Influence of NEP activity on the alcohol-preference ratio in transgenic mice.

<p>(<b>A</b>) No significant differences between NEP-deficient (hatched bars) and NEP wild-type mice (white bars) in absolute alcohol consumption (left panel), total fluid consumption (second left panel), alcohol/total fluid ratio (second right panel), and in alcohol degradation (measured 2 h after alcohol i.p. injection; right panel) under stress-free conditions; (<b>B</b>) Development of stress-induced differences in the preference ratio between the both genotypes; (<b>C</b>) Development of stress-induced differences in the preference ratio between the both genotypes between days 22 and 70 of the experiment; (<b>D</b>) No fading of stress-induced differences after second stress phase in the preference ratio of NEP-deficient and NEP wild-type mice at 3 consecutive periods of approx 30 days. All data sets include at least measurements at 8 independent successive time points. **<i>P</i><0.01, ***<i>P</i><0.001 vs. wild-type.</p

Influence of pharmacological inhibition of NEP activity on the alcohol-preference ratio.

<p>(<b>A</b>) Effects of candoxatril (200 mg/kg/day) on central and peripheral NEP activity; (<b>B</b>) Candoxatril-induced differences in the alcohol preference ratio and (<b>C</b>) total fluid intake. All data sets include at least measurements at 8 independent successive time points. **<i>P</i><0.01, ***<i>P</i><0.001 vs. control.</p

Response of mice with NEP deficiency in two independent tests of emotional behavior.

<p>NEP deficient mice (hatched bars) did not show alterations in the time spent in the (<b>A</b>) closed arm and (<b>B</b>) in the time travelling, (<b>C</b>) but number of transitions between the chambers crossed the area between the arms significantly more frequent. (<b>D</b>) Using a motility monitor system, NEP-deficient mice did show significant reduction in travelling time, (<b>E</b>) distance travelled, and (<b>F</b>) time in the middle (open field) of the monitor system. All data sets include at least measurements at 8 independent successive time points. *<i>P</i><0.05, **<i>P</i><0.01 vs. control.</p

Age-related obesity in NEP-deficient mice.

<p>(<b>a</b>) Comparison of a NEP-knockout (NEP −/−) mouse with an age-matched wild-type animal (NEP +/+). (<b>b</b>) Steatosis of thorax and heart in a NEP-knockout mouse. (<b>c</b>) Abdominal fat accumulation in a NEP-deficient animal. Age-dependent development of body weight in (<b>d</b>) females and (<b>e</b>) males. Data is presented as means ± SEM. Where not shown, error bars lie within the dimensions of the symbols. Average per group 22 mice. Genotype effects ***<i>P</i><0.001 by two-way ANOVA. Significant differences at specific times are calculated by Bonferroni <i>post-hoc</i> test, **<i>P</i><0.01 and ***<i>P</i><0.001. (<b>f</b>) Daily food consumption in 7 and >11 month-old female NEP-knockout (NEP −/−) mice compared with age-matched wild-type animal (NEP +/+), *<i>P</i><0.05 and **<i>P</i><0.01.</p

Body composition and diet-depending weight development in NEP-deficient mice.

<p>NMR-monitored influence of feeding on age-dependent body composition in mice fed with low fat diet divided in (<b>a</b>) muscle masses, (<b>b</b>) free body fluid, (<b>c</b>) fat masses, and (<b>d</b>) fat masses per body weight. Effects of (<b>e</b>) low fat and (<b>f</b>) high fat diet on the development of body mass. Data is presented as means ± SEM of at least 10 animals per group. Two-way ANOVA **<i>P</i><0.01; ***<i>P</i><0.001. Where not shown, error bars lie within the dimensions of the symbols. Genotype effects are calculated by two-way ANOVA (**<i>P</i><0.01; ***<i>P</i><0.001). Significant differences at specific time-points are calculated by Bonferroni <i>post-hoc</i> test, *<i>P</i><0.05 and **<i>P</i><0.01.</p

Effects of candoxatril (Pfizer, UK79,300) on body mass development.

<p>(<b>a</b>) Development of body weight in male C57BL/6 mice starting in an age of 6 months fed with standard diet, supplemented with placebo (solid line and squares) or of the NEP inhibitor candoxatril [consumption 200 mg/kg/day] (broken line and triangles). Treatment day 0 is the first day of treatment. Treatment effect ***<i>P</i><0.001 by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni <i>post-hoc</i> test, *<i>P</i><0.05 and **<i>P</i><0.01. (<b>b</b>) Abdominal fat in male C57BL/6 mice after feeding with standard food (open column) or with food supplemented with the NEP inhibitor candoxatril (black column) for two months [consumption 200 mg/kg/day]. Student's <i>t</i>-test **<i>P</i><0.01 versus placebo. (<b>c</b>) Development of food intake in male C57BL/6 mice fed with standard food (open columns) or the same food supplemented with the NEP inhibitor candoxatril (black columns) [consumption 200 mg/kg/day]. Student's <i>t</i>-test **<i>P</i><0.01, ***<i>P</i><0.001 versus placebo. (<b>d</b>) Development of NEP activity in kidney (left panel) and brain (right panel) of mice after oral treatment with candoxatril (1) Placebo, (2) 100 mg/kg/day candoxatril, (3) 200 mg/kg/day candoxatril. Student's <i>t</i>-test *<i>P</i><0.05, **<i>P</i><0.01 versus placebo. (<b>e</b>) Development of body weight in male tumor-bearing mice (Pancreas carcinoma, PSN-1) fed with standard food (solid line and squares) or standard food supplemented with the NEP inhibitor candoxatril (200 mg/kg/day, broken line and triangles). Treatment effect <i>*P<0.05</i>, by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni post-hoc test, *<i>P</i><0.05 and **<i>P</i><0.01.</p

Biochemical parameters in obese NEP-deficient mice under low fat diet.

<p>(<b>a</b>) Serum triglycerides in one-year old NEP-deficient animals (−/−) and their age-matched wild-type controls (+/+). Student's <i>t</i>-test ***<i>P</i><0.001 versus wild-type. (<b>b</b>) Serum HDL (left panel) and VLDL (right panel) in both genotypes. Data is presented as means ± SEM. Student's <i>t</i>-test ***<i>P</i><0.001 versus wild-type. (<b>c</b>) Significant correlation of serum leptin levels in NEP-deficient mice with increasing body weight (r² 0.92 [Pearson correlation]). (<b>d</b>) Comparison of basic glucose values (before glucose tolerance test) in plasma of NEP-knockout mice with wild-type animals. Student's <i>t</i>-test **<i>P</i><0.01 versus wild-type. (<b>e</b>) Comparison of NEP-knockout mice (dotted line) with wild-type animals (solid line) in their response on a glucose tolerance test. Treatment differences are calculated by two-way ANOVA *<i>P</i><0.05.</p

Assessment of Pre- and Pro-haptens Using Nonanimal Test Methods for Skin Sensitization

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to <i>in vivo</i> methods, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten–peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in the keratinocyte and dendritic cell based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (WoE) approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of <i>in chemico</i> and <i>in vitro</i> test methods is suitable to identify pre-haptens and the majority of pro-haptens

Cortical Aβ deposition in brains of humans and mice.

<p>Representative examples of immunostaining for Aβ in brain sections obtained from 6-month-old (b) and 24-month-old NEP-deficient mice (d) and corresponding age-matched wildtype controls (a,c), and from post mortem human brain material of a 75-year-old Alzheimer patient (f) and a 31-year-old human control (e). Images shown in a–d represent the mouse somatosensory cortex (barrel field), while human brain sections (e,f) were obtained from the temporal cortex. All sections were immunostained under the same conditions and in the same experimental session with the biotinylated primary antiserum 4G8 which is known to react with both human and murine Aβ peptides. Scale bar represents 200 µm.</p

Identification of Small Molecule Modulators of Gene Transcription with Anticancer Activity

Epigenetic regulation of gene expression is essential in many biological processes, and its deregulation contributes to pathology including tumor formation. We used an image-based cell assay that measures the induction of a silenced GFP-estrogen receptor reporter to identify novel classes of small molecules involved in the regulation of gene expression. Using this Locus Derepression assay, we queried 283,122 compounds by quantitative high-throughput screening evaluating compounds at multiple concentrations. After confirmation and independent validation, the Locus Derepression assay identified 19 small molecules as new actives that induce the GFP message over 2-fold. Viability assays demonstrated that 17 of these actives have anti-proliferative activity, and two of them show selectivity for cancer versus patient-matched normal cells and cause unique changes in gene expression patterns in cancer cells by altering histone marks. Hence, these compounds represent chemical tools for understanding the molecular mechanisms of epigenetic control of transcription and for modulating cell growth pathways