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Abstract: Although luteinizing hormone (LH) affects androstenedione (A4) and progesterone (P4) production in theca cells, it is still unknown how LH influences molecular mechanism of A4 and P4 production. To examine the relationship between LH and transcription factors involved in A4 and P4 production, ovarian theca cells were cultured in the presence or absence of high concentrations of LH for 24h (pre-treatment with high concentration of LH) and then cultured in the presence or absence of low concentration of LH for 48h. Low LH enhanced production of A4 and P4, and expressions of CYP17 and StAR mRNA in theca cells without pre-treatment with high LH. In addition, low LH stimulated the expression of SF-1 protein in nuclear fractions from theca cells with or without pre-treatment with high LH. The binding of SF-1 to the CYP17 and StAR promoter regions increased in theca cells treated with low LH. Although GATA-4 and GATA-6 are both found in the nuclear fraction but not in the cytosol of theca cells, low LH enhanced the binding of GATA-6, but not of GATA-4, to the CYP17 promoter region without pre-treatment with high LH. Acetylation histone H3 in StAR and CYP17 promoter regions were changed by different LH-dosage. Overall, we showed that LH regulates the production of A4 and P4 by affecting the nuclear localization and switching of transcription factors in theca cells and that target transcription factors involved in steroid production in theca cells are changed by different LH concentration. [Copyright &y& Elsevier]

Increased levels of dialkylphosphates (DAP) in maternal urine are associated with a variety of adverse developmental outcomes in children. Although urinary DAP levels are usually considered to be a marker of exposure to organophosphate (OP) pesticides, excretion of DAP may also increase by ingesting preformed DAP. To date, no study has quantitatively assessed the possible contribution of the dietary intake of preformed DAP and OP pesticides to urinary levels of DAP. Therefore, we aimed to estimate the levels of 6 DAPs and 84 OP pesticides in duplicate diet samples and urine samples collected from 73 women living in urban areas of Japan in 2018. DAP and OP pesticides were detected in 94% and 45% of diet samples, while DAP was detected in 100% of urinary samples, respectively. The average daily intake of preformed DAP was significantly higher than that of parent OP pesticides in our participants. Dimethylphosphate and diethylphosphate were predominant in the preformed DAP, and the estimated average daily intake of total amount of DAP was 78.3 nmol. Fruits and vegetables were the major dietary sources of DAP. Dietary intake of DAP was positively associated with urinary DAP levels, suggesting that a considerable amount of urinary DAP was derived from ingesting preformed DAP. Our results show that attributing urinary DAP levels exclusively to OP pesticide exposure would result in a substantial overestimation of the exposure level. Therefore, the urinary levels of DAP may not be suitable for evaluating OP pesticide exposure in the general urban population. [Display omitted] • Duplicate diets were collected from Japanese women of reproductive age. • Diet samples were analyzed for the presence of OP pesticides and DAPs. • The average dietary intake of DAP was much higher than that of OP pesticides. • Dietary DAP intake is associated with urine DAP levels. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity. • Glucose and pyruvate support sperm motility primarily via mitochondrial activity. • Sperm penetration ability into IPVL relies on glucose rather than pyruvate. • Low penetration under pyruvate was not relevant to changes in ROS and pHi. • Glycolysis and mitochondrial oxidation play distinct roles in rooster sperm. [ABSTRACT FROM AUTHOR]

• A simultaneous LC/MS/MS-method for 12 lethal plant toxins in human serum was developed. • The method was fully validated and can quantitate the toxins with high sensitivity. • We applied it to pseudo poisoning serum using animal models, showing its practicality. We developed a highly sensitive quantification method using liquid chromatography tandem mass spectrometry (LC/MS/MS) for 12 plant toxins in human serum. In this paper, we selected lycorine, galanthamine, protoveratrine A, protoveratrine B, veratramine, veratridine, jervine, cyclopamine, cevadine, α-solanine, α-chaconine, and solanidine as targeted analytes. The ADME column was utilized for LC separation and a Monolithic SPE column (MonoSpin® C18) for analyte extraction. The total time for SPE clean-up and LC/MS/MS analysis was completed within 30 min. The method validation results were as follows: the linearity (r2) of each calibration curve was over 0.99; the inter- and intra-day accuracies were 92.7 %–116 % and 91.6 %–106 %, respectively; and the inter- and intra-day precisions were below 14 % and 11 %, respectively. Also, the lower limits of detection and quantification were 0.0071–0.15 and 0.022–0.46 ng/mL, respectively, indicating the method's high sensitivity. Finally, to confirm its feasibility, our method was applied to two model samples: (1) commercially available human serum and (2) pseudo poisoning serum via dilution of mouse serum with human serum. We were able to quantify α-chaconine at 0.84 ± 0.02 ng/mL in the serum (Case 1) and protoveratrine A at 0.15 ± 0.032 ng/mL in the pseudo poisoning serum (Case 2), demonstrating our method's practicality. This is the first time that the 12 plant toxins in human serum were simultaneously quantitated. Our method can investigate accidental poisonings involving toxic plants, enabling prompt decisions on patient treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simple and rapid method for determining propionic acid in food was developed. • Propionic acid in the vapour phase was directly captured on a Solid phase extraction (SPE) column. • The method achieved satisfactory recovery, reproducibility and selectivity. • The applicability of the method was verified in various types of food. • New SPE method shows promise in the analysis of volatile compounds. Solid phase extraction (SPE) is a technique widely used in food analysis for the isolation of analytes. Herein, we proposed a novel application of SPE to extract vaporised propionic acid, a common preservative, from a heated sample solution. A sample was heated under acidified conditions and the resulting steam was directly passed through an SPE column to extract the propionic acid, followed by elution and HPLC analysis. Here, the extraction on the SPE column ensures direct capture of propionic acid. The results demonstrated excellent linearity (R2 greater than 0.999) and recoveries of 89.9%–97.6% with intra- and inter-day precisions lower than 3.9%. To the best of our knowledge, no study has investigated the applicability of SPE to an analyte vaporised in the headspace of food products. The proposed method is promising in its application to various volatile compounds and in the routine analysis of propionic acid in food. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Indoxyl sulfate, a uremic toxin, is considered a risk factor for arteriosclerosis in patients with chronic kidney disease (CKD). We previously reported the actions of indoxyl sulfate including crosstalk with platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells (VSMCs). The present study examines whether indoxyl sulfate enhances angiotensin II (Ang II) signaling because serum levels of Ang II are elevated in patients with CKD. Main methods: The effect of indoxyl sulfate and Ang II on phosphorylation of ERK and epidermal growth factor receptor (EGFR), and migration were determined using VSMCs. The expression of EGFR was determined using not only VSMCs but also artery of normal, uremic, and indoxyl sulfate-administrated uremic rats. Key findings: Ang II-dependent phosphorylation of ERK and EGFR, and migration of VSMCs were augmented by a prior 24-h incubation with indoxyl sulfate even in the absence of indoxyl sulfate during Ang II stimulation. The expression of EGFR was increased in indoxyl sulfate-stimulated cultured VSMCs. In arterial VSMCs of rats, serum levels of indoxyl sulfate reflected the expression level of EGFR. The upregulated EGFR expression by indoxyl sulfate was suppressed by the antioxidant, N-acetylcysteine. An EGFR inhibitor, AG1478, repressed the enhancement of Ang II-induced cellular effects by indoxyl sulfate. Taken together, these findings indicate that indoxyl sulfate enhances Ang II signaling through reactive oxygen species-induced EGFR expression. Significance: The actions of indoxyl sulfate including crosstalk with Ang II signaling may be closely involved in the pathogenesis of CKD associated with arteriosclerosis. [Copyright &y& Elsevier]

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKa kinase and the degradation of IκκBα, suppressing the NFκB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity. [ABSTRACT FROM AUTHOR]

Objective: To examine the effect of vascular endothelial growth factor (VEGF) and growth differentiation factor-9 (GDF-9) on follicular development of the ovaries in immature female rats. Design: Superovulation and gene injection. Setting: Animal reproduction laboratory in Tohoku University, Sendai, Japan. Animal(s): Wister-Imamichi female rats. Intervention(s): The ovulated oocytes from rats with injected VEGF and GDF-9 gene fragments were counted, and the ovaries removed from those rats were used in the histologic observation. Main Outcome Measure(s): Follicular dynamics and angiogenesis after VEGF and GDF-9 gene fragments injection. Result(s): A single injection of the VEGF gene led to the production of a large number of oocytes (approximately 110 oocytes) from an individual animal that was injected with the gene at 21 days after birth, and after mating most of the oocytes were fertilized. Direct ovarian injection of GDF-9 stimulated the development of medium-sized antral follicles. The number of ovulated oocytes after injection of the VEGF plus GDF-9 gene fragments was the same as with a single injection of the VEGF gene. Conclusion(s): A single injection of the VEGF or GDF-9 gene stimulated follicular development, and injection of both genes did increase the number of ovulated oocytes from individual animals. An exogenous gene fragments injection promoted the maximum potential of ovarian function in immature female rats. [Copyright &y& Elsevier]

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Noxi small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1- generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes. [ABSTRACT FROM AUTHOR]

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and coimmunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism. [ABSTRACT FROM AUTHOR]