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Find video protocols related to scientific articles indexed in Pubmed.
Effects of lead exposure on d-serine metabolism in the hippocampus of mice at the early developmental stages.
Toxicology
PUBLISHED: 08-12-2014
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The aim of this study was to explore the mechanisms of lead neurotoxicity by focusing on the alteration of d-serine metabolism in the hippocampus of mice at the early life. Mother mice and their offspring were exposed to 0, 0.5, 1.0 and 2.0g/L lead in lead acetate via drinking water from the first day of gestation until the postnatal day (PND) 40. Morris water maze was used to measure the spatial learning and memory ability of PND 40 mice. Expressions of serine racemase (SR), d-amino acid oxidase (DAAO), alanine-serine- cysteine transporter-1 (asc-1) and subunits of N-methyl-d-aspartate receptor (NMDAR) in the hippocampus of PND 10, 20 and 40 mice were examined by western blot and real time RT-PCR. Findings from this study disclosed that the spatial learning ability of mice tested by place trial could be significantly impaired by 0.5g/L lead exposure, and the spatial memory ability tested by probe trail could be impaired by 1.0g/L lead exposure. Exposure to 2.0g/L lead in the water could significantly inhibit the protein and mRNA expression of SR; conversely enhance the expression of DAAO protein and mRNA in the hippocampus during the early developmental stages. However, the protein expressions of DAAO and asc-1 in the hippocampus were significantly enhanced by 0.5g/L lead exposure at different developmental stages. On the other hand, the protein and mRNA expressions of both NR1 and NR2A were inhibited significantly by 1.0g/L lead exposure since PND 10, and by 0.5g/L lead exposure since PND 20. Noteworthy, the protein expression of NR2B was inhibited significantly by 0.5g/L lead exposure in PND 10 mice, and by 1.0g/L lead exposure in PND 20 mice, but there was no significant group difference in PND 40 mice. Meanwhile, expressions of asc-1 and NR2B mRNA were not affected obviously by lead exposure. In conclusion, chronic lead exposure during brain development might affect d-serine metabolism by enhancing its degradation, which might be related to the inhibited expression of NMDAR subunits, and furthermore contribute to deficits in learning and memory ability in mice.
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Roles of aquaporins and matrix metalloproteinases in mouse brain edema formation induced by subacute exposure to 1,2-dichloroethane.
Neurotoxicol Teratol
PUBLISHED: 05-06-2014
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The aim of this study was to explore the effects of 1,2-dichloroethane (1,2-DCE) on expression of aquaporins (AQPs) and matrix metalloproteinases (MMPs) in the process of brain edema formation. Two parts were included in this study, establishment of animal model of brain edema, and mechanism of brain edema induced by subacute exposure to 1,2-DCE. In part one, mice were exposed to 0, 1.1, 1.2 or 1.3g/m(3) 1,2-DCE, 3.5h per day for 3days. Pathological analysis and water content detection in the brain were examined. In part two, mice were exposed to 1.2g/m(3) 1,2-DCE, 3.5h per day for 1, 2 or 3days, named group D, E and F, respectively. Expression of AQP4, MMP2 and MMP9 in the brain was determined by immunochemical staining, western blot and real time PCR. According to the results of part one, the 1.2g/m(3) dose was chosen for part two, a follow-up time-course study. In part two, protein expression of MMP2 and MMP9 in group F, and AQP4 in group E and F significantly increased compared to the control. Similarly, mRNA levels of AQP4 in group F, and MMP9 in group E and F significantly increased. Our results suggested that exposure to 1,2-DCE might up-regulate the expression of AQP4 protein and MMP9 mRNA at the early phase of brain edema, and AQP4 may play an important role in the brain edema formation.
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CREBZF expression and hormonal regulation in the mouse uterus.
Reprod. Biol. Endocrinol.
PUBLISHED: 10-07-2013
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CREBZF is a member of the mammalian ATF/CREB family of the basic region-leucine zipper (bZIP) transcription factors. Two isoforms of CREBZF have been identified from the alternative usage of initiation codons, SMILE (long isoform of CREBZF) and Zhangfei (short isoform of CREBZF). Until recently, the physiological function of CREBZF in mammalian reproductions has not been reported.
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GRP78 expression and regulation in the mouse uterus during embryo implantation.
J. Mol. Histol.
PUBLISHED: 09-18-2013
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The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1-4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6-8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.
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The expression and localization of LRF in the female reproductive tract of cycling mice throughout the estrous cycle.
J Immunoassay Immunochem
PUBLISHED: 07-18-2013
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In this article, the expression patterns of LRF in the mouse oviduct, uterus, and ovary were checked during estrous cycle. The expression of LRF mRNA and protein were remarkably changed in the ovary, oviduct, and uterus at four phases. LRF immunostaining was detected in the follicle from primary to antral follicle, luminal and glandular epithelial cells of the uterus, and cilia of the ciliated cells of the oviduct at all phase. Our findings suggested that LRF may be related to the processes of development and maturation of oocyte, gamete transport, and the development of early embryo.
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Comparison of Visual Recovery Following Ex-PRESS Versus Trabeculectomy: Results of a Prospective Randomized Controlled Trial.
J. Glaucoma
PUBLISHED: 06-29-2013
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To compare the rate of visual recovery after Ex-PRESS implantation versus standard trabeculectomy.
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Identification of a histone acetyltransferase as a novel regulator of Drosophila intestinal stem cells.
FEBS Lett.
PUBLISHED: 03-11-2013
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One of the major challenges in stem cell research is to decipher the controlling mechanisms/genes of stem cell homeostasis. Through an RNAi mediated genetic screen of living animals, we identified an evolutionarily conserved histone acetyltransferase Atac2 as a novel regulator of Drosophila intestinal stem cells (ISCs). Expression of Atac2-RNAi or a dominant negative allele of Atac2 generated more ISCs, while excessive Atac2 or a histone deacetylase inhibitor promoted ISC differentiation without affecting ISC survival or lineage specification. These findings extend our knowledge of epigenetic mechanisms in stem cell regulation.
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Expression and localization of Luman RNA and protein during mouse implantation and decidualization.
Theriogenology
PUBLISHED: 02-25-2013
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Luman (also known as LZIP and CREB3) is a basic leucine zipper transcription factor of the cAMP response element-binding protein/activating transcription factor gene family. Although Luman had specific roles near termination of Drosophila embryogenesis, the physiological functions of Luman in female mammals have apparently not been reported. Therefore, our objective was to investigate the spatiotemporal expression and regulation of Luman in the mouse uterus during the peri-implantation period. Luman protein was clearly present in the luminal and glandular epithelium on days 1 to 4 of pregnancy (day 1, presence of a vaginal plug) and was observed in decidual cells on day 6 of pregnancy. Expression had progressively increased to day 7 when the second decidual zone was formed. On day 8, apoptosis of the decidualized cells was present, and Luman protein expression was decreased (in close association with decidualization). Luman protein was also present in decidual cells of the artificially decidualized uterus. The expression of Luman was regulated by an activated embryo (according to its expression patterns during pseudopregnancy and delayed implantation). Furthermore, expression of Luman was induced by estrogen in ovariectomized mice. We have concluded that Luman might have important roles in embryo implantation and decidualization.
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Expression pattern implicates a potential role for luman recruitment factor in the process of implantation in uteri and development of preimplantation embryos in mice.
J. Reprod. Dev.
PUBLISHED: 02-12-2013
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Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.
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Establishment and Evaluation of a Stable Cattle Type II Alveolar Epithelial Cell Line.
PLoS ONE
PUBLISHED: 01-01-2013
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Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs) also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells) by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II) that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-? and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.
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Genetic characterization of the Drosophila birt-hogg-dubé syndrome gene.
PLoS ONE
PUBLISHED: 01-01-2013
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Folliculin (FLCN) is a conserved tumor suppressor gene whose loss is associated with the human Birt-Hogg-Dubé (BHD) syndrome. However, its molecular functions remain largely unknown. In this work, we generated a Drosophila BHD model through genomic deletion of the FLCN gene (DBHD(-) ). The DBHD mutant larvae grew slowly and stopped development before pupation, displaying various characteristics of malnutrition. We found the growth delay was sensitive to the nutrient supplies. It became more severe upon restrictions of the dietary yeast; while high levels of yeast significantly restored the normal growth, but not viability. We further demonstrated that leucine was able to substitute for yeast to provide similar rescues. Moreover, the human FLCN could partially rescue the DBHD(-) phenotypes, indicating the two genes are involved in certain common mechanisms. Our work provides a new animal model of the BHD syndrome and suggests that modulation of the local nutrient condition might be a potential treatment of the BHD lesions.
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Hormonal regulation of ovarian bursa fluid in mice and involvement of aquaporins.
PLoS ONE
PUBLISHED: 01-01-2013
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In rodent species, the ovary and the end of oviduct are encapsulated by a thin membrane called ovarian bursa. The biological functions of ovarian bursa remain unexplored despite its structural arrangement in facilitating oocytes transport into oviduct. In the present study, we observed a rapid fluid accumulation and reabsorption within the ovarian bursa after ovarian stimulation (PMSG-primed hCG injection), suggesting that the ovarian bursa might play an active role in regulating local fluid homeostasis around the timing of ovulation. We hypothesized that the aquaporin proteins, which are specialized channels for water transport, might be involved in this process. By screening the expression of aquaporin family members (Aqp1-9) in the ovarian tissue and isolated ovarian bursa (0, 1, 2 and 5 h after hCG injection), we found that AQP2 and AQP5 mRNA showed dynamic changes after hCG treatment, showing upregulation at 1-2 h followed by gradually decrease at 5 h, which is closely related with the intra-bursa fluid dynamics. Further immunofluorescence examinations of AQP2 and AQP5 in the ovarian bursa revealed that AQP2 is specifically localized in the outer layer (peritoneal side) while AQP5 localized in the inner layer (ovarian side) of the bursa, such cell type specific and spatial-temporal expressions of AQP2 and 5 support our hypothesis that they might be involved in efficient water transport through ovarian bursa under ovulation related hormonal regulation. The physiological significance of aquaporin-mediated water transport in the context of ovarian bursa still awaits further clarification.
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Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.
PLoS ONE
PUBLISHED: 01-01-2013
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The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.
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Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer.
Mol. Biol. Rep.
PUBLISHED: 06-02-2011
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Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08-73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83-70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x(2) = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.
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Effects of arsenite on glutamate metabolism in primary cultured astrocytes.
Toxicol In Vitro
PUBLISHED: 05-25-2011
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The aim of this study was to explore the mechanisms that contribute to neurotoxicity induced by arsenite exposure focusing on the alteration of glutamate metabolism in primary cultured astrocytes. The cells were exposed to 0-30?M arsenite for 24h, and then cell viability, intracellular nonprotein sulfhydryl (NPSH) levels, mitochondrial membrane potential, activity of Na(+)/K(+)-ATPase, glutamine synthetase (GS) and glutamate transporter (GLAST and GLT-1), and protein expression of GS, GLAST and GLT-1 were examined. Compared with those in control, exposure to arsenite resulted in damages of astrocytes in a concentration dependent manner, which were shown by cell viabilities, and supported by morphological observation, mitochondrial membrane potential and intracellular NPSH levels. On the other hand, activities and protein expression of GS, GLAST and GLT-1 were significantly inhibited by arsenite exposure. Moreover, protein expression of GLAST and activities of GS were much more sensitive to arsenite. However, activities of Na(+)/K(+)-ATPase were not influenced obviously by arsenite exposure. In conclusion, findings from this study indicated that exposure to arsenite could inhibit glutamate metabolism in astrocytes, which might be related to arsenic-induced neurotoxicity.
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Effects of exogenous methionine on arsenic burden and NO metabolism in brain of mice exposed to arsenite through drinking water.
Environ. Toxicol.
PUBLISHED: 05-10-2011
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The aim of this study was to explore the effects of exogenous methionine (Met) on arsenic burden and metabolism of nitric oxide (NO) in the brain of mice exposed to arsenite via drinking water. Mice were exposed to sodium arsenite through drinking water contaminated with 50 mg/L arsenic for four consecutive weeks, and treated intraperitoneally with saline solution, 100 mg/kg body weight (b.w), 200 mg/kg b.w or 400 mg/kg b.w of Met, respectively at the fourth week. Levels of inorganic arsenic (iAs), monomethylarsenic acid (MMAs), and dimethylarsenic acid (DMAs) in the liver, blood and brain were determined by method of hydride generation coupled with atomic absorption spectrophotometry. Nitric oxide synthase (NOS) activities and NO levels in the brain were determined by colorimetric method. Compared with mice exposed to arsenite alone, administration of Met increased significantly the primary methylation ratio in the liver, which resulted in decrease of percent iAs and increase of percent DMAs in the liver, and decrease of iAs, MMAs and total arsenic levels (TAs) in the blood and DMAs and TAs in the brain. NOS activities and NO levels in the brain of mice exposed to arsenite alone were significantly lower than those in control, however administration of Met could increase significantly NO levels. Findings from this study suggested that exogenous Met could benefit the primary arsenic methylation in the liver, which might increase the production of methylated arsenicals and facilitate arsenic excretion. As a consequence, arsenic burden in both blood and brain was reduced, and toxic effects on NO metabolism in the brain were ameliorated.
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Therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mild lead-poisoning.
Environ. Toxicol. Pharmacol.
PUBLISHED: 06-04-2010
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The aim of this study was to explore therapeutic efficiency of succimer used with calcium and ascorbic acid in the treatment of mildly lead-poisoned mice and preschool children. Mice were exposed to lead by drinking water, and then treated with saline solution, 50mg/kg body weight (b.w.) succimer, 100mg/kg b.w. succimer, or 50mg/kg b.w. succimer plus calcium and ascorbic acid by gavage. Seventy-two children aged 48-72 months were randomly assigned into combined treatment or nutritional intervention group. Lead levels in blood and bone were analyzed by atomic absorption spectrophotometry. Activities of aminolevulinic acid dehydratase (ALAD) in blood were determined by colorimetric method. Results of animal experiment showed that succimer used alone could reduce lead levels in blood and bone and reverse activities of ALAD in blood, however, a better therapeutic efficiency in mobilizing bone lead could be achieved by succimer used with calcium and ascorbic acid. Findings from the clinical study showed that reduction of blood lead levels (BLLs) between the end and initiation of therapy in the combined treatment group was significantly greater than that in the nutritional intervention group. Percentage of children with BLLs less than 10?g/dL at the end of therapy and the eighth week after therapy in the combined treatment group was significantly higher than that in the nutritional intervention group. In conclusion, combined use of succimer with calcium and ascorbic acid seemed to be a choice in the treatment of mildly lead poisoned children.
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Effects of exogenous glutathione on arsenic burden and NO metabolism in brain of mice exposed to arsenite through drinking water.
Arch. Toxicol.
PUBLISHED: 04-27-2010
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Chronic exposure to inorganic arsenic (iAs) is associated with neurotoxicity. Studies to date have disclosed that methylation of ingested iAs is the main metabolic pathway, and it is a process relying on reduced glutathione (GSH). The aim of this study was to explore the effects of exogenous GSH on arsenic burden and metabolism of nitric oxide (NO) in the brain of mice exposed to arsenite via drinking water. Mice were exposed to sodium arsenite through drinking water contaminated with 50 mg/L arsenic for 4 weeks and treated intraperitoneally with saline solution, 200 mg/kg body weight (b.w), 400 mg/kg b.w, or 800 mg/kg b.w GSH, respectively, at the 4th week. Levels of iAs, monomethylarsenic acid, and dimethylarsenic acid (DMAs) in the liver, blood, and brain were determined by method of hydride generation coupled with atomic absorption spectrophotometry. Activities of nitric oxide synthase (NOS) and contents of NO in the brain were determined by colorimetric method. Compared with mice exposed to arsenite alone, administration of GSH increased dose-dependently the primary and secondary methylation ratio in the liver, which caused the decrease in percent iAs and increase in percent DMAs in the liver, as a consequence, resulted in significant decrease in iAs levels in the blood and total arsenic levels in both blood and brain. NOS activities and NO levels in the brain of mice in iAs group were significantly lower than those in control; however, administration of GSH could increase significantly activities of NOS and contents of NO. Findings from this study suggested that exogenous GSH could promote both primary and secondary arsenic methylation capacity in the liver, which might facilitate excretion of arsenicals, and consequently reduce arsenic burden in both blood and brain and furthermore ameliorate the effects of arsenicals on NO metabolism in the brain.
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Prenatal and early life arsenic exposure induced oxidative damage and altered activities and mRNA expressions of neurotransmitter metabolic enzymes in offspring rat brain.
J. Biochem. Mol. Toxicol.
PUBLISHED: 04-09-2010
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To better understand the effect of arsenic on central nervous system by prenatal and early life exposure, the oxidative stress and neurotransmitter metabolic enzymes were determined in offspring rats brain cortex and hippocampus. Forty-eight pregnant rats were randomly divided into four groups, each group was given free access to drinking water that contained 0, 10, 50, and 100 mg/L NaAsO(2) from gestation day 6 (GD 6) until postnatal day 42 (PND 42). Once pups were weaned, they started to drink the same arsenic (As)-containing water as the dams. The level of malondialdehyde in 100 mg/L As-exposed pups brain on PND 0 and cortex on PND 28 and 42 were significantly higher than in the control group (p < 0.05). Reduced glutathione (GSH) levels showed a clear decreasing trend in pups cortex and hippocampus on PND 42. Activity of acetylcholinesterase was significantly higher in 100 mg/L As-exposed pups hippocampus than in control group on PND 28 and 42. mRNA expression of glutamate decarboxylase (GAD(65) and GAD(67)) in 100 mg/L As-exposed pups cortex or hippocampus on PND 28 and 42 were significantly higher than in control (p < 0.05). These alterations in the neurotransmitters and reduced antioxidant defence may lead to neurobehavioral and learning and memory changes in offspring rats.
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The Canadian Neurological Scale and the NIHSS: development and validation of a simple conversion model.
Cerebrovasc. Dis.
PUBLISHED: 03-12-2010
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The Canadian Neurological Scale (CNS) and the National Institutes of Health Stroke Scale (NIHSS) are among the most reliable stroke severity assessment scales. The CNS requires less extensive neurological evaluation and is quicker and simpler to administer.
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Distribution of speciated arsenicals in mice exposed to arsenite at the early life.
Ecotoxicol. Environ. Saf.
PUBLISHED: 02-08-2010
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The aim of this study was to explore distribution of speciated arsenicals in mice exposed to arsenite at early developmental stages. Levels of speciated arsenicals in both liver and brain of mice were analyzed by hydride generation of volatile arsines, and determined by atomic absorption spectrophotometry (HG-AAS). In the liver, levels of inorganic arsenic (iAs) increased on postnatal day (PND) 15, and monomethylarsonic acid (MMA) increased on PND 21, however, levels of dimethylarsinic acid (DMA) in newborn mice were significantly higher than those on PND 10 and 15. In the brain, levels of iAs on PND 21 were the highest; iAs levels on PND 15 were also significantly higher than those on PND 35. Our results suggested transplacental transfer of arsenicals from pregnant mice into their fetus was relatively efficient, lactational transfer from mother mice into their offspring was inefficient, and transfer of iAs from blood into brain at early developmental stages was efficient.
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Amino acid deprivation induces CREBZF/Zhangfei expression via an AARE-like element in the promoter.
Biochem. Biophys. Res. Commun.
PUBLISHED: 11-17-2009
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CREBZF (also called ZF or Zhangfei) is a basic region-leucine zipper transcription factor that has been implicated in the herpesvirus infection cycle and related cellular processes. Since ATF4 is known to play a key role in cellular responses to various ER stresses as well as amino acid deprivation, we sought to examine the potential involvement of CREBZF in the amino acid response (AAR). We found that the CREBZF protein was induced by amino acid deprivation in the canine MDCK cells. We subsequently cloned a canine CREBZF promoter region (-1767bp to +1bp) that responds to amino acid limitation. Using deletion mapping and site-directed mutagenesis, we identified a 9-bp sequence 5-ATTCACTCA-3 in the promoter (-1227 to -1219), deletion of which resulted in a complete loss of inducibility by amino acid deprivation. This sequence is similar to the known amino acid response elements (AAREs) found in other AAR-inducible genes, such as CHOP (C/EBP homologous protein, also known as GADD153). These results suggest that CREBZF may be an amino acid stress sensor. Considering the AARE-like sequence found in CREBZF and other similarities between CREBZF and CHOP, we postulate that CREBZF and CHOP may be two sensors that regulate different yet related signaling pathways governing the AAR.
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Identification of changes in the composition of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens.
Vet. Microbiol.
PUBLISHED: 03-31-2009
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We previously reported that Clostridium perfringens in vivo proliferation and alpha-toxin gene expression were highly correlated, both progressing in a parabolic curve pattern during the development of necrotic enteritis (NE). The present study investigated the response of dominant ileal bacteria in abundance to C. perfringens infection using PCR-based denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (QPCR) techniques. Chickens were fed antibiotic-medicated (bacitracin, 55 mg/kg) or non-medicated diets, and were challenged with C. perfringens through the diet at 18 days of age. Ileal digesta was collected daily before and after the challenge for 5 days. Bacterial profiles of PCR-DGGE from both bacitracin-treated and untreated chickens responding to clostridial infection were analyzed by the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Treatment-related differences in PCR-DGGE bacterial profiles on day 2 post-infection (PI) were detected. Subsequent QPCR assays identified changes in the abundance of lactobacilli, L. aviarius in particular. Changes in both populations correlated negatively with the population of C. perfringens in bacitracin-untreated group of chickens that displayed a high incidence of NE lesions. The results indicated that L. aviarius was suppressed by C. perfringens infection. This observation warrants further studies on the mechanisms underlying the ecological change and to assist in further development of novel probiotics to control NE disease.
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Distribution and speciation of arsenic by transplacental and early life exposure to inorganic arsenic in offspring rats.
Biol Trace Elem Res
PUBLISHED: 03-03-2009
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The amount of arsenic compounds was determined in the liver and brain of pups and in breast milk in the pups stomach in relation to the route of exposure: transplacental, breast milk, or drinking water. Forty-eight pregnant rats were randomly divided into four groups, each group was given free access to drinking water that contained 0, 10, 50, and 100 mg/L NaAsO(2) from gestation day 6 (GD 6) until postnatal day 42 (PND 42). Once pups were weaned, they started to drink the same arsenic-containing water as the dams. Contents of inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and trimethylarsenic acid (TMA) in livers and brains of the pups on PND 0, 15, 28, and 42 and breast milk taken from the pups stomach on PND 0 and 15 were detected using the hydride generation atomic absorption spectroscopy method. Concentrations of iAs, MMA, and DMA in the breast milk, the brain, and the liver of the pups increased with the concentration of arsenic in drinking water on PND 0, 15, 28, and 42. Compared to the liver or brain, breast milk had the lowest arsenic concentrations. There was a significant decrease in the levels of arsenic species on PND 15 compared to PND 0, 28, or 42. It was confirmed that arsenic species can pass through the placental barrier from dams to offspring and across the blood-brain barrier in the pups, and breast milk from dams exposed to arsenic in drinking water contains less arsenic than the liver and brain of pups.
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Transplacental and early life exposure to inorganic arsenic affected development and behavior in offspring rats.
Arch. Toxicol.
PUBLISHED: 01-15-2009
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To evaluate the developmental neurotoxicity of arsenic in offspring rats by transplacental and early life exposure to sodium arsenite in drinking water, the pregnant rats or lactating dams, and weaned pups were given free access to drinking water, which contained arsenic at concentrations of 0, 10, 50, 100 mg/L from GD 6 until PND 42. A battery of physical and behavioral tests was applied to evaluate the functional outcome of pups. Pups in arsenic exposed groups weighed less than controls throughout lactation and weaning. Body weight of 10, 50 and 100 mg/L arsenic exposed groups decreased significantly on PND 42, 16 and 12, respectively. Physical development (pinna unfolding, fur appearance, incisor eruption, or eye opening) in pups displayed no significant differences between control and arsenic treated groups. The number of incidences within the 100 mg/L arsenic treated group, in tail hung, auditory startle and visual placing showed significant decrease compared to the control group (p < 0.05). In square water maze test, the trained numbers to finish the trials successfully in 50 and 100 mg/L arsenic exposed groups increased remarkably compared to control group, and there was a dose-related increase (p < 0.01) observed. Taken together, these data show that exposure of inorganic arsenite to pregnant dams and offspring pups at levels up to 100 mg/L in drinking water may affect their learning and memory functions and neuromotor reflex.
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Discrepancy between results and abstract conclusions in industry- vs nonindustry-funded studies comparing topical prostaglandins.
Am. J. Ophthalmol.
PUBLISHED: 01-09-2009
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To investigate the relationship between industry- vs nonindustry-funded publications comparing the efficacy of topical prostaglandin analogs by evaluating the correspondence between the statistical significance of the publications main outcome measure and its abstract conclusions.
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Luman recruiting factor regulates endoplasmic reticulum stress in mouse ovarian granulosa cell apoptosis.
Theriogenology
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Follicular atresia is primarily induced by granulosa cell apoptosis; however, the molecular mechanisms that control apoptotic cell death in granulosa cells remain poorly understood. The present studies were undertaken to investigate the role of a novel endoplasmic reticulum stress-regulated gene Luman recruiting factor (LRF) in granulosa cell apoptosis during mouse follicular atresia. Based on immunohistochemistry and confocal laser scanning microscope analysis, LRF protein was localized in the cytoplasm of apoptotic granulosa cells, similar to localization of the LRF, Luman, CCAAT/enhancer-binding protein homologous protein and caspase-12 proteins were localized in apoptotic granulosa cells. However, glucose-regulated protein 78 protein was only present in healthy cells of the mural granulosa cell layers. A spontaneous onset of apoptotic cell death of granulosa cells was induced by thapsigargin or tunicamycin treatment in vitro, which was closely related to the increase of LRF, Luman, CCAAT/enhancer-binding protein homologous protein, and caspase-12 mRNA. Taken together, LRF might be involved in inducing apoptosis of granulosa cells through the endoplasmic reticulum stress pathway and might have a key role in mouse follicular selection.
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Challenges in mass spectrometry-based quantification of bioactive peptides: a case study exploring the neuropeptide Y family.
Biopolymers
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The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges.
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Caprine endometrial stromal cells modulate the effects of steroid hormones on cytokine secretion by endometrial epithelial cells in vitro.
Reprod Biol
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The main purpose of this study was to examine the effects of 17?-estradiol (E(2)) and progesterone (P(4)) on cytokine secretion by caprine endometrial epithelial cells (EEC) in vitro. Epithelial cells grown alone or in co-culture with stromal cells (ESC) were treated with E(2) or P(4), or both. Homogeneity of the endometrial cell populations was ascertained immunocytochemically. The quantities of cytokines secreted in this system were assessed by ELISA and their protein expression by Western blot. The exposure of EEC to P(4) alone or in combination with E(2) significantly increased the amount of TGF-?1, TNF-? and IL-18 secretion, whereas E(2) had no effect on the synthesis of these cytokines. When epithelial cells were co-cultured with ESC, the secretion of TGF-?1, TNF-? and IL-18 by EEC significantly increased compared to that by EEC alone. However, the treatment with both steroids decreased the secretion of TNF-?, IL-18 and TGF-?1 by EEC in the presence of ESC. In contrast to TGF-?1, TNF-? and IL-18, the secretion of leukemia inhibitory factor (LIF) by EEC was not affected by E(2) and/or P(4) either directly or indirectly. The present results indicate that the interactions between caprine endometrial stromal and epithelial cells can modulate the secretion of TGF-?1, TNF-? and IL-18 by EEC exposed to E(2) and/or P(4)in vitro.
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Effects of arsenite in astrocytes on neuronal signaling transduction.
Toxicology
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The main purpose of this study was to test the hypothesis that arsenite induces neurotoxicity via effects on astrocytes. Astrocytes were exposed to 0, 5 or 10 ?M arsenite in medium for 24 h, and then astrocyte-conditioned medium (ACM) was collected after incubation with fresh medium for 6 h. Primary neuron cultures were divided into four groups due to ACM, which were neurons without ACM exposure (group I) and neurons exposed to ACM from 0, 5 or 10 ?M arsenite treated astrocytes (group II-IV). Protein expression of N-methyl-d-aspartate receptors (NR1, NR2A, NR2B), calmodulin-dependent protein kinase II (CaMKII) and adenylate cyclase (AC) in neurons were measured after incubation with ACM for 4, 8 or 12 h. Morphological changes and synaptic formation were observed after a 72 h-incubation with ACM. Compared to group II, synaptic formation and protein expression of NR2A, NR2B, CaMKII and AC in group III and IV were significantly suppressed. Moreover, synaptic formation and protein expression of CaMKII and AC in group II were significantly enhanced when compared with group I. Taken together, findings from this study suggested that arsenic in astrocytes might impair synaptic formation through disturbing astrocytic effects on neuronal signal transduction.
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In vitro potency and efficacy favor later generation fluoroquinolones for treatment of canine and feline Escherichia coli uropathogens in the United States.
World J. Microbiol. Biotechnol.
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Information regarding in vitro activity of newer fluoroquinolones (FQs) is limited despite increasing resistance in canine or feline pathogenic Escherichia coli (E. coli). This study describes in vitro potency and efficacy toward E. coli of seven FQs grouped according to similarities in chemical structure: enrofloxacin, ciprofloxacin, orbifloxacin (first-group), levofloxacin, marbofloxacin (second-group) and pradofloxacin, moxifloxacin (third-group; latest S, S-pyrrolidino-piperidine at C-7). Potency measures included minimum inhibitory concentration (MIC) (geometric mean MIC, MIC(50), MIC(90)); and mutant prevention concentration (MPC) for FQ susceptible isolates only. In vitro efficacy measures included relative susceptibility (MIC(BP-S):MIC) or resistance (MIC:MIC(BP-R)) and mutant selection window (MSW) (MPC:MIC). For enrofloxacin susceptible isolates, mean MIC (?g/ml) was least for each third-group drug and ciprofloxacin and greatest for enrofloxacin and orbifloxacin (P = 0.006). For enrofloxacin susceptible isolates, MPC were below MIC:MIC(BP-R) and least for pradofloxacin (0.29 ± 0.16 ?g/ml) and greatest for enrofloxacin (1.55 ± 0.55 ?g/ml) (P = 0.006). MSW was least for pradofloxacin (55 ± 30) and greatest for ciprofloxacin (152 ± 76) (P = 0.0024). MIC(BP-S):MIC was greatest (P = 0.025) for pradofloxacin (190.1 ± 0.61) and least for enrofloxacin (23.53 ± 0.83). For FQ susceptible isolates, FQs MIC:MIC(BP-R) may serve as a surrogate for MPC. Because in vitro efficacy was greatest for pradofloxacin; it might be preferred for treatment of urinary tract infections (UTIs) associated with FQ susceptible E. coli uropathogens.
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Comparison of speciated arsenic levels in the liver and brain of mice between arsenate and arsenite exposure at the early life.
Environ. Toxicol.
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The aim of this study was to compare the risk from exposure to arsenate (iAs(V) ) or arsenite (iAs(III) ) at the early life. Mother mice were exposed to equimolar dose of iAs(V) and iAs(III) via drinking water during gestation and lactation. Their offspring continually drank the same water after weaning. Levels of speciated arsenic in both liver and brain were analyzed by hydride generation of volatile arsines and atomic absorption spectrophotometry (HG-AAS). In the liver, inorganic arsenic (iAs) levels significantly increased from postnatal day (PND) 15, and those on PND 35 were significantly higher than on PND 15 and 21 in iAs(III) exposed mice, but iAs levels did not significantly differ until PND 35 in iAs(V) exposed mice; Furthermore, all speciated arsenic levels on PND 35 and dimethylarsinic acid (DMA) levels on PND 1 were significantly higher in iAs(III) exposed mice than those in iAs(V) exposed mice. In the brain, iAs levels increased significantly on PND 21, but those declined sharply on PND 35 in either iAs(III) or iAs(V) exposed mice, however the mean difference between the two exposure groups was not significant; whereas DMA levels in iAs(III) exposed mice were significantly higher than those in iAs(V) exposed mice on both PND 1 and 35. In conclusion, findings from this study suggested that iAs(III) was preferentially accumulated into liver, and expected to result in more efficient methylation capacity than iAs(V) ; either iAs(V) or iAs(III) might be accumulated in the brain readily, when immature blood brain barrier can not limit it into brain. Hence, exposure to either iAs(V) or iAs(III) at the early life may increase the risk of iAs exposure in the brain. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
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Endoplasmic reticulum stress is involved in granulosa cell apoptosis during follicular atresia in goat ovaries.
Mol. Reprod. Dev.
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Follicular atresia is primarily induced by granulosa cell apoptosis, but description of the apoptotic pathway in granulosa cells is incomplete. In this study, we explored the possibility that endoplasmic reticulum (ER) stress could be involved in granulosa cell apoptosis during goat follicular atresia. Immunohistochemical analysis revealed that DNA damage-inducible transcript 3 (DDIT3) and glucose-regulated protein 78 (Grp78) were observed in scattered apoptotic granulosa cells of atretic follicles. Grp78 and DDIT3 mRNA and protein were upregulated in granulosa cells during follicular atresia, although DDIT3 was not significantly different between early atretic and progressed atretic follicles. Spontaneous apoptosis was also observed in vitro in granulosa cells induced by serum deprivation or by the ER stress agent tunicamycin, both inducing similar increases in DDIT3 mRNA. Activating transcription factor-6 (ATF6) and ATF4 mRNAs were significantly increased during granulosa cell apoptosis in vivo; in contrast to ATF6, ATF4 mRNA was attenuated after 16 hr of culture despite the persistence of ER stress. Taken together, ER stress-dependent DDIT3 pathways may play an important role in the regulation of selective granulosa cell apoptosis in goat ovaries during early follicular atresia. Serum deprivation could also increase apoptosis of cultured granulosa cells through the ER stress pathway as both ATF6 and PERK/eIF2?/ATF4 signaling have been implicated in the granulosa cell apoptosis of atretic follicles.
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