255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
T. T. B. Vo ◽  
E. B. Jeung
Keyword(s):  
Gene Expression ◽  
Adverse Effects ◽  
Treated Group ◽  
Environmental Chemicals ◽  
Female Rats ◽  
Protein Levels ◽  
Calbindin D9k ◽  
The Difference

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERα and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

scholarly journals Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 331
Author(s):  
Jung-Yun Lee ◽  
Tae Yang Kim ◽  
Hanna Kang ◽  
Jungbae Oh ◽  
Joo Woong Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Density Lipoprotein ◽  
Treated Group ◽  
Control Group ◽  
Chitosan Oligosaccharide ◽  
Sd Rats ◽  
Adipogenic Gene

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

PDGFR-Mediated Cytomegalovirus Infection of Megakaryocytes Induces Thrombocytopenia Via Demethylation of AML1 and IEX-1 in the TPO/c-Mpl Signaling Pathway Following Allo-HSCT

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3937-3937
Author(s):  
Xiao-Hui Zhang ◽  
Feng Fei-er ◽  
Qian-ming Wang ◽  
Xiao-lu Zhu ◽  
Lan-ping Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Hcmv Infection ◽  
Treated Group ◽  
In Vitro Studies ◽  
Significant Difference

Abstract Introduction: Human cytomegalovirus (HCMV) infection is a common complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is associated with high morbidity and mortality. Thrombocytopenia is one of the major hematological complications of HCMV infection. Possible causes include direct HCMV injury to hematopoietic progenitor cells and the microenvironment, as well as HCMV-related immune thrombocytopenia. Previous in vitro studies demonstrate that HCMV could directly infect megakaryocytes(MKs) and their progenitors, resulting in decreased CFU-MK and increased apoptosis, but the underlying mechanisms remain uncertain. It remains unknown whether HCMV can directly target MKs in vivo, how MK function changes after infection, why HCMV selectively infects certain patients and what inhibits MK maturation and results in apoptosis. It has been reported that patients with HCMV-related thrombocytopenia showed poor response to rhTPO, implying blockage of the TPO/c-Mpl signaling pathway. Our previous research indicated that PDGFR+CXCR4lowCCR5lowMKs are correlated with HCMV infection.We hypothesized that PDGFR+CXCR4lowCCR5lowMKs are more susceptible to HCMV infection. HCMV could directly target MKs both in vitro and in vivo, resulting in increased apoptosis and decreased MK ploidy. HCMV infection could possibly disturb the downstream TPO/c-Mpl signaling pathway, thereby inhibiting MK differentiation and maturation. Methods: We collected bone marrow from HCMV DNAemia patients post allo-HSCT for in vivo study. Transmission electron microscopy(TEM) was used to detect HCMV particles inside MKs. MKs were identified as CD41+vWF+cells by flow cytometry(FCM). To analyze the susceptibility of MKs to HCMV, expression levels of PDGFR, αvβ3, TLR2, CCR5 and CXCR4 in different groups were tested. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. We also measured c-Mpl expression in MKs.In vitro study, we used plasma from HCMV-infected patients post allo-HSCT to infect MKs cultured from bone marrow CD34+ cells. We validated cell susceptibility with the same markers used in vivo. Next, inhibitors of the positive markers were co-cultured with MKs. We analyzed pp65 expression in the inhibitor-treated group and control group to explore potential prevention of HCMV infection. We investigated AML1 and IEX-1 in the downstream TPO/c-Mpl signaling pathway by PCR and Western Blot. We used bisulfite sequencing PCR (BSP) to study the methylation status in different gene expression profiles of AML1 and IEX-1. 5-ara-dC is a type of DNA methylation inhibitor. After incubation with MKs, we analyzed changes in gene expression and MKs function. Results: Using TEM, we managed to find HCMV particles in MKs from HCMV-infected patient bone marrow samples. The proportion of apoptosis markedly increased compared with HCMV-negative MKs, whereas the mean ploidy slightly decreased. C-Mpl expression showed no significant difference between the two groups. Pp65 positive cells showed elevated expression in PDGFR and reduced expression in CXCR4 and CCR5. In vitro studies revealed similar results. After treating with the PDGFR inhibitor IMC-3G3, the pp65 positive cell population was slightly decreased, but the Gleevec-treated group showed no difference. We found a decrease in both IEX-1 and AML1 on both the molecular and protein levels. Both gene promoters were hypermethylated in the HCMV-infected group. After demethylation with 5-ara-dC, IEX-1 and AML1expression levels were both up-regulated, and cell apoptosis was reduced. Conclusion: (1)HCMV inhibited megakaryocytic differentiation and maturation and reduced MKs polyploidy both in vivo and in vitro. (2)MKs positive for PDGFR and low in CXCR4 and CCR5 were more susceptible to HCMV infection. The PDGFR inhibitor IMC-3G3 protected MKs from HCMV infection. (3)The mechanism of HCMV-associated thrombocytopenia may be a disturbance of the TPO/c-Mpl signaling pathway in MKs through hypermethylation of the AML1 and IEX-1 promoters. Demethylation with 5-ara-dC could reverse cell apoptosis. Therefore, we illustrated the possible mechanism of HCMV-induced thrombocytopenia, highlighting new insights for future potential therapeutic approaches. Disclosures No relevant conflicts of interest to declare.

Testosterone treatment increases thromboxane function in rat cerebral arteries

2005 ◽  
Vol 289 (2) ◽  
pp. H578-H585 ◽  
Author(s):  
Rayna J. Gonzales ◽  
Amir A. Ghaffari ◽  
Sue P. Duckles ◽  
Diana N. Krause
Keyword(s):  
Cerebral Arteries ◽  
Underlying Mechanism ◽  
Cerebral Vessel ◽  
Testosterone Treatment ◽  
Protein Levels ◽  
Tp Receptors ◽  
The Difference ◽  
Synthase Inhibitor

We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA2) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on TxA2 synthase levels and the contribution of TxA2 to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that TxA2 synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that TxA2 synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX+T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA2 synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA2 mimetic U-46619 were not different between the ORX+T and ORX groups. However, dilator responses to either the selective TxA2 synthase inhibitor furegrelate or the TxA2-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX+T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX+T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA2-mediated tone in rat cerebral arteries by increasing endothelial TxA2 synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA2 synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.

scholarly journals Investigation of exposure to lifestyle and environmental factors on cumulus-oocyte complex function

2021 ◽  
Author(s):  
◽  
Kelly Anne Campen
Keyword(s):  
Gene Expression ◽  
Oocyte Quality ◽  
Protein Levels ◽  
Time Interaction ◽  
Cumulus Oocyte Complex ◽  
In Vitro Exposure ◽  
In Vitro Effects ◽  
In Vivo Effects

<p>Pathways involved in bi-directional communication within the cumulus-oocyte complex (COC) include gap junction (GJ) communication, oocyte growth factor production, and glucose metabolism and are essential for oocyte health. Perturbation of these pathways may result in reduced oocyte quality due to altered COC function. Using rats as a model, in vitro effects of exposure to bisphenol A (BPA), caffeine, nicotine, ethanol, methylenedioxymeth- amphetamine (MDMA), or Δ⁹-tetrahydrocannabinol (THC) on COC function were investigated. Furthermore, MDMA was administered to rats to compare in vitro with in vivo effects.  The transfer of a fluorescent dye (calcein) from cumulus cells (CC) to the oocyte was used as a measure of GJ communication. Expression of CC-derived (Atr, Cx43, Cycs, Gfpt1, Pfkp) and oocyte-derived (Atr, Bmp15, Cx37, Gdf9) genes were measured using multiplex TaqMan quantitative PCR. Levels of CX43 and GDF9 proteins were quantified using Western blotting.  Optimisation of the GJ bioassay included the addition of phosphodiesterase inhibitors (rolipram and dipyridamole), and a 1 hour post-calcein incubation period to allow dye transfer. Quantification of gene expression in calcein-treated CC and oocytes was validated, enabling direct comparisons between GJ communication and gene expression.  To determine the in vitro effects, COC were incubated with test factors at high physiological concentrations over 25 hours. GJ communication decreased over time in control COC. This reduction was attenuated after exposure to BPA and nicotine, and partially by caffeine. Furthermore, exposure to ethanol maintained oocyte meiotic arrest, whereas MDMA and THC promoted meiotic resumption.  Oocyte-derived gene expression was mostly unaffected by in vitro exposure to the lifestyle and environmental factors, although a treatment x time interaction for Cx37 levels following nicotine exposure was observed. Of the CC-derived genes, Cx43 was the most sensitive where BPA, MDMA, and THC increased, and caffeine and ethanol decreased, expression. In CC, exposure to MDMA and THC increased Gfpt1 levels and exposure to MDMA resulted in a treatment x time interaction in Cycs and Pfkp expression.  In COC, caffeine increased CX43 protein levels after 1 hour. Nicotine initially reduced, but with time increased CX43 levels. Furthermore, CX43 levels decreased and increased after 25 hour exposures to ethanol and MDMA, respectively. GDF9 protein levels in COC exhibited wide within-treatment variation. Overall, BPA and caffeine reduced GDF9 levels after 1 hour whereas GDF9 levels were increased following exposure to BPA, caffeine, MDMA, and THC for 25 hours.  To determine in vivo effects, female rats were administered saline or 5 mg/kg/day MDMA for 3 days. COC from MDMA-treated rats had higher levels of CX43 protein but gene expression and meiotic reactivation were unaffected.  In conclusion, COC function was altered by in vitro exposure to BPA, caffeine, ethanol, nicotine, MDMA, and THC. Furthermore, in vivo exposure to MDMA elicits similar, albeit reduced, effects on COC function. A role for CC in protecting the oocyte from harmful contaminants is proposed. Perturbation of the bi-directional communication pathway is likely to influence oocyte quality due to alterations in nutrient availability and timing of follicular events, although these may not be associated with negative outcomes. This study provides evidence that exposure to lifestyle factors and environmental contaminants affect COC function.</p>

scholarly journals Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

2009 ◽  
Vol 296 (6) ◽  
pp. C1321-C1328 ◽  
Author(s):  
R. P. Rhoads ◽  
R. M. Johnson ◽  
C. R. Rathbone ◽  
X. Liu ◽  
C. Temm-Grove ◽  
...  
Keyword(s):  
Gene Expression ◽  
Satellite Cell ◽  
Hypoxia Inducible Factor ◽  
Collagen Gel ◽  
Culture Conditions ◽  
Cell Physiology ◽  
Vegf Gene ◽  
Protein Levels

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1α activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction

2017 ◽  
Vol 126 (04) ◽  
pp. 255-262 ◽  
Author(s):  
Katarína Chalásová ◽  
Lukáš Pácal ◽  
Anna Pleskačová ◽  
Lucia Knopfová ◽  
Jitka Řehořová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Disease ◽  
Pentose Phosphate ◽  
Protective Mechanism ◽  
Protein Levels ◽  
Human Experiments ◽  
Key Enzyme ◽  
Vitro Analysis

Abstract Aim Pentose phosphate pathway (PPP) with key enzyme transketolase (TKT), represents a potentially ‘protective’ mechanism in hyperglycaemia. Diabetic kidney disease (DKD), a common complication of both type 1 and type 2 diabetes associated with significant morbidity and mortality, represents the most common cause of chronic kidney disease (CKD). We hypothesized that protective PPP action in diabetes and eventually even more severely in concomitant DKD might be compromised by limited intracellular availability of an active TKT cofactor thiamine diphosphate (TDP). Methods Effect of hyperglycaemia on gene expression and protein levels of key PPP loci was studied in vitro using human cell lines relevant to diabetes (HUVEC and HRGEC) and (together with measurement of TKT activity, plasma thiamine and erythrocyte TDP concentration) in vivo in diabetic vs. non-diabetic subjects with comparable renal function (n=83 in total). Results Hyperglycaemia significantly decreased protein levels of RFC-1, THTR1, THTR2 and TKT (P<0.05) in vitro. Analysis of blood samples from CKD patients with and without diabetes and from controls did not reveal any difference in gene expression and protein levels of thiamine transporters while TKT activity and TDP in erythrocytes gradually increased with decreasing kidney function being highest in patients with CKD3-4 of both diabetic and non-diabetic aetiology. Hyperglycaemia and uremic serum mimicking CKD in diabetes did not affect TKT activity in vitro (P<0.05). Conclusion Both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while TKT activity in parallel with intracellular TDP was increased in CKD patients with or without diabetes. Therefore, lack of adaptive increase of thiamine transmembrane transport allowing further increase of TKT activity might contribute to compromised PPP function in diabetes and CKD and to the development of glycotoxic injury.

Transcriptional regulation of pulmonary elastin gene expression in elastase-induced injury

2003 ◽  
Vol 285 (2) ◽  
pp. L354-L362 ◽  
Author(s):  
Celeste B. Rich ◽  
Isabel Carreras ◽  
Edgar C. Lucey ◽  
Julie A. Jaworski ◽  
Jo Ann Buczek-Thomas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Injury ◽  
Gene Promoter ◽  
Activity Levels ◽  
Intratracheal Administration ◽  
Protein Levels ◽  
Elastin Gene ◽  
Pulmonary Fibroblast

Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase. In addition, we extended our studies to mice that were given an intratracheal dose of elastase; the effects on lung elastin mRNA and elastin promoter activity levels were measured and compared with results from in vitro cell models. The results demonstrate that upregulation of elastin gene expression during the transition of subconfluent to confluent cultures and after elastase injury is associated with an increase in the level of transcription both in vitro and in vivo. Furthermore, intratracheal administration of elastase to transgenic mice illustrates that the increased levels of elastin mRNA are accompanied by increased activity of the elastin gene promoter in cells spatially positioned near major sites of tissue injury.

scholarly journals miR-29a and miR-30c negatively regulate DNMT 3a in cardiac ischemic tissues: implications for cardiac remodelling

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Carolina Gambacciani ◽  
Claudia Kusmic ◽  
Elena Chiavacci ◽  
Francesco Meghini ◽  
Milena Rizzo ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Ischemia Reperfusion ◽  
Chromatin Accessibility ◽  
Response To Therapy ◽  
Vitro Assay ◽  
Protein Levels ◽  
Transcriptional Reprogramming

AbstractRecent evidences indicate that epigenetic changes play an important role in the transcriptional reprogramming of gene expression that characterizes cardiac hypertrophy and failure and may dictate response to therapy. Several data demonstrate that microRNAs (miRNAs) play critical roles both in normal cardiac function and under pathological conditions. Here we assessed, in in vivo rat models of myocardial infarction (MI) and ischemia-reperfusion (IR), the relationship between two miRNAs (miR-29a and miR-30c) and de novo methyltransferase (DNMT3a) which, altering the chromatin accessibility for transcription factors, deeply impacts gene expression. We showed that the levels of members of miR-29 and miR- 30 families were down regulated in ischemic tissues whilst the protein levels of DNMT3a were increased, such a relation was not present in healthy tissues. Furthermore, by an in vitro assay, we demonstrated that both miRNAs are able to down regulate DNMT3a by directly interacting with DNMT3a 3’UTR and that miR-29a or miR-30c overexpression in the cardiac HL1 cell line causes decrease of DNMT3a enzyme both at the mRNA and protein levels. Our data, besides confirming the down regulation of the miR-29a and miR-30c in infarcted tissues, envisage a cross-talk between microRNAs and chromatin modifying enzymes suggesting a new mechanism that might generate the alterations of DNA methylation often observed in myocardial pathophysiology.

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