Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

Inhalation developmental neurotoxicity study of ethylbenzene in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of whole-body inhalation exposure of F0 and F1 parental animals from a 2-generation reproduction study of ethylbenzene on nervous system functional and/or morphologic end points in the F2 offspring from four groups of male and female Crl:CD (SD)IGS BR rats. METHODS: Thirty rats/sex/group for F0 and 25/sex/group for F1 were exposed to 0, 25, 100, and 500 ppm ethylbenzene for six hours daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure for the F0 and F1 females continued throughout mating and gestation through Gestation Day (GD) 20. On lactation days (LD) 1-4, the F0 and F1 females received no inhalation exposure, but instead were administered ethylbenzene in corn oil via oral gavage at dosages estimated to result in similar internal maternal exposure based upon PBPK modeling estimates (0, 26, 90, and 342 mg/kg/day, respectively, divided into three equal doses, approximately two hours apart). Inhalation exposure of the F0 and F1 females was reinitiated on LD 5 and continued through weaning on postnatal day (PND) 21. Survival, body weights, and physical landmarks were assessed in selected F2 offspring. Neurobehavioral development of one F2-generation treatment derived offspring/sex/litter was assessed in a functional observational battery (FOB; PND 4, 11, 22, 45, and 60), motor activity sessions (PND 13, 17, 21, and 61), acoustic startle testing (PND 20 and 60), a Biel water maze learning and memory task (initiated on PND 26 or 62), and in evaluations of whole-brain measurements and brain morphometric and histologic assessments (PND 21 and 72). RESULTS: There were no adverse effects on reproductive performance in either the F0 or F1 parental generations exposed to up to 500 ppm ethylbenzene [Faber et al. Birth Defects Res Part B 77:10-21, 2006]. In the current developmental neurotoxicity component, parental ethylbenzene exposure did not adversely affect offspring survival, clinical condition, body weight parameters, or acquisition of developmental landmarks of the F2-generation treatment derived offspring. There were no alterations in FOB parameters, motor activity counts, acoustic startle endpoints, or Biel water maze performance in offspring attributed to parental ethylbenzene exposure. A few isolated instances of statistically significant differences obtained in the treatment-derived groups occurred sporadically, and were attributed to unusual patterns of development and/or behavior in the concurrent control group. There were no exposure-related differences in any neuropathology parameters in the F2-generation treatment derived offspring. CONCLUSIONS: The no observed adverse effect level (NOAEL) for maternal reproductive toxicity, developmental toxicity, and developmental neurotoxicity in this study was considered to be 500 ppm/342 mg/kg/day ethylbenzene, the highest exposure level tested in the study.     

Two-generation reproduction study of di-2-ethylhexyl terephthalate in Crl:CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of di-2-ethylhexyl terephthalate (DEHT) on reproductive capability from exposure of F(0) and F(1) parental animals. METHODS: Four groups of male and female Crl:CD (SD)IGS BR rats (30/gender/group) were exposed to 0, 0.3%, 0.6%, and 1.0% DEHT in the feed for at least 70 consecutive days before mating for the F(0) and F(1) generations. Exposure for the F(0) and F(1) males continued throughout the mating period until euthanasia. Exposure for the F(0) and F(1) females continued throughout mating, gestation, and lactation. The F(1) and F(2) pups were weaned on postnatal day (PND) 21. Assessments included gonadal function, estrous cyclicity, mating behavior, conception rate, gestation, parturition, lactation, and weaning in the F(0) and F(1) generations, and F(1) generation offspring growth and development. RESULTS: DEHT exposure did not affect clinical observations. However, lethality was observed in F(0) and F(1) dams consuming the 1.0% diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3% or 0.6% DEHT. Male rats consuming the 1.0% diet in both parental generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either the F(0) or F(1) generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, developmental landmarks, and postnatal survival were similar in all exposure groups. Additionally, ovarian follicle counts for the F(1) females in the high-exposure group were similar to the control values. No adverse exposure-related macroscopic pathology was noted at any exposure level in the F(0) and F(1) generations. CONCLUSIONS: Increases in liver weights were found in the male and female animals exposed to 0.6% or 1.0% DEHT in the diet. Because there were no accompanying histopathologic changes, this effect was not considered adverse. Significant decreases in feed consumption in the female animals from the groups consuming 1.0% DEHT in the diet during lactation accompanied reduced postnatal pup body weights and rate of weight gain. Reductions in pup body weights later in lactation may also have been due to direct consumption of the treated feed by the pups or taste aversion to the same. Reduced relative spleen weight was found in male weanling pups from the 1.0% group in both generations and reduced relative spleen and thymus weights were found in female pups from the 1.0% group in the F(2) generation at necropsy on PND 21. Therefore, for parental and pup systemic toxicity, 0.3% DEHT in the diet (182 mg/kg/day) was considered no-observed-effect level (NOEL). The 1.0% DEHT (614 mg/kg/day) in the diet exposure concentration was considered a NOEL for F(0) and F(1) reproductive toxicity endpoints.     

Genetic selection for modulators of the MAP kinase and beta-catenin growth-control pathways in mammalian cells

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.