Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation.

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors.

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis.

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an alpha and a beta subunit. There are 2 main alpha subunits, HIF-1 alpha and HIF-2 alpha. Recently, a HIF-2 alpha Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2 alpha in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase.

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.

Sex differences in response to red and blue light in human primary visual cortex: a bold fMRI study.

Studies using a variety of investigative methods, including functional brain imaging and electroencephalography (EEG), have suggested that changes in central nervous system (CNS) dopamine function result in altered visual system processing. The discovery of abnormal retinal blue cone, but not red cone, electroretinogram in association with cocaine withdrawal and Parkinson's disease suggests that visual system response to blue light might be a marker for CNS dopamine tone. As there are numerous sex-related differences in central nervous system dopamine function, we predicted that blue and red light stimulation would produce sex-specific patterns of response in primary visual cortex when studied using the blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) technique. We analyzed the BOLD response to red and blue light in male and female human volunteers (N=20). Red and blue light responses in primary visual cortex (V1) to stepped intensities of red and blue light were compared by sex for threshold to detectable BOLD signal increase and for stimulus intensity vs. BOLD signal response. Near threshold, males and females showed similar BOLD signal change to red light, but males showed a threefold greater increase (0.52%) to blue light stimulation when compared to females (0.14%). Log-linear regression modeling revealed that the slope coefficients for the red light stimulus intensity vs. signal change curve were not significantly different for males and females (z=0.995, P=0.320), whereas the slope coefficients for the blue light stimulus intensity vs. signal change curve were significantly larger in males (z=2.251, P=0.024). These findings support a sex and color-dependent differential pattern of primary visual cortical response to photic stimulation and suggest a method for assessing the influence of specific dopamine agonist/antagonist medications on visual function.

Dopamine is required for hyperphagia in Lep(ob/ob) mice.

Feeding is a complex process responsive to sensory information related to sight and smell of food, previous feeding experiences, satiety signals elicited by ingestion and hormonal signals related to energy balance. Dopamine released in specific brain regions is associated with pleasurable and rewarding events and may reinforce positive aspects of feeding. Dopamine also influences initiation and coordination of motor activity and is required for sensorimotor functions. Thus, dopamine may facilitate integration of sensory cues related to hunger, initiating the search for food and its consumption. Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens, where they modulate movement and reward. There are projections from the nucleus accumbens to the lateral hypothalamus that regulate feeding. Dopamine-deficient mice (Dbh(Th/+), Th-/-; hereafter DD mice) cannot synthesize dopamine in dopaminergic neurons. They gradually become aphagic and die of starvation. Daily treatment of DD mice with L-3,4-dihydroxyphenylalanine (L-DOPA) transiently restores brain dopamine, locomotion and feeding. Leptin-null (Lep(ob/ob)) mice exhibit obesity, decreased energy expenditure and hyperphagia. As the hypothalamic leptin-melanocortin pathway appears to regulate appetite and metabolism, we generated mice lacking both dopamine and leptin (DD x Lep(ob/ob)) to determine if leptin deficiency overcomes the aphagia of DD mice. DD x Lep(ob/ob) mice became obese when treated daily with L-DOPA, but when L-DOPA treatment was terminated the double mutants were capable of movement, but did not feed. Our data show that dopamine is required for feeding in leptin-null mice.

Feeding behavior in dopamine-deficient mice.

Mice that cannot make dopamine (DA), a condition caused by the selective inactivation of tyrosine hydroxylase in dopaminergic neurons, are born normal but gradually become hypoactive and hypophagic, and die at 3 weeks of age. We characterized the feeding and locomotor responses of these DA-deficient (DA-/-) mice to 3, 4-dihyroxy-L-phenylalanine (L-DOPA) to investigate the relationship between brain DA levels and these complex behaviors. Daily administration of L-DOPA to DA-/- mice stimulated locomotor activity that lasted 6 to 9 hr; during that time the mice consumed most of their daily food and water. The minimal dose of L-DOPA that was sufficient to elicit normal feeding behavior in the DA-/- mice also restored their striatal DA to 9.1% of that in the wild-type (WT) mice at 3 hr; then DA content declined to <1% of WT levels by 24 hr. This dose of L-DOPA induced locomotor activity that exceeded that of treated WT mice by 5- to 7-fold, suggesting that DA-/- mice are supersensitive to DA. Unexpectedly, DA-/- mice manifested a second wave of activity 24 to 48 hr after L-DOPA treatment that was equivalent in magnitude to that of WT mice and independent of DA receptor activation. The DA-/- mice approached, sniffed, and chewed food during this second period of activity, but they ate <10% of that required for sustenance. Therefore, DA-/- mice can execute behaviors necessary to seek and ingest food, but they do not eat enough to survive.

The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I.

BACKGROUND: The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised. The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction. What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS: We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons. Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria. Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p. Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus. Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome. CONCLUSIONS: CDC9 encodes two distinct forms of DNA ligase I. The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.

Oral squamous cell carcinoma after allogeneic bone marrow transplantation for Fanconi anaemia.

We report a case of oral squamous cell carcinoma (SCC) originating in the buccal mucosa of an 18-year-old female patient with chronic graft-versus-host disease (GVHD) 9 years after HLA-identical sibling bone marrow transplantation (BMT) for Fanconi anaemia (FA). The case highlights the problems of malignant change in FA and also the increased risk of second malignancy after BMT. The literature is reviewed with regard to previous cases and the possible aetiology of tumour formation. A high index of suspicion to any epithelial lesion in FA is appropriate so that early diagnosis may lead to improved prognosis.

Using a simple diffusion model to predict residual monomer migration--considerations and limitations.

A simple mathematical equation based on a diffusion model has been utilized recently to estimate migration of both acrylonitrile and styrene from polymers produced from these monomers which are used under a wide variety of food-contact applications. These calculated migration values have subsequently been used to estimate the US consumer's exposure to acrylonitrile and styrene from food stored in these materials. The basic assumptions integral to the model are discussed in relation to potential errors in migration estimates that could be experienced if the assumptions are not true. In addition to the discussion of the basic assumptions, factors affecting the migration predictions such as polymer 'ageing', temperature changes during the lifetime of the polymeric article, the effects of polymer-modifying materials (plasticizers, impact modifiers), and the physical form of the article or test sample are discussed.

Spontaneous graft-versus-host disease following autologous peripheral blood progenitor cell transplantation in chronic myeloid leukaemia.

Myeloablation followed by haemopoietic reconstitution using autologous peripheral blood progenitor cells (PBPC) is applicable to some patients with CML, particularly where there is no allogeneic stem cell donor available, and interferon alpha has failed to achieve a significant cytogenetic response. Cells lacking the Philadelphia (Ph) chromosome can be collected at the early phase of myeloid recovery after intensive chemotherapy, and reconstitution after autografting can be associated with prolonged suppression of the Ph positive clone. It is possible that mechanisms other than this "in vivo purge' may contribute to disease control, for example an autologous graft-versus-leukaemia effect. We report two patients in whom significant autologous graft-versus-host disease (auto-GVHD) has occurred, which has not previously been described as a spontaneous event after PBPC autograft for CML. We postulate that mononuclear cells collected in an early phase of recovery after intense myelosuppression have the capacity to produce self-reactivity after autografting. These cells, which may include autoreactive T lymphocytes or antigen-presenting dendritic cells, might mediate a useful graft-versus-leukaemia effect.

A model for estimating the daily dietary intake of a substance from food-contact articles: styrene from polystyrene food-contact polymers.

An approach has been developed to estimate the exposure of consumers to styrene from polystyrene food-contact articles which incorporates published literature on the diffusion of styrene through polymeric materials and industry survey data on uses of polystyrene in food-contact applications. The approach has been shown to be quite practical and has yielded an exposure estimate of 3 ppb (9 micrograms/day) styrene, on average, in the daily diet of people in the United States. This value compares reasonably well with the value of 1-4 micrograms/day for residents of the United Kingdom in 1983. The value is also four orders of magnitude less than the acceptable daily intake calculated by the Styrene Information and Research Center.

The Executive Skills Profile: a method for assessing development needs among family medicine faculty.

BACKGROUND AND OBJECTIVES: The purpose of this paper is to report on how the Executive Skills Profile, a measure of job demands and employee skills, can be used as a faculty development tool in academic family medicine departments. METHODS: The Executive Skills Profile (ESP) identifies 72 work activities and groups them into 12 scales, each of which is displayed graphically. Faculty identify the work activities that are relevant to their jobs and then identify the personal skills they must have to carry out those work activities. RESULTS: The ESP successfully characterized the job demands and personal skills of one family medicine department. The evaluation showed that overall perceived job demands were greater than the faculty's perceived personal skills, particularly in the areas of interpersonal and behavioral skills. CONCLUSIONS: The ESP can be used to characterize the job demands and personal skills of faculty.

The migration of acrylonitrile from acrylonitrile/butadiene/styrene polymers into food-simulating liquids.

The correlation of residual acrylonitrile (AN) monomer concentration in AN-containing polymers with AN migration into food simulants is of interest because the US FDA regulates the use of these polymers on the basis of the amount of AN that may migrate into food simulants. Studies of the migration of AN into water from seven acrylonitrile/butadiene/styrene polymers with varying composition and residual AN levels showed that a linear relationship exists between the concentration of AN in the polymer and the amount of AN migrating, for a given set of exposure conditions. A linear relationship was also observed between the diffusion coefficients generated from the experimental data using a simple Fickian diffusion model and the inverse of the absolute temperature of exposure.

Prepubertal exposure to dietary zearalenone alters hypothalamo-hypophysial function but does not impair postpubertal reproductive function of gilts.

At an average age of 70 d, 60 Yorkshire gilts born either in July (Trial 1; n = 30) or August (Trial 2; n = 30) received a diet containing zearalenone for 0 (control), 45 or 90 d. The concentration of zearalenone in diets was 2 ppm for 2 wk and 1.5 ppm for the remainder of the study. Vulval swelling and reddening was evident within 7 d after zearalenone was first fed. Zearalenone consumption had no effect on BW or backfat depth. Puberty occurred in Trial 1 at 219 +/- 6 d and was not influenced by zearalenone. Gilts in Trial 2 were divided into two groups; blood samples were taken from 12 gilts to assess pulsatile LH patterns and LH response to estradiol benzoate (EB) and 18 were handled similarly to those in Trial 1. Of this latter subgroup, age at puberty was younger (P less than .05) with zearalenone (217 +/- 7.0, 193 +/- 9.1 and 185.6 +/- 8.2 d for 0-, 45-, and 90-d treatments). Prepubertal consumption of zearalenone did not affect conception rates, ovulation rates, number of fetuses or percentage of embryo survival following mating at pubertal estrus. Two days before the 90-d experimental period ended for Trial 2, blood samples were taken from 12 gilts (four/treatment) every 15 min for 4 h prior to injection of EB (10 micrograms/kg) and every 6 h for 108 h after EB. Analysis of pulsatile patterns of LH revealed no influence of zearalenone on the number of peaks/4 h, baseline concentration or peak height.(ABSTRACT TRUNCATED AT 250 WORDS)

Premedical and medical school performance in predicting first-year residency performance.

In the study reported here the authors examined the relationships among 40 measures of undergraduate college and medical school performance and competence in 18 medical care tasks during the first year of residency. A rating form was developed for the study to assess residents' competency in the medical care tasks and was sent to the directors of the residency programs entered by the graduates of a medical school. Stepwise multiple regression procedures were used to analyze the relationship between these ratings of residency performance and the residents' premedical and medical school performance and to identify the best predictors of residency performance for the 1982 and 1984 classes. A Rasch model analysis of the residency performance ratings indicated the ease or difficulty of each of the 18 tasks. The results provide information that would allow medical educators to use premedical and medical school performance to predict residents' competencies. The task of "clinically evaluates research and clinical data" was the most difficult for the graduates; that is, they were rated lower on it than on any other task. Two groups of measures of undergraduate and medical school performance were significantly related to performance in the residency: the Part II examination of the National Board of Medical Examiners (particularly the scores on the obstetrics-gynecology, medicine, surgery, and pediatrics subtests and the overall score) and the clerkships (particularly the third-year medicine clerkship, the fourth-year medicine clerkship, and the surgery clerkship).