Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Do G-protein coupled estrogen receptor and bisphenol A analogs influence on Leydig cell epigenetic regulation in immature boar testis ex vivo?

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

Compounds of PAH mixtures dependent interaction between multiple signaling pathways in granulosa tumour cells.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Interplay between estrogen-related receptors and steroidogenesis-controlling molecules in adrenals. In vivo and in vitro study.

Estrogen-related receptors (ERRs) α, ß and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRß/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 µg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 µg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRß/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Young Accreting Compact Objects in M31: The Combined Power of NuSTAR, Chandra, and Hubble.

We present 15 high-mass X-ray binary (HMXB) candidates in the disk of M31 for which we are able to infer compact object type, spectral type of the donor star, and age using multiwavelength observations from NuSTAR, Chandra, and the Hubble Space Telescope. The hard X-ray colors and luminosities from NuSTAR permit the tentative classification of accreting X-ray binary systems by compact object type, distinguishing black hole from neutron star systems. We find hard-state black holes, pulsars, and non-magnetized neutron stars associated with optical point-source counterparts with similar frequency. We also find nine non-magnetized neutron stars coincident with globular clusters and an equal number of pulsars with and without point-source optical counterparts. We perform spectral energy distribution (SED) fitting for the most likely optical counterparts to the HMXB candidates, finding seven likely high-mass stars and one possible red helium-burning star. The remaining seven HMXB optical counterparts have poor SED fits, so their companion stars remain unclassified. Using published star formation histories, we find that the majority of HMXB candidates-X-ray sources with UV-bright point-source optical counterpart candidates-are found in regions with star formation bursts less than 50 Myr ago, and three are associated with young stellar ages (<10Myr). This is consistent with similar studies of HMXB populations in the Magellanic Clouds, M33, NGC 300, and NGC 2403.

Expression of peroxisome proliferator-activated receptors is regulated by gonadotropins and steroid hormones in in vitro porcine ovarian follicles.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors which are involved in the regulation of different processes such as lipid metabolism, inflammation, angiogenesis, tissue remodeling and steroidogenesis. Our previous data described the ovarian expression of PPAR isoforms (-α, -γ and -ß), and the increase of PPAR-α and PPAR-γ in porcine ovarian follicles during the estrous cycle. The current studies were undertaken to test the hypothesis that gonadotropin and steroid hormones can regulate the expression of PPAR isoforms in the ovary. Medium follicles (4 - 6 mm) at 10 - 12 days of the estrous cycle were obtained from mature crossbred gilts. Ovarian follicles were exposed to gonadotropins FSH and LH at 50, 100 and 150 ng/ml, and to steroid hormones such as progesterone (P4), testosterone (T) and estradiol (E2) at 10-8, 10-7, 10-6 M for 24 hours. Then mRNA and protein expression of PPAR-α/γ/ß via real time PCR and Western blot, respectively, were measured. We observed that FSH increased both mRNA and protein expression of all PPAR isoforms, while LH only increased PPAR-α/γ. We have also noted that P4 and E2 significantly increased expression of PPAR-α/γ without having an effect on ß isoform, while T had no effect on all PPARs expression. Our study clearly showed that local regulators of ovarian activity, both gonadotropin and steroid hormones are regulators of PPAR isoforms expression in porcine ovarian follicles.

Primary and tumor mouse Leydig cells exposed to polychlorinated naphthalenes mixture: Effect on estrogen related-receptors expression, intracellular calcium level and sex hormones secretion.

We report the effects of polychlorinated napthalanes (PCNs) on the mRNA expression of estrogen-related receptors (ERRs) α, ß and γ, calcium (Ca2+) concentration, and sex steroid secretion in mouse primary and tumor Leydig cells. The cells were exposed to a mixture of PCNs (10nM) alone or in combination with one of sex steroid receptor antagonists; 182,780 (ICI; 10µM); hydroxyflutamide (HF; 10(-4)M) and G-coupled estrogen receptor antagonist (G15; 10nM) respectively. The expression of mRNAs and protein for ERRα, ß, and γ was detected in primary and tumor Leydig cells. The expression of ERRs was always lower in primary Leydig cells. Exposure of Leydig cells to PCNs significantly increased the expression of ERRs mRNA irrespective of the cell type. Concomitantly, an increased concentration of Ca2+ and sex steroids was revealed in exposed cells. After ICI, HF or G15 was added no changes in expression of ERRs was found. In Leydig cells changes in ERRs expression at mRNA level are clearly linked to changes in Ca2+ level and steroid secretion. Estrogen and androgen receptors are not involved in PCNs action in Leydig cells. The effect of PCNs on mouse Leydig cells is independent on the cell of origin (primary or tumor).

Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue.

Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2 ng/ml HCBz or PeCBz for 6-72 h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

The influence of short-term kinesiology taping on force-velocity parameters of the rectus abdominis muscle.

PURPOSE: The purpose of the study was to evaluate the influence of Kinesio-Tex tape on force-velocity parameters (F-V) of the rectus abdominis muscle immediately following application. METHODS: The study group selected consisted of 52 women with a BMI below 23 [kg/m(2)]. The study involved measuring the force-velocity parameters of trunk flexor muscles twice using the Biodex System 3 Pro Set. Analyzed parameters included peak torque [N/m], total work [J], power [W] and percentage ratio of trunk flexor and extensor peak torque. RESULTS: Comparing the results obtained in both tests, for a majority there were no statistically significant differences found in both the study group and control group. CONCLUSIONS: 1. Short-term Kinesiology Taping on the rectus abdominis muscle does not cause a change in force-velocity parameters of trunk flexors. 2. Isolated application on the rectus abdominis muscle may be insufficient to cause a change in force-velocity parameters following such a short application time.

The effect of microbial phytase and myo-inositol on performance and blood biochemistry of broiler chickens fed wheat/corn-based diets.

A total of 1,200 Ross broiler chickens were used in 2 separate feeding studies to explore the effect of myo-inositol (MYO) and phytase on performance and blood biochemistry of broilers fed diets formulated to be either adequate or insufficient in Ca and digestible P (dP). Supplementation of diets that were formulated to be insufficient in Ca and dP with MYO resulted in improved BW gain and feed conversion ratio in both experiments. However, these effects were most pronounced in the finisher phase, and moderate negative effects were observed during the starter period. Supplementation of the diet with microbial phytase improved BW gain and feed conversion ratio to a similar extent as was observed with MYO, and there was a degree of subadditivity between the 2 additives. Blood glucose concentrations were increased by both MYO and phytase, though possibly by different mechanisms, because insulin concentrations were not directly relatable to circulating glucose levels, especially when both MYO and phytase were applied simultaneously. The increase in blood glucose concentrations with MYO and phytase was most pronounced in the diet with a lower Ca and dP concentration. It can be concluded that dietary supplementation with MYO or phytase was effective in improving performance of commercial broiler chickens. However, further work is required to explore complex ontogenetic effects of MYO and possible involvement of both MYO and phytase in Na-dependent transport mechanisms.

Efficient room-temperature nuclear spin hyperpolarization of a defect atom in a semiconductor.

Nuclear spin hyperpolarization is essential to future solid-state quantum computation using nuclear spin qubits and in highly sensitive magnetic resonance imaging. Though efficient dynamic nuclear polarization in semiconductors has been demonstrated at low temperatures for decades, its realization at room temperature is largely lacking. Here we demonstrate that a combined effect of efficient spin-dependent recombination and hyperfine coupling can facilitate strong dynamic nuclear polarization of a defect atom in a semiconductor at room temperature. We provide direct evidence that a sizeable nuclear field (~150 Gauss) and nuclear spin polarization (~15%) sensed by conduction electrons in GaNAs originates from dynamic nuclear polarization of a Ga interstitial defect. We further show that the dynamic nuclear polarization process is remarkably fast and is completed in <5 µs at room temperature. The proposed new concept could pave a way to overcome a major obstacle in achieving strong dynamic nuclear polarization at room temperature, desirable for practical device applications.

Magnetic-field-induced delocalized to localized transformation in GaAs:N.

The use of a high magnetic field (57 T) to study the formation and evolution of nitrogen (N) cluster and supercluster states in GaAs:N is demonstrated. A magnetic field is used to lift the conduction band edge and expose resonant N cluster states so that they can be directly experimentally investigated. The reduction of the exciton Bohr radius also results in the fragmentation of N supercluster states, enabling a magnetic field induced delocalized to localized transition. The application of very high magnetic fields thus presents a powerful way to probe percolation phenomena in semiconductors with bound and resonant isoelectronic cluster states.

Activation of the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism by 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in the pig ovary.

The aim of the current study was to determine metabolism of polybrominated diphenyl ether (BDE-47) in the porcine ovary. We analyzed the activity and expression of enzymes involved in phase I (CYP1A1 and CYP2B1/2) and phase II (SULT1A and COMT) of BDE-47 metabolism. Basal CYP1A1 and CYP2B1/2 activity increased during culture. BDE-47 had no effect on CYP1A1, however increased CYP2B1/2 activity after exposure for 6h. Basal SULT1A activity was 2.5 fold lower than that of COMT, and both proteins were stable during culture. BDE-47 increased SULT1A after exposure for 6 h, and COMT activity after exposure for 24 and 48 h. BDE-47 had no effect on the expression of all investigated enzymes. In conclusion, fast activation of CYP2B1/2 and late activation of COMT (with a very low basal SULT1A activity) indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification.

Hydroxylated estrogens (2-OH-E2 AND 4-OH-E2) do not activate cAMP/PKA and ERK1/2 pathways activation in a breast cancer MCF-7 cell line.

OBJECTIVE: The current study was undertaken to determine the involvement of cAMP/PKA and MAPK-mediated signalling pathways in the regulation of cell proliferation by hydroxylated metabolites of 17ß-estradiol (E2). METHODS: MCF-7, human breast cancer cells, were cultured in phenol red-free DMEM and treated with 1 nM 2-OH-E2 or 4-OH-E2. E2 was used as a positive control. Cell proliferation was measured using the BrdU incorporation assay. Cellular levels of cAMP and PKA were determined using ELISA kits. ERK1/2 protein expression was evaluated by Western Blot analysis. To determine the involvement of different intracellular pathways in the regulation of cell proliferation appropriate activators or inhibitors were used. RESULTS: Hydroxylated estrogens, as E2, exhibited no influence on cAMP accumulation and PKA activation. In concomitant treatments with forskolin, cell proliferation decreased to the amount noted under the influence of forskolin alone. A PKA inhibitor (PKI) had no statistically significant effect on proliferation stimulated by E2 and its hydroxylated metabolites. Phospho-ERK1/2 protein expression in cells stimulated with E2, 2-OH-E2 and 4-OH-E2 was not significantly different from the control. However, co-treatment with both PD98059 and E2 or its hydroxylated metabolites reversed the effect of tested compounds on cell proliferation. CONCLUSIONS: We have shown that E2 hydroxylated metabolites do not activate cAMP/PKA in breast cancer cells and confirm previously published data, which showed a lack of ERK1/2 activation in a breast cancer cell line. The observed reversible action of PD98059 on cell proliferation can be explained by the fact that hydroxylated estrogens, as E2, stimulate secretion of a number of growth factors, which affect MAPK activity, suggested by Lobenhofer et al. (2000).

The ability of hydroxylated estrogens (2-OH-E2 and 4-OH-E2) to increase of SHBG gene, protein expression and intracellular levels in MCF-7 cells line.

OBJECTIVE: Sex Hormone-Binding Globulin (SHBG) - specific carrier for sex steroids - regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. This study was conducted to elucidate the effects of hydroxylated estrogen (E2) metabolites (2-OH-E2 and 4-OH-E2) on sex hormone binding globulin (SHBG) mRNA and protein expression as well as on intracellular and extracellular SHBG levels. METHODS: MCF-7 human breast cancer cells were cultured with 2-OH-E2 or 4-OH-E2 in concentration of 1, 10 and 100 nM for 24 h, 17ß-estradiol being used as a positive control. SHBG levels were measured in medium and cells by ELISA, SHBG mRNA expression was evaluated by real-time-PCR and protein expression by Western blot analysis. RESULTS: 4-OH-E2 in high doses and 2-OH-E2 in the highest dose, while 17ß-estradiol in all doses used increased intracellular but not extracellular SHBG levels. Both metabolites increased SHBG mRNA expression, the rank order of potency being E2 > 4-OH-E2 > 2-OH-E2. Both E2 and its metabolites increased SHBG protein expression. CONCLUSION: Although the metabolites showed a lower potency than 17ß-estradiol, further studies are needed to clarify whether hydroxylated metabolites of E2 are potent ligands for SHBG.

Involvement of caspase-9 but not caspase-8 in the anti-apoptotic effects of estradiol and 4-OH-Estradiol in MCF-7 human breast cancer cells.

OBJECTIVES: Evidence is accumulating that certain estradiol metabolites may play a more important role in enhancing breast cancer risk than their parent substance - 17 ß-estradiol (E2). Of special interest are the metabolites 2-hydroxyestradiol (2-OH-E2), which can show anticarcinogenic effect, while that of 4-hydroxyestradiol (4-OH-E2) may be rather procarcinogenic. We suggest that local activation of cytochrome P450 enzymes - CYP1A1 and/or CYP1B1 - by E2 could generate active metabolites that affect the apoptosis and thereby promote mammary carcinogenesis. Over the last several years, there has been accumulating evidence that, apart from the receptor-mediated (extrinsic) pathway, also the mitochondrial (intrinsic) pathway plays a role in E2-induced apoptosis. In the present study, we have compared the effect of these metabolites and their parent substance E2 on caspase-8 and caspase-9 activity as well as on the end step of apoptosis DNA fragmentation. METHODS: MCF-7 human breast cancer cells (ATCC) were routinely cultured in DMEM supplemented with 10 % heat-inactivated FBS. Forty-eight hours before experiments, the medium was removed and replaced by DMEM without phenol red supplemented with 5 % heat-inactivated fetal bovine serum. For determination of caspase-8 and caspase-9 activities, MCF-7 cells were seeded in 48-well culture plates at a density of 15 x 104 cells/well and incubated with 1 nM E2 and its metabolites for 24 h. DNA fragmentation, caspase-8 and caspase-9 activities were determined in cell lysates by ELISAs. The CYP1A1 and CYP1B1 protein expression was evaluated by Western blotting. RESULTS: E2 had no effect on CYP1A1 protein levels. However an increase in CYP1B1 protein expression was observed within 48 hrs of exposure. None of the compounds tested changed caspase-8 activity as compared to the controls. Statistically significant decrease in caspase-9 activity and DNA fragmentation was observed in the presence of E2 and 4-OH-E2, but no significant effect was found for the metabolite 2-OH-E2. CONCLUSIONS: It was found that local activation of cytochrome P450 enzyme CYP1B1 by E2 may change the local metabolic activation pathway into 4-OH-E2 as well as the activation of caspase-9 (a part of the intrinsic mitochondrial apoptotic pathway) in the antiapoptotic effect of E2 and 4-OH-E2.

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

Multi-carbohydrase and phytase supplementation improves growth performance and liver insulin receptor sensitivity in broiler chickens fed diets containing full-fat rapeseed.

The effect of a combination of carbohydrase and phytase enzymes on growth performance, insulin-like growth factor 1 gene expression, insulin status, and insulin receptor sensitivity in broiler chickens fed wheat-soybean meal diets containing 6% (starter) and 12% (grower-finisher) of full-fat rapeseed (canola type; low glucosinolate, low erucic acid) from 1 to 42 d of age was studied. A total of 510 one-day-old male broiler chickens were randomly assigned to 3 dietary treatments, with 17 pens per treatment and 10 birds per pen. The dietary treatments consisted of a control diet and P- and Ca-deficient diets supplemented with either phytase (500 U/kg) or a combination of phytase and a multi-carbohydrase enzyme (Superzyme OM). The diets were pelleted at 78 degrees C and were fed ad libitum throughout the starter (9 d), grower (18 d), and finisher (15 d) phases of the experiment. Over the entire trial, growth performance of birds fed the phytase-supplemented diet did not differ from birds fed the control diet. The use of phytase in combination with a multicarbohydrase enzyme improved (P = 0.007) the feed conversion ratio from 1.90 to 1.84. Insulin liver receptor sensitivity increased by 9.3 and 12.3% (P = 0.004) for the phytase- and the carbohydrase-phytase-supplemented diets, respectively. There was no effect of phytase alone or carbohydrase and phytase supplementation on total plasma cholesterol, high-density lipoprotein cholesterol, and blood glucose levels. However, low-density lipoprotein cholesterol decreased (P = 0.007) for the phytase-carbohydrase treatment. Gene expression of insulin-like growth factor 1 tended to decrease by 32% (P = 0.083) after phytase-carbohydrase supplementation. The combination of carbohydrase and phytase enzymes may serve as an attractive means of facilitating nutrient availability for digestion and thus enhance the feeding value of wheat-soybean meal-based diets containing full-fat rapeseed. However, the extent to which the effects of enzyme addition on insulin receptors are associated with growth performance of broiler chicken requires further research.

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration.

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Microelectrical characterizations of junctions in solar cell devices by scanning Kelvin probe force microscopy.

Scanning Kelvin probe force microscopy was applied to the microelectrical characterizations of junctions in solar cell devices. Surface Fermi-level pinning effects on the surface potential measurement were avoided by applying a bias voltage (V(b)) to the device and taking the V(b)-induced potential and electric field changes. Two characterizations are presented: the first is a direct measurement of Bi-induced junction shift in GaInNAs(Bi) cells; the second is a junction-uniformity measurement in a-Si:H devices. In the first characterization, using Bi as a surfactant during the molecular beam epitaxy growth of GaInNAs(Bi) makes the epitaxial layer smoother. However, the electrical potential measurement exhibits a clear Bi-induced junction shift to the back side of the absorber layer, which results in significant device degradation. In the second characterization, the potential measurement reveals highly non-uniform electric field distributions across the n-i-p junction of a-Si:H devices; the electric field concentrates much more at both n/i and i/p interfaces than in the middle of the i-layer. This non-uniform electric field is due possibly to high defect concentrations at the interfaces. The potential measurements further showed a significant improvement in the electric field uniformity by depositing buffer layers at the interfaces, and this indeed improved the device performance.