Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Differentiation of subnucleus-sized oligomers and nucleation-competent assemblies of the Aß peptide.

A significant feature of Alzheimer's disease is the formation of amyloid deposits in the brain consisting mainly of misfolded derivatives of proteolytic cleavage products of the amyloid precursor protein amyloid-ß (Aß) peptide. While high-resolution structures already exist for both the monomer and the amyloid fibril of the Aß peptide, the mechanism of amyloid formation itself still defies precise characterization. In this study, low and high molecular weight oligomers (LMWOs and HMWOs) were identified by sedimentation velocity analysis, and for the first time, the temporal evolution of oligomer size distributions was correlated with the kinetics of amyloid formation as determined by thioflavin T-binding studies. LMWOs of subnucleus size contain fewer than seven monomer units and exist alongside a heterogeneous group of HMWOs with 20-160 monomer units that represent potential centers of nucleus formation due to high local monomer concentrations. These HMWOs already have slightly increased ß-strand content and appear structurally similar regardless of size, as shown by examination with a range of fluorescent dyes. Once fibril nuclei are formed, the monomer concentration begins to decrease, followed by a decrease in oligomer concentration, starting with LMWOs, which are the least stable species. The observed behavior classifies the two LMWOs as off pathway. In contrast, we consider HMWOs to be on-pathway, prefibrillar intermediates, representing structures in which nucleated conformational conversion is facilitated by high local concentrations. Aß40 and Aß42 M35ox take much longer to form nuclei and enter the growth phase than Aß42 under identical reaction conditions, presumably because both the size and the concentration of HMWOs formed are much smaller.

Met/Val129 polymorphism of the full-length human prion protein dictates distinct pathways of amyloid formation.

Methionine/valine polymorphism at position 129 of the human prion protein, huPrP, is tightly associated with the pathogenic phenotype, disease progress, and age of onset of neurodegenerative diseases such as Creutzfeldt-Jakob disease or Fatal Familial Insomnia. This raises the question of whether and how the amino acid type at position 129 influences the structural properties of huPrP, affecting its folding, stability, and amyloid formation behavior. Here, our detailed biophysical characterization of the 129M and 129V variants of recombinant full-length huPrP(23-230) by amyloid formation kinetics, CD spectroscopy, molecular dynamics simulations, and sedimentation velocity analysis reveals differences in their aggregation propensity and oligomer content, leading to deviating pathways for the conversion into amyloid at acidic pH. We determined that the 129M variant exhibits less secondary structure content before amyloid formation and higher resistance to thermal denaturation compared to the 129V variant, whereas the amyloid conformation of both variants shows similar thermal stability. Additionally, our molecular dynamics simulations and rigidity analyses at the atomistic level identify intramolecular interactions responsible for the enhanced monomer stability of the 129M variant, involving more frequent minimum distances between E196 and R156, forming a salt bridge. Removal of the N-terminal half of the 129M full-length variant diminishes its differences compared to the 129V full-length variant and highlights the relevance of the flexible N terminus in huPrP. Taken together, our findings provide insight into structural properties of huPrP and the effects of the amino acid identity at position 129 on amyloid formation behavior.

Tryptophan metabolism in rheumatoid arthritis is associated with rheumatoid factor and predicts joint pathology evaluated by the Rheumatoid Arthritis MRI Score (RAMRIS).

OBJECTIVES: Tryptophan and its metabolites have been suggested to play a role in inflammatory processes. However, studies in rheumatoid arthritis (RA) are scarce, which prompted us to investigate two cohorts of RA patients to better understand the importance of tryptophan metabolism in this disease. METHODS: Tryptophan and its metabolites were characterised by ELISA in a cross-sectional cohort 1 (81 RA, 55 OA) and a longitudinal cohort 2 (25 RA, 3 visits over 6 months) to investigate discriminatory power between diseases and predicitive value for radiologic outcome, respectively. Radiologic outcome was monitored by RA MRI Score (RAMRIS), including grading of synovitis, bone oedema and erosion, as well as delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) index assessing cartilage quality of the MCP II joint. RESULTS: RA patients showed higher levels of serum serotonin (RA: 206.8 ng/ml ± 156.7; OA: 81.2 ng/ml ± 63.6) and estimated indoleamine (2,3)-dioxygenase (IDO) activity (kynurenine / tryptophan ratio; RA: 0.065±0.067; OA: 0.021±0.010). IDO activity showed similar, or better discriminatory power between RA and OA (AUC 0.914) than anti-CCP antibody level (AUC 0.922) and rheumatoid factor (RF, AUC 0.783), respectively. In cohort 2, regression analysis revealed a predictive value of baseline serotonin levels and IDO activity for changes in RAMRIS score and erosions at month six, respectively. CONCLUSIONS: This study supports the hypothesis that tryptophan and its metabolites can be used as biomarkers predicting radiologic outcome and discriminate between RA and OA patients. Overall, our results strengthen the notion that tryptophan metabolism is closely linked to RA disease mechanisms.

Inflammation and vascularisation markers of arthroscopically-guided finger joint synovial biospies reflect global disease activity in rheumatoid arthritis.

OBJECTIVES: To analyse whether synovial markers of the clinically dominant metacarpophalangeal (MCP) joint reflect global disease activity measures in rheumatoid arthritis (RA). METHODS: Arthroscopically-guided synovial biopsies from the dominant metacarpophalangeal (MCP) joint of 10 patients with RA (DAS28 >3.2) were stained for determination of the synovitis score, CD68, vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α). MRI and ultrasound were used to calculate the RAMRIS and US7 score respectively. Arthroscopy of the same joint was repeated in 6 patients after 6 months. RESULTS: The synovitis score significantly correlated to DAS28 (Spearman r=0.74), CRP (r=0.69), and US7 (r=0.66); sublining CD68 macrophages to CRP (r=0.6); HIF-1α to DAS28 (r=0.77), CRP (r=0.73); and VEGF to DAS28 (r=0.753) and RAMRIS (r=0.663). All patients showed a reduction of the DAS28 after 6 months (mean±SD: 5.2±1.5 vs. 2.75±1.1; p<0.05). There were three patients with a good EULAR response, and only these showed declining sublining CD68 macrophages in the control biopsy (χ2 test: LR 8.3, p=0.05). Two of the remaining patients with increasing CD68 sublining macrophages showed a deterioration of the RAMRIS. CONCLUSIONS: Some histological findings in arthroscopically-guided biopsies of the dominantly affected MCP joint reflect global disease activity measures and their changes in RA patients. Moreover, repeated MCP synovial biopsy may distinguish true responders from individuals with residual disease activity, who are not readily recognized by clinical means.

Fermentation Quality of Ensiled Water Hyacinth (Eichhornia crassipes) as Affected by Additives.

A lab-scale ensiling study was carried out to investigate the fermentation quality of water hyacinth (WH) supplemented with molasses, rice bran, as an absorbent, and an inoculant in the form of fermented vegetable juice and their combinations. After wilting the water hyacinths for 7 h to a dry matter (DM) content of 240 to 250 g/kg, the following treatments were applied: i) Control (C), WH only; ii) WH with sugarcane molasses at 40 g/kg WH (CM); iii) WH inoculated with fermented vegetable juice at 10 ml/kg WH (CI); iv) CM and CI (CMI) combined; v) WH with 150 g rice bran/kg WH (CA); vi) CA and CI combined (CAI); vii) CA and CM combined (CAM); and viii) CA, CM and CI combined (CAMI). After application of additives, the differently treated forages were mixed and ensiled in triplicates in 1,500-ml polyethylene jars. After ensiling for 3 d, pH values in all treatments, except C and CI, had decreased to approximately 4.0 and remained low till 14 d. After 56 d, pH had increased between 0.4 to 0.9 pH-units compared to those at 14 d. The ammonia nitrogen (NH3-N) concentration ranged from an acceptable level in treatment CM (8 g/kg N) to a high NH3-N value in treatment CMI (16 g/kg N). Lactic acid formation was higher in CI than in all other treatments. Butyric acid contents, which indicate badly fermented silages, were low in all silages (<2 g/kg DM). There were two-way interactions (p-values from <0.001 to 0.045) for almost all fermentation end-products and pH, except for the molasses×inoculant interaction on NH3-N (p = 0.26). Significant 3-way interactions were found on all observed variables except for weight losses of silages. It is concluded that conserving wilted WH as silage for ruminants may be improved by the addition of molasses or rice bran.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Versatile Route toward Hydrophobically Polymer-Grafted Gold Nanoparticles from Aqueous Dispersions.

Stabilization of gold nanoparticles in organic solvents is a key challenge in making them available for a wider range of material applications. Polymers are often used as stabilizing ligands because they also allow for the introduction of new properties and functionalities. Many of the established synthesis protocols for gold nanoparticles are water-based. However, the insolubility of many synthetic polymers in water renders the direct functionalization of aqueous particle dispersions with these ligands difficult. Here, we report on an approach for the functionalization of gold nanoparticles, which were prepared by aqueous synthesis, with hydrophobic polymer ligands and their characterization in nonpolar, organic dispersions. Our method employs an auxiliary ligand to first transfer gold nanoparticles from an aqueous to an organic medium. In the organic phase, the auxiliary ligand is then displaced by thiolated polystyrene ligands to form a dense polymer brush on the particle surface. We characterize the structure of the ligand shell using electron microscopy, scattering techniques, and ultracentrifugation and analyze the influence of the molecular weight of the polystyrene ligands on the structure of the polymer brush. We further investigate the colloidal stability of polystyrene-functionalized gold nanoparticles in various organic solvents. Finally, we extend the use of our protocol from small, spherical gold nanoparticles to larger gold nanorods and nanocubes.

Thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV: structure-based drug design, synthesis, and biological evaluation.

A novel series of potent thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV was discovered using structure-based drug design. Synthesis, structure-activity relationship, and optimization of physicochemical properties are described. Low nanomolar potency was achieved, and selected compounds with improved thermodynamic solubility showed promising in vitro inhibition of carbonic anhydrase activity in rabbit iris ciliary body homogenate.

A comparative health care inventory for primary hip arthroplasty between Germany versus the Netherlands. Is there a downside effect to fast-track surgery with regard to patient satisfaction and functional outcome?

BACKGROUND: Treatment and rehabilitation protocol for hip arthroplasty differs between Germany and the Netherlands. The Dutch system promotes fast-track surgery whereas in Germany conventional care is provided with a longer hospital stay including rehabilitation. Clinical outcome, patient satisfaction and costs in both treatment protocols were compared in a prospective setup. MATERIAL AND METHODS: This prospective cohort study included patients allocated for primary THA in 3 German and 1 Dutch hospital in the border region. Patient-reported outcome scores (PROMS) were measured pre- and postoperatively at 6 and 12 months including the Oxford Hip Score, SF12 survey, visual analogue scale for satisfaction and pain. Length of hospitalisation and availability of postoperative rehabilitation were recorded. In addition, a total cost estimation was calculated using health insurers data. RESULTS: A total of 360 consecutive patients were included; 175 THA in Germany compared to 185 THA in the Netherlands. No cross-border healthcare was encountered in both cohorts. Mean length of hospitalisation was 11.3 (range 6-23) days in Germany, compared to 4.4 (range 3-25) days in the Netherlands. In Germany 92% of the patients was discharged with inpatient (72%) or outpatient (20%) rehabilitation, compared to 21% with only inpatient rehabilitation in the Netherlands. No significant differences were measured regarding the PROMS and patient satisfaction between both countries. Due to profound differences in health care financing only a global cost estimation could be made and no major differences were encountered. CONCLUSION: Germany and the Netherlands both offer highly protocolled care for THA with comparable functional outcome and patient satisfaction with treatment after 12 months. Despite the length of hospitalisation in Germany is significantly longer including a more intensive rehabilitation programme, no significant differences were recorded regarding functional outcome nor patient satisfaction compared to fast-track surgery performed in the Netherlands.

Author Correction: Activating low-temperature diesel oxidation by single-atom Pt on TiO2 nanowire array.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activating low-temperature diesel oxidation by single-atom Pt on TiO2 nanowire array.

Supported metal single atom catalysts (SACs) present an emerging class of low-temperature catalysts with high reactivity and selectivity, which, however, face challenges on both durability and practicality. Herein, we report a single-atom Pt catalyst that is strongly anchored on a robust nanowire forest of mesoporous rutile titania grown on the channeled walls of full-size cordierite honeycombs. This Pt SAC exhibits remarkable activity for oxidation of CO and hydrocarbons with 90% conversion at temperatures as low as ~160 oC under simulated diesel exhaust conditions while using 5 times less Pt-group metals than a commercial oxidation catalyst. Such an excellent low-temperature performance is sustained over hydrothermal aging and sulfation as a result of highly dispersed and isolated active single Pt ions bonded at the Ti vacancy sites with 5 or 6 oxygen ions on titania nanowire surfaces.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11beta-hydroxysteroid dehydrogenase type 1: Discovery of PF-915275.

N-(Pyridin-2-yl) arylsulfonamides are identified as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), an enzyme that catalyzes the reduction of the glucocorticoid cortisone to cortisol. Dysregulation of glucocorticoids has been implicated in the pathogenesis of diabetes and the metabolic syndrome. In this Letter, we present the development of an initial lead to an efficient ligand with improved physiochemical properties using a deletion strategy. This strategy allowed for further optimization of potency leading to the discovery of the clinical candidate PF-915275.

Stoichiometric Zn2+ interferes with the self-association of Aß42: Insights from size distribution analysis.

The abnormal aggregation of amyloid ß (Aß) peptides in the brain has been recognized as a central event in Alzheimer's disease (AD). Divalent metal ions such as Zn2+ have been shown to be closely involved in modulating Aß self-association. Although the link between Zn2+ dyshomeostasis and brain Aß deposition has been established, the effect of Zn2+ on the aggregation of Aß is still incompletely clarified. By combining analytical ultracentrifugation (AUC), circular dichroism (CD) spectroscopy, thioflavin T (ThT) assay and atomic force microscopy (AFM) imaging, we analyzed the impact of stoichiometric Zn2+ on the aggregation process of Aß42, the main toxic isoform of Aß species in the brain. Aß42 aggregates found in the presence of Zn2+ were smaller in size, non-fibrillary and showed less ß-sheet structures than aggregates formed in absence of Zn2+. AUC showed that Zn2+ was capable of retaining monomeric Aß42 in solution. Zn2+ chelation by EDTA totally reversed the inhibitory effect of Zn2+ on Aß42 fibrillation. Our results provide further evidence that Zn2+ shifts the self-association of Aß42 toward a non-fibrillary pathway by interfering with the aggregation process at multiple levels.

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase.

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.

Inhomogeneity of immune cell composition in the synovial sublining: linear mixed modelling indicates differences in distribution and spatial decline of CD68+ macrophages in osteoarthritis and rheumatoid arthritis.

BACKGROUND: Inhomogeneity of immune cell distribution in the synovial sublining layer was analyzed in order to improve our mechanistic understanding of synovial inflammation and explore potential refinements for histological biomarkers in rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial tissue of 20 patients (11 RA, 9 OA) was immunohistochemically stained for macrophages (CD68), synovial fibroblasts (CD55), T cells (CD3), plasma cells (CD38), endothelial cells (vWF) and mast cells (MCT). The synovial sublining layer was divided into predefined adjacent zones and fractions of the stained area (SA) were determined by digital image analysis for each cell marker. RESULTS: Distribution of CD68, CD55, CD38 and MCT staining of the sublining area was heterogeneous (Friedman ANOVA p < 0.05). The highest expression for all markers was observed in the upper layer close to the lining layer with a decrease in the middle and lower sublining. The SA of CD68, CD55 and CD38 was significantly higher in all layers of RA tissue compared to OA (p < 0.05), except the CD38 fraction of the lower sublining. Based on receiver operating characteristics analysis, CD68 SA of the total sublining resulted in the highest area under the curve (AUC 0.944, CI 95 % 0.844-1.0, p = 0.001) followed by CD68 in the upper and middle layer respectively (both AUC 0.933, CI 95 % 0.816-1.0, p = 0.001) in both RA and OA. Linear mixed modelling confirmed significant differences in the SA of sublining CD68 between OA and RA (p = 0.0042) with a higher concentration of CD68+ towards the lining layer and more rapid decline towards the periphery of the sublining in RA compared to OA (p = 0.0022). CONCLUSIONS: Immune cells are inhomogeneously distributed within the sublining layer. RA and OA tissue display differences in the number of CD68 macrophages and differences in CD68 decline within the synovial sublining.

Structure-activity analysis of the purine binding site of human liver glycogen phosphorylase.

Human liver glycogen phosphorylase (HLGP) catalyzes the breakdown of glycogen to maintain serum glucose levels and is a therapeutic target for diabetes. HLGP is regulated by multiple interacting allosteric sites, each of which is a potential drug binding site. We used surface plasmon resonance (SPR) to screen for compounds that bind to the purine allosteric inhibitor site. We determined the affinities of a series of compounds and solved the crystal structures of three representative ligands with K(D) values from 17-550 microM. The crystal structures reveal that the affinities are partly determined by ligand-specific water-mediated hydrogen bonds and side chain movements. These effects could not be predicted; both crystallographic and SPR studies were required to understand the important features of binding and together provide a basis for the design of new allosteric inhibitors targeting this site.

Elevated plasma 4-pyridoxic acid in renal insufficiency.

BACKGROUND: Renal insufficiency is associated with altered vitamin B-6 metabolism. We have observed high concentrations of 4-pyridoxic acid, the major catabolite of vitamin B-6 metabolism, in plasma during renal insufficiency. OBJECTIVE: The objective was to evaluate the renal handling of 4-pyridoxic acid and the effects of renal dysfunction on vitamin B-6 metabolism. DESIGN: We measured the renal clearance of 4-pyridoxic acid and creatinine in 17 nonpregnant, 17 pregnant, and 16 lactating women. We then examined the influence of vitamin B-6 or alkaline phosphatase activity on the ratio of 4-pyridoxic acid to pyridoxal (PA:PL) in plasma in 10 men receiving a low (0.4 mg pyridoxine.HCl/d) or high (200 mg pyridoxine.HCl/d) vitamin B-6 intake for 6 wk, in 10 healthy subjects during a 21-d fast, in 1235 plasma samples from 799 people screened for hypophosphatasia, and in 67 subjects with a range of serum creatinine concentrations. RESULTS: Renal clearance of 4-pyridoxic acid was 232 +/- 94 mL/min in nonpregnant women, 337 +/- 140 mL/min in pregnant women, and 215 +/- 103 mL/min in lactating healthy women. These values were approximately twice the creatinine clearance, indicating that 4-pyridoxic acid is at least partially eliminated by tubular secretion. Elevated plasma creatinine concentrations were associated with marked elevations in 4-pyridoxic acid and PA:PL. PA:PL was not affected by wide variations in vitamin B-6 intake or by the wide range of pyridoxal-P concentrations encountered while screening for hypophosphatasia. CONCLUSIONS: Plasma 4-pyridoxic acid concentrations are markedly elevated in renal insufficiency. Plasma PA:PL can distinguish between increases in 4-pyridoxic acid concentrations due to increased dietary intake and those due to renal insufficiency.

In utero nicotine exposure causes persistent, gender-dependant changes in locomotor activity and sensitivity to nicotine in C57Bl/6 mice.

Maternal cigarette smoking during pregnancy can result in a wide variety of adverse fetal outcomes, ranging from preterm delivery and low birth weight, to sudden infant death syndrome. In addition, in utero tobacco smoke exposure is associated with delayed or impaired neuropsychological development. Although the causative agent in tobacco smoke that leads to these aberrations is not known, some studies have concluded that nicotine may play an important role. Many studies using animal models of prenatal nicotine exposure have supported the hypothesis that nicotine may directly and/or indirectly cause impairments in fetal and neonatal development. However, in many of the animal studies nicotine has been administered acutely to naive dams, which could lead to significant fetal hypoxia; some routes of drug administration are also very stressful to pregnant dams, and changes in stress hormones could also create an unfavorable fetal environment. In this study, pregnant mice were exposed to chronic nicotine via the drinking solution; locomotor activity and sensitivity to nicotine were evaluated in the offspring. We have previously shown that oral nicotine administration produces behavioral and physiological changes that resemble those seen following other routes of nicotine administration. Although oral nicotine exposure did not significantly alter any aspect of the pregnancy, dams drinking a nicotine-containing solution consumed approximately 20% less volume, compared to saccharin controls. All animals were cross fostered to nicotine naïve lactating dams, immediately after birth. On PN40 and PN60, male mice exposed to in utero nicotine demonstrated significant locomotor hyperactivity in an open filed arena. Although female animals did not show any signs of hyperactivity, they did have a significant attenuation of their hypothermic response to acute nicotine challenge. These results suggest that oral nicotine delivery to pregnant mice causes persistent, gender-dependant changes in behavior and sensitivity to nicotine. This model may be very useful for future studies that try to more accurately define the windows of sensitivity for nicotine exposure and the possible underlying neurochemical mechanisms involved.