Mode-of-action and human relevance framework analysis for rat Leydig cell tumors associated with sulfoxaflor

Abstract

Sulfoxaflor, a molecule that targets sap-feeding insects, was assessed for carcinogenic potential in groups of 50 Fischer rats fed with diets containing 0, 25, 100, 500 (males), or 750 (females) ppm sulfoxaflor for 2 years according to OECD 453. Sulfoxaflor did not alter the number of rats with Leydig cell tumors (LCTs: 88% of controls and 90–92% in treated groups). The size of LCT was increased at 100 and 500 ppm. The spontaneous incidence of LCT in Fischer rat is 75–100% compared with less than 0.01% in humans. These fundamental interspecies differences in spontaneous incidence of LCT are the result of quantitative and qualitative differences in Leydig cell response to hormonal stimuli. There are nine known modes of actions (MoA) for LCT induction. Analysis sulfoxaflor data suggested a hormone-based dopamine enhancement MoA causing the LCT effect through: 1) increased neuronal dopamine release via specific dopaminergic neuron-based nicotinic acetylcholine receptor (nAChR) agonism, leading to 2) decreased serum prolactin (Prl) levels, 3) downregulation of luteinizing hormone receptor (LHR) gene expression in Leydig cells, 4) transient decreases in serum testosterone, 5) increased serum LH levels, and 6) promotion of LCTs. The analysis suggested that sulfoxaflor promoted LCTs through a subtle stimulation of dopamine release. The MoA for LCT promotion in the carcinogenicity study is considered to have no relevance to humans due to qualitative and quantitative differences between rat and human Leydig cells. Therefore, the Fischer 344 rat LCT promotion associated with lifetime administration of high-dose levels of sulfoxaflor would not pose a cancer hazard to humans.     

Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.     

Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His

Antithrombin III (AT-III) Rouen is a hereditary abnormal antithrombin with normal progressive inhibitory activity and reduced heparin cofactor activity. It was isolated from the plasma of a woman who suffered a sudden idiopathic sensorineural hearing loss and balance impairment. There was no familial history of thrombosis. By heparin- Sepharose chromatography, AT-III Rouen was separated from the normal antithrombin on elution with increasing concentrations of NaCl. AT-III Rouen eluted earlier than is normal at both pH 7.4 and pH 6.0. At the lower pH, the antithrombins bound more avidly to the column, with the abnormal AT-III eluting closer to the normal than at the higher pH. Two- dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests of carboxymethylated antithrombins was performed on thin-layer silica plates. The abnormal peptide was located by tryptophan staining, and amino acid analysis and sequence studies demonstrated a substitution of an arginine at residue 47 for a histidine. Results from this study suggest that replacement of arginine 47 by a partially positively charged histidine has less effect on the heparin binding affinity than dose replacing it with a neutral cysteine side chain as in AT-III Toyama, in which no heparin binding was observed. In addition, heparin binding per se is not a sufficient condition to activate AT-III.     

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s⁻¹ at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k_cat ∼6 s⁻¹) was similar to the actin-detachment rate (k_det = 6.2 s⁻¹) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.Unconventional myosin 15 is expressed by inner ear hair cells and accumulates at the tips of mechanosensory stereocilia (1, 2), actin-based organelles that are stimulated by sound and accelerations. A missense substitution in the myosin 15 motor domain results in abnormally short stereocilia in the Myo15^sh2/sh2 (shaker 2) mouse, indicating that myosin 15 motor activity is essential for elongation of the core actin filaments during development (1, 3). Emphasizing the critical importance of myosin 15 for sensory function, mutations of the orthologous MYO15A also cause profound nonsyndromic autosomal recessive deafness (DFNB3) in humans (4, 5). Myosin 15 is hypothesized to be a molecular motor that transports the scaffolding protein whirlin (DFNB31) and epidermal growth factor receptor kinase substrate 8 (EPS8) to the stereocilia tips, where these cargos regulate elongation of the developing actin core (6, 7). Although myosin 15 motility has not been visualized in live hair cells, it similarly transports whirlin and EPS8 to the tips of filopodia, finger-like protrusions of bundled actin filaments (6, 7). Little is known about the functional properties of myosin 15, its mechanism of motility along stereocilia or filopodial actin filaments, or how this might be altered by mutations associated with DFNB3 human deafness.Myosins are a superfamily of actin-activated P-loop ATPases that produce force to power fundamental cellular processes, such as cytokinesis and vesicle trafficking (8). Biochemical and biophysical analyses of purified isozymes have been critical to deciphering their cellular functions. Most myosins share a common catalytic mechanism, but kinetic tuning of reaction rates can result in very different characteristics (9, 10). An important property is the duty ratio; defined as the fraction of the ATPase cycle that myosin spends strongly bound to actin. Low-duty ratio motors, exemplified by muscle myosins, spend a small fraction (∼0.05) of their cycle strongly bound to actin and act in large ensembles. In contrast, processive motors like myosin 5a have a duty ratio of >0.7 at saturating [actin], and thus spend most of their ATPase cycle in strongly actin-bound states (11). For myosin 5a, the high-duty ratio supports the processive movement of dimers by increasing the likelihood that one head is always bound to actin, thus preventing diffusion away from the filament track.Myosin 15 is the largest myosin heavy chain in the mammalian proteome and is highly expressed in the inner ear and endocrine organs (2). Phylogenetic analysis of motor domain sequences reveals its close relationship with unconventional class VII and X myosins, in addition to sharing similar myosin tail homology 4 (MyTH4), Src homology 3 (SH3) and band 4.1, ezrin, radixin, moesin (FERM) domains in the C-terminal tail (Fig. 1A). Alternate splicing of exon 2 (1, 2), creates two protein isoforms that are identical except for a large (133 kDa) proline-rich N-terminal extension that is unique to isoform 1 (Fig. 1A). The function of isoform 1 is unknown, whereas isoform 2 targets to stereocilia and filopodia tips, and is sufficient to rescue stereocilia development in Myo15^sh2/sh2 hair cells (1, 6). Myosin 15 has three presumed light-chain binding sites (IQ motifs) that are predicted to form a lever arm; however, the light chains that potentially bind these in vivo are unknown.Open in a separate windowFig. 1.Purification of mouse myosin 15 from Sf9 cells by chaperone coexpression. (A) Schematic domain structure of mouse myosin 15 isoforms. (B) Motor domain constructs used in this study. All expressed proteins have a C-terminal FLAG epitope for affinity purification. (C) ClustalW alignments of the second IQ of myosin 15 show similarity to the ELC binding site (first IQ) of class II myosins. (D) Coexpression with UNC45B/HSP90AA1 increases the solubility of EGFP-Myo15-3IQ in Sf9 insect cells (abbreviated to 3IQ for D–F). Western blot probed with antibody against FLAG (green) and alpha-tubulin (red). Total and soluble fractions were prepared from equal volumes of Sf9 cell cultures expressing EGFP-Myo15-3IQ, plus combinations of chaperones and light chains as shown. EGFP-Myo15-3IQ sedimented when coexpressed with calmodulin (CALM) alone, or with CALM + ELC + RLC. Expression of UNC45B + HSP90AA1 increased EGFP-Myo15-3IQ in the supernatant (compare arrowheads), but did not change heavy chain expression overall (compare stars). (E) Supernatants from D were bound to FLAG-M2 affinity resin, eluted with FLAG peptide (0.5-mL fractions), and assayed for EGFP fluorescence (representative of three experiments). Individual images (outlined in white) for each condition were captured with identical settings and consolidated. Samples coexpressing RLC + ELC in addition to UNC45B + HSP90AA1 eluted in earlier fractions (third row), compared with coexpressing UNC45B/HSP90AA1 and CALM (second row). (F) SDS/PAGE of FLAG-purified proteins from Sf9 cells expressing UNC45B + HSP90AA1 and combinations of Myo15 IQ mutants and lights chains as indicated. Note increased mobility of Myo15-1IQ (arrowhead) without an EGFP fusion.To understand how myosin 15 operates within the stereocilia organelle, we have purified the motor domain and used a combination of single molecule and ensemble kinetic/mechanical analyses to determine its properties. While attempting to purify myosin 15 from Spodoptera frugiperda (Sf9) insect cells, we discovered that coexpression of heat-shock protein 90 (HSP90) and its cochaperone UNC45B significantly improved the yield of soluble protein. We demonstrate that purified myosin 15 is a mechanically active, barbed-end–directed molecular motor that exhibits a high-duty ratio. These data suggest that myosin 15 might be capable of processive motility if dimerized, consistent with its proposed role as a stereocilia transporter.     

Modification of conventional glass-ionomer cements with N-vinylpyrrolidone containing polyacids, nano-hydroxy and fluoroapatite to improve mechanical properties

OBJECTIVE: The objective of this study was to enhance the mechanical strength of glass-ionomer cements, while preserving their unique clinical properties. METHODS: Copolymers incorporating several different segments including N-vinylpyrrolidone (NVP) in different molar ratios were synthesized. The synthesized polymers were copolymers of acrylic acid and NVP with side chains containing itaconic acid. In addition, nano-hydroxyapatite and fluoroapatite were synthesized using an ethanol-based sol-gel technique. The synthesized polymers were used in glass-ionomer cement formulations (Fuji II commercial GIC) and the synthesized nanoceramic particles (nano-hydroxy or fluoroapatite) were also incorporated into commercial glass-ionomer powder, respectively. The synthesized materials were characterized using FTIR and Raman spectroscopy and scanning electron microscopy. Compressive, diametral tensile and biaxial flexural strengths of the modified glass-ionomer cements were evaluated. RESULTS: After 24h setting, the NVP modified glass-ionomer cements exhibited higher compressive strength (163-167MPa), higher diametral tensile strength (DTS) (13-17MPa) and much higher biaxial flexural strength (23-26MPa) in comparison to Fuji II GIC (160MPa in CS, 12MPa in DTS and 15MPa in biaxial flexural strength). The nano-hydroxyapatite/fluoroapatite added cements also exhibited higher CS (177-179MPa), higher DTS (19-20MPa) and much higher biaxial flexural strength (28-30MPa) as compared to the control group. The highest values for CS, DTS and BFS were found for NVP-nanoceramic powder modified cements (184MPa for CS, 22MPa for DTS and 33MPa for BFS) which were statistically higher than control group. CONCLUSION: It was concluded that, both NVP modified and nano-HA/FA added glass-ionomer cements are promising restorative dental materials with improved mechanical properties.     

The effect of maturation on in-vitro erosion of glass-ionomer and other dental cements.

The clinical durability of restorations made using water-based materials depends upon the type of material used. Current specification tests cannot predict durability, possibly because these are carried out on samples matured for 24 hours whereas longer maturation times may be more relevant. The lactic acid jet test was selected as a test method, being believed capable of ranking materials in the same order of erosion resistance in vitro as found in vivo. The powder/liquid interaction (glass or zinc oxide with polymeric or phosphoric acid) was investigated by selecting glass-ionomer, zinc polycarboxylate, silicate and zinc phosphate cements, with emphasis placed on glass ionomers. Two test times, 24 hours and 2 months, were chosen, with eight samples for each material tested at each time. All glass ionomers showed a significant reduction in erosion rate with time; two zinc polycarboxylates also showed some reduction but not to a significant degree. The silicate and zinc phosphate cements increased in erosion rate but not significantly. It was concluded that materials using polymeric acids had erosion rates which reduced with time, significantly so for glass ionomers. This might explain their longevity over silicate cements.     

Urinary bladder neoplasms: evaluation with contrast-enhanced MR imaging

Forty-eight patients with urinary bladder neoplasms were examined with magnetic resonance imaging before and after intravenous administration of gadolinium diethylene-triaminepentaacetic acid (DTPA). Spin-echo sequences with short repetition and echo times were used in all patients; in 20 a gradient-echo technique was used to perform sequential imaging. In 31 patients ratios of tumor signal intensity to that of fat, muscle, and bone marrow were calculated before and after Gd-DTPA enhancement on T1-weighted spin-echo images. Increases in tumor signal intensity on T1-weighted spin-echo images were statistically significant after contrast enhancement (alpha = 1%, P less than .0001). The average rise in relative signal intensity after contrast enhancement was 120% for the tumor-fat ratio (tumor-marrow ratio, 105%; tumor-muscle ratio, 85%). Tumor signal intensity peaked within 120 seconds and remained on a plateau for up to 45 minutes. Necrotic tissue within the tumor, seen in three cases, was detectable only on contrast-enhanced images.