternal blood flow in the placental cotyledons was decreased by the first heat exposure, and remained depressed during the period of periodic heating. Reduction in placental blood flow on the maternal side could be part of the mechanism by which fetal development is retarded in heat-stressed sheep. Acclimation to heat was indicated by a decrease in the rate of rise in rectal temperature during the first 2 h of daily exposure to heat. Concomitantly, there was a progressive increase in the blood flow in extremity skin and nasal mucosa, which are tissues concerned with facilitation of heat loss during acute heat stress. However, in respiratory muscles which are also concerned with heat-loss, there was no further increase in flow after the initial response to to acute heat, indicating that heat acclimation is not due to an increased ability to pant. Blood flow in other tissues such as gut, pancreas and adipose tissue progressively decreased. These changes in blood flow have possible adaptive significance.

A rosette inhibition test was developed using pig lymphocytes and sheep red blood cells. Antilymphocyte serum (ALS) in the presence of complement inhibited rosette formation by greater than 95% at 1/250 declining to no inhibition at 1/8000. Sera obtained from a total of 14 pregnant sows before and 1, 2, 3 and 4 wk after mating were tested for their ability to augment the rosette depression caused by ALS. In one experiment in which the responses of 4 pregnant sows were compared to 4 non-pregnant sows by discriminant analysis, sera were classified correctly in 83% of the samples taken from either pregnant or non-pregnant sows. When the more usual method of calculating the rosette inhibition titre was used, the responses of sera from pregnant pigs were classified with 31% accuracy and those from non-pregnant pigs with 80% accuracy. In a second experiment, sera from 10 pregnant sows were classified with 25% accuracy using the rosette inhibition titre. Thus 4 of these pigs were classified as non-pregnant by this method. Data from the second experiment were not suitable for discriminant analysis. It was concluded that there was some factor present in the sera of pregnant pigs, particularly by 3 or 4 wk post-mating, which could be detected by the rosette inhibition test. However, the test is not sensitive enough to allow specific diagnosis of early pregnancy in pigs.

Human forehead skin incubated in vitro is known to metabolize testosterone to 17-oxosteroids faster than the reverse reaction, while axillary skin rapidly metabolizes androstenedione to 17 beta-hydroxysteroids, such as testosterone and 5 alpha-dihydrotestosterone. While this has been confirmed using a larger number of patients, some indication has been found that 17 beta-hydroxysteroid oxidoreductase activity declines with age in the axilla. The relative rates of 17 beta-oxidation and reduction (direction of operation of skin 17 beta-hydroxysteroid oxidoreductase activity) were not altered by variety of incubation conditions. Large amounts of a membrane-bound 17 beta-hydroxysteroid oxidoreductase, showing preference for NAD as coenzyme and testosterone (rather than androstenedione) as steroid substrate, were found in forehead skin from one patient. On the other hand, the main axillary skin enzyme in skin from another patient was soluble and showed preference for NADP and androstenedione. It is postulated that 17 beta-oxidation and reduction in skin is controlled by the relative amount, the coenzyme preferences and the kinetic properties of these two enzymes.