Identification of a Novel Substrate-Derived Spermine Oxidase Inhibitor.

Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in µM-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N1-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met2-Spm) has, under standard assay conditions, an IC50 value of 169 µM towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.

Sampling large geographic areas for rare species using environmental DNA: a study of bull trout Salvelinus confluentus occupancy in western Montana.

This study tested the efficacy of environmental DNA (eDNA) sampling to delineate the distribution of bull trout Salvelinus confluentus in headwater streams in western Montana, U.S.A. Surveys proved fast, reliable and sensitive: 124 samples were collected across five basins by a single crew in c. 8 days. Results were largely consistent with past electrofishing, but, in a basin where S. confluentus were known to be scarce, eDNA samples indicated that S. confluentus were more broadly distributed than previously thought.

Moderators and Predictors of Response to Eating Disorder Risk Factor Reduction Programs in Collegiate Female Athletes.

OBJECTIVE: The primary aim of this paper was to investigate moderators and predictors of response to two programs designed to reduce eating disorder risk factors in collegiate female athletes. This study served as an ancillary study to a parent trial that investigated the feasibility of an athlete modified cognitive dissonance-based program (AM-DBP) and an athlete modified healthy weight intervention program (AM-HWI). DESIGN: 157 female collegiate athletes were randomized to either the AM-DBP or the AM-HWI program. Participants completed surveys at baseline, post-intervention, 6 weeks, and 1 year. METHODS: After classifying sports as either lean or non-lean, we investigated if sport type acted as a moderator of program response to AM-DBP and AM-HWI using ANOVAs. Next, we examined whether baseline thin-ideal internalization, weight concern, shape concern, bulimic pathology, dietary restraint, and negative affect acted as predictors of changes in bulimic pathology using linear regression models. RESULTS: Athletes in non-lean sports who received AM-DBP showed more improvement in negative affect versus non-lean sport athletes in AM-HWI. Higher baseline scores of bulimic pathology predicted greater response in bulimic pathology to both programs at 6-weeks. In contrast, athletes with higher dietary restraint and negative affect baseline scores showed decreased response to both interventions at 6-weeks. Finally, athletes with higher baseline shape concern showed a decreased response to the AM-HWI intervention at the post intervention time point. CONCLUSION: Results from the present study indicate that lean/non-lean sport may not play a strong role in determining response to efficacious programs. Further, factors such as pre-existing bulimic pathology, dietary restraint, negative affect, and shape concern may affect general response to intervention versus specific responses to specific interventions.

Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE Phase 2) screening and recruitment: methods and results.

The Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE) study is a systematic investigation of sustained 25% calorie restriction (CR) in non-obese humans. CALERIE is a multicenter (3 clinical sites, one coordinating center), parallel group, randomized controlled trial. Participants were recruited, screened, and randomized to the CR or control group with a 2:1 allocation. Inclusion criteria included ages 21-50 years for men and 21-47 years for women, and a body mass index (BMI) of 22.0 ≤ BMI < 28.0 kg/m(2). Exclusion criteria included abnormal laboratory markers, significant medical conditions, psychiatric/behavioral problems, and an inability to adhere to the rigors of the evaluation/intervention schedule. A multi-stage screening process (telephone screen and 3 in-clinic visits) was applied to identify eligible participants. Recruitment was effective and enrollment targets were met on time. 10,856 individuals contacted the clinical sites, of whom 9787 (90%) failed one or more eligibility criteria. Of the 1069 volunteers who started the in-clinic screening, 831 (78%) were either ineligible or dropped. 238 volunteers were enrolled (i.e., initiated the baseline evaluations), 220 were randomized, and 218 started the assigned intervention (2% from the first screening step). This study offered lessons for future multi-center trials engaging non-disease populations. Recruitment strategies must be tailored to specific sites. A multi-disciplinary screening process should be applied to address medical, physical, and psychological/behavioral suitability of participants. Finally, a multi-step screening process with simple criteria first, followed by more elaborate procedures has the potential to reduce the use of study resources.

Recruitment bias in studies of semen and other factors affecting pregnancy rates in fertile men.

BACKGROUND: Recruitment bias is possible in population studies of semen quality because few men volunteer. We examine differences between Australian couples with natural conceptions who agreed or declined to participate in such a study. METHODS: Women pregnant between 16 and 32 weeks gestation participating in a retrospective time to pregnancy (TTP) study were each requested to recruit their eligible (on the basis of age, place of his birth and of his mother's birth) male partner to complete additional questionnaires, have a physical examination and provide blood and two semen samples. RESULTS: From 2061 women who completed the TTP questionnaire (response rate, 98%) there were 928 eligible male partners of whom 225 (24%) were responders. There were significant socio-demographic and self-reported exposure differences between responders and non-responders in particular, female professional occupation, knowledge of the fertile phase, pelvic inflammatory disease, non-smoker at time of conception and wine consumption per week were more frequent in the responders. There was no evidence of a bias for the subfertile being more likely to volunteer for the study. Mean TTP for planned pregnancies for responders and non-responders were 3.3 and 3.8 cycles (P = 0.319), respectively, and the cycle specific pregnancy rates were not significantly different after covariate adjustment by Cox regression. CONCLUSIONS: The present study confirms that participation rates are low in studies of semen quality. Although the expected higher participation of subfertile couples was not confirmed, there remains considerable potential for bias and other problems that could invalidate this type of study.

Associations between andrological measures, hormones and semen quality in fertile Australian men: inverse relationship between obesity and sperm output.

BACKGROUND: The World Health Organization developed a time to pregnancy (TTP) study (number of menstrual cycles taken to conceive) to determine whether the average TTP is increasing and semen quality decreasing with time. The present study describes clinical, semen and hormone characteristics obtained from male partners of pregnant women in Melbourne, Australia, and examines the associations between these characteristics. METHODS: Male partners (n = 225) of pregnant women (16-32 weeks) who conceived naturally had physical examination, health and lifestyle questionnaires, semen and hormone (FSH, LH, sex hormone-binding globulin, testosterone and Inhibin B) analyses. RESULTS: Previously known associations between semen, hormone and clinical variables were confirmed as significant: sperm numbers (concentration and total sperm count) correlated positively with Inhibin B and inversely with FSH and left varicocele, while total testicular volume correlated positively with sperm numbers and Inhibin B and inversely with FSH. However, only abstinence, total testicular volume, varicocele grade and obesity (BMI > 30 kg/m2) were independently significantly related to total sperm count. Compared with those with BMI < 30 (n = 188), obese subjects (n = 35) had significantly lower total sperm count (mean 324 versus 231 million, P = 0.013) and Inhibin B (187 versus 140 pg/ml, P < 0.001) but not FSH (3.4 versus 4.0 IU/l, P = 0.6). CONCLUSIONS: Obese fertile men appear to have reduced testicular function. Whether this is cause or effect, i.e. adiposity impairing spermatogenesis or reduced testicular function promoting fat deposition, remains to be determined.

Association of depression with Body Mass Index, sedentary behavior, and maladaptive eating attitudes and behaviors in 11 to 13-year old children.

We examined the relation of different behavioral dimensions of depression with weight-related variables (BMI percentile, sedentary behavior, eating attitudes, and weight control behaviors) in children aged 11 to 13 years. Depression was assessed using the Children's Depression Inventory (CDI). Sedentary behavior was measured in 45 sixth grade students (23 boys and 22 girls) using a validated 24-hour recall instrument, the Self-Administered Physical Activity Checklist. BMI was calculated directly from measured height and weight (kg/m2). The Children's Eating Attitudes Test (ChEAT) was used to measure eating attitudes and weight control behaviors. There were not significant gender differences in reported minutes (142 vs. 91 minutes for boys vs. girls; p=0.25) of sedentary behavior (i.e., television watching and video game playing). The major finding of this study was that certain aspects of depression (i.e., interpersonal problems and feelings of ineffectiveness) were correlated with higher levels of sedentary behavior in children aged 11 to 13. A factor analysis of the study variables indicated that most dimensions of depression, sedentary behavior, and body size represent distinct but correlated behavioral dimensions. This study provides support for a link between specific aspects of depression (i.e., interpersonal problems and feelings of ineffectiveness) and sedentary behavior in children.

Reformulation of the Children's Eating Attitudes Test (ChEAT): factor structure and scoring method in a non-clinical population.

The primary aims of this study were to empirically test the factor structure of the Children's Eating Attitudes Test (ChEAT) through both exploratory and confirmatory factor analyses and to interpret the factor structure of the ChEAT within the context of a new scoring method. The ChEAT was administered to 728 children in the 2nd through 6th grades (from five schools) at two different time points. Exactly half the students were male and half were female. To the best of our knowledge, this is the first study to empirically test the merits of an alternative 6-point scoring system as compared to the traditionally used 4-point scoring system. With the new scoring procedure, the skewness for all factor scores decreased, which resulted in increased variance in the item scores, as well as the total ChEAT score. Since the internal consistency of two factors in a recently proposed model was not acceptable (<0.60), this model did not adequately fit our data. Thus, exploratory and confirmatory factor analyses were conducted. A 6-factor solution based on a 20-item version was found to best fit the data and have the best internal reliability. The six factors were labeled: 1) overconcern with body size, 2) dieting, 3) food preoccupation, 4) social pressure to gain weight, 5) vomiting, and 6) caloric awareness and control. The obtained factor solution had considerable overlap with the original factor analysis performed on the 26-item Eating Attitudes Test and with the factor structure of the ChEAT reported by previous investigations. Intercorrelations among the factors suggested three higher order constructs. These findings indicate that the ChEAT subscales may be sufficiently stable to allow use in non-clinical samples of children.

Association of body size estimation and age in African-American females.

This cross-sectional research study tested the hypothesis that body image estimates of African-American females differ as a function of age. To test this hypothesis, body image estimates of 379 African-American females, ranging in age from 16 to 96 years, were contrasted as a function of age group, while statistically controlling body mass index. Three body size estimates, current body size, ideal body size, and reasonable body size were measured using the Body Image Assessment for Obesity. The discrepancies between current and ideal body size estimates and between current and reasonable body size estimates were also analyzed to assess for differences in body size dissatisfaction. The study found that younger African-American women (16 to 35 years) differed from older African-American women (>35 years) on measures of body size dissatisfaction. Women in the age range of 26 to 35 years reported higher estimates of current body size in comparison to women older than 35 years. The youngest age group (16 to 25 years) reported thinner ideal body size goals in comparison to women who were slightly older (26 to 35 years) and women who were older than 75 years. The pattern of body image estimates across a large age range suggests that younger African-American women, in comparison to older African-American women, may have body images that may make them more susceptible to eating disorders.

Polyunsaturated fatty acid supplementation in MS.

This article focuses on polyunsaturated fatty acid (PUFA) supplementation, which is a popular form of complementary and alternative therapy among people with MS. Owing to their popularity, clinicians should be knowledgeable about the PUFA supplements that are widely available, and the efficacy and safety data from clinical studies. Small-scale studies have demonstrated trends towards some beneficial effects. PUFA supplementation is generally well tolerated, although some specific supplements are best avoided and some clinical situations warrant caution. A review of the efficacy and safety data suggests that PUFA supplementation may be a promising approach. Large-scale trials are required to confirm the benefits.

The role of polyamine catabolism in anti-tumour drug response.

Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.

Psychological adjustment in the children of mothers with a history of eating disorders.

In order to test the impact of mothers' eating disorders (EDs) on their children's psychological adjustment, we recruited mothers belonging to three different populations: women with eating disorders, women with depression, and normal controls. The parents responded to self-report inventories relating to psychological adjustment of the parent and child. The study found that the psychological adjustment of the children of mothers with a history of ED was not different from that of the children of mothers in the normal control group, although mothers described significant pregnancy and birth complications, parenting stress, and symptoms of clinical depression. The children of mothers with a history of depression had significantly greater psychological problems in comparison with those of the children of mothers in the other two groups. The results are interpreted in the context of the protective factors that may have buffered the effects of maternal psychopathology in children of mothers with a history of ED.

Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure.

Mammalian polyamine catabolism is under the control of two enzymes, spermidine/spermine N1-acetyltransferase and the flavin adenine dinucleotide-dependent polyamine oxidase (PAO). In this study, the cloning and initial characterization of human PAO is reported. A 1894-bp cDNA with an open reading frame of 1668-bp codes for a protein of 555 amino acids. In vitro transcription/translation of this cDNA clone produces the expected M(r) 61,900 protein with PAO activity. The PAO activity of this clone is inhibited by MDL 72,527, a specific inhibitor of mammalian PAO. However, neither pargyline, a specific monoamine oxidase inhibitor, nor semicarbazide, a specific diamine oxidase inhibitor, inhibits the PAO activity of this clone. PAO has been referred to as being constitutively expressed. However, 24-h exposure of a non-small cell lung carcinoma cell line, NCI H157, to 10 microM of N1,N"-bis(ethyl)norspermine results in approximately 5-fold induction of PAO mRNA and a >3-fold induction of PAO activity. These results demonstrate that in at least one cell type, PAO is up-regulated in response to polyamine analogue exposure. The PAO clone described here should provide a useful tool, which will facilitate the dissection of the role of polyamine catabolism in normal growth and in response to the antitumor polyamine analogues.

Body Morph Assessment: preliminary report on the development of a computerized measure of body image.

OBJECTIVE: To develop a prototype of the Body Morph Assessment (BMA), and to test the reliability and validity of this new measure of body image. The BMA is a realistic and relatively simple procedure that uses computer morphing for the assessment of body image. For the purposes of this preliminary study, a prototype of the BMA was developed for usage with white women ranging from very thin to obese. RESEARCH METHODS AND PROCEDURES: A total of 72 subjects participated in tests of reliability, content, and convergent validity of the BMA. RESULTS: The reliability and validity of the BMA was supported by the results of this study. In a test of convergent validity, the measures of current, ideal, and reasonable body size were positively correlated with their equivalents from a similar body image assessment procedure. In addition, reliability coefficients were found to be satisfactory for all variables. Participants found the human figural stimuli to be realistic. DISCUSSION: These preliminary findings support the reliability and validity of the BMA with white women. Given these positive findings, we plan to extend the procedure to males and to other racial and ethnic groups.

Characterization of the interaction between the transcription factors human polyamine modulated factor (PMF-1) and NF-E2-related factor 2 (Nrf-2) in the transcriptional regulation of the spermidine/spermine N1-acetyltransferase (SSAT) gene.

Polyamines and polyamine analogues have been demonstrated to modulate the transcription of various genes. Spermidine/spermine N(1)-acetyltransferase (SSAT) is transcriptionally regulated through the interaction of at least two trans-acting transcription factors, NF-E2-related factor 2 (Nrf-2) and PMF-1 (polyamine modulated factor-1). Nrf-2 has previously been shown to regulate transcription of other genes through interactions between its C-terminal leucine zipper and the leucine-zipper region of other members of the small Maf protein family (the term "Maf" is derived from MusculoAponeurotic-Fibrosarcoma virus). Here it is demonstrated that the interaction between Nrf-2 and PMF-1 is mediated through the binding of the leucine-zipper region of Nrf-2 and a C-terminal coiled-coil region of PMF-1 that does not contain a leucine zipper. Mutations that interrupt either the leucine zipper of Nrf-2 or the coiled-coil region of PMF-1 are demonstrated to alter the ability of these factors to interact, thus their ability to regulate the transcription of the SSAT gene is lost.

Feasibility of surveillance of changes in human fertility and semen quality.

There is concern that male fertility is declining, but this is difficult to study because few men volunteer for studies of semen quality, and recruitment bias may over-represent the subfertile. The Human Reproduction Programme of the World Health Organization developed a protocol for multicentre studies of fertility involving a questionnaire for pregnant women to obtain time to pregnancy (TTP): the number of menstrual cycles taken to conceive. Male characteristics and semen quality will be determined in a subset of the partners. Our aim was to validate the TTP questionnaire, and to examine potential recruitment bias and feasibility of conducting large-scale surveillance of fertility. The questionnaire was administered to 120 pregnant women (16-32 weeks). Validation included internal reliability by consistency of responses, test-re-test reliability by repeat administration (20 women) and accuracy by comparison of gestational age from first antenatal ultrasound and menstrual dates. Internal reliability was high. Agreement between categorical responses on re-testing was very good (k > 0.8). In both the re-test and gestational age analysis, differences in TTP of 1 cycle were found (standard deviation <0.25 cycles). In this small pilot study there was no evidence of recruitment bias. Response rates indicate the feasibility of surveillance of fertility in large maternity centres.

Feasibility of surveillance of changes in human fertility and semen quality.

To show that male fertility is declining is not simple. Few men volunteer and recruitment bias may lead to over-representation of the subfertile. Semen analysis has errors arising from counting and poorly standardized criteria, which may be overcome by automation. Time to pregnancy (TTP)-the number of menstrual cycles taken to conceive-measures fertility and allows male recruitment bias to be estimated. We review automated measurement of sperm concentration, motility and morphology and present a preliminary report on a study to assess a retrospective TTP questionnaire, recruitment bias and feasibility for large-scale surveillance of fertility.

Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene.

The increased transcription and ultimate superinduction of the spermidine/spermine N(1)-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of approximately 1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of approximately 20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.

The primary visual system of adult lizards demonstrates that neurogenesis is not obligatorily linked to central nerve regeneration but may be a prerequisite for the restoration of maps in the brain.

Following optic nerve crush in the adult lizard Ctenophorus ornatus, most retinal ganglion cells regrow their axons into visual brain centres: however, the regenerated projections lack retinotopic order and the animals are blind via the experimental eye. Here we have used 3H-thymidine autoradiography to demonstrate that cell division is no longer taking place in the retina of normal adult lizards. We conclude that the optic nerve can regenerate in lizard even though cells are no longer being added to the retina. However, continued retinal neurogenesis may be linked to the ability to restore topographic maps.