Search

The development of new technologies and industry increases the number and variety of electromagnetic field (EMF) sources. Researcher are increasingly interested in the effects of EMF on brain health. The brain's function is largely dependent on electrical excitability, so it would be expected to be vulnerable to EMF. We therefore investigated the effects of brain development in the fetus, histopathological changes in female rats and the hippocampal level of MAPK proteins in male rats after exposed to pre and postnatal 2450 MHz continuous wave (CW) radiofrequency radiation (RFR) over four generations. Four groups; sham, irradiated female, irradiated male, irradiated male and female, with each consisting of four rats (one male and three females) were created. Rats in the exposure groups were whole-body exposed to 2450 MHz CW-RFR for 12 h/day during the experiment. Irradiation started one month before fertilization in the experimental group. On the 18th day of the gestational period, one pregnant rat from each group was decapitated under general anesthesia and the fetuses were taken. The remaining two pregnant rats completed the normal gestation period. When the offspring were two months old, four rats, one male and three female, were allocated for the second generation study. Next generation animals were also experienced the same processes as the first generation rats. This study were evaluated development of brain in fetuses and histopathological changes in brain of female rats using haematoxylin eosin staining, and the hippocampal level of MAPK proteins in brain of male rats by Western Bloting. We observed hemorrhagic areas, irregular cellular localization and vascular structures in the brain of fetal and adult female rat of exposed groups in the all generations. pERK, ptau, pJNK and pP38 were increased in the brain of adult male rat of exposed groups in the all generations (p < 0.005). Pre and postnatal 2450 MHz continuous wave radiofrequency radiation exposure may cause changes in the function of the MAPK pathway affecting cognitive processes such as learning and memory and may cause damage to both the fetus and adult brain tissue. Also, EMF may have potential to affect brain of future generations. © 2022 Elsevier B.V., TYL-2017-7110, The authors acknowledge the financial support from Erciyes University-The Scientific Research Projects of Turkey (ERUBAP); Project number: TYL-2017-7110 .

Zebra fish is an important species in genetics and considering the proximity of its genome to the human genome, investigating the expression of some of the growth and appetite genes during its larvae development is essential. Genes coding growth and appetite (GH and ghrelin) hormones are involved in the synthesis and release of growth hormone, which can be considered to be economic genes in pisciculture. Given the importance of these genes during the early larvae development stages, this study was performed to assess their activity. Samples were collected at 4, 7, 10, 15, 30 and 45 post-hatching days. Samples were immediately placed in liquid nitrogen (-196 degree centigrade) and then stored in a freezer at -80 degree centigrade until RNA extraction (using RNX-Plus kit). To analyze normal expression of target genes, reference gene β-actin was used by 2-∆∆Ct method. The expression of genes associated with the growth and appetite was significantly different at various stages of the development of zebra fish, as the gene expression of GH on day 4 and ghrelin gene on day 10 after hatching were significantly higher compared with other samples (P

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment., Author summary Racemose neurocysticercosis is the most aggressive and lethal form of the disease characterized by progressive expansion of the larval stage of Taenia solium within the subarachnoid spaces of the brain. Currently, the cellular or molecular events that promote its formation and development are unknown. Our observations, employing tissue samples, revealed the presence of mitotically active cells in the bladder wall of the racemose larvae. Finally, we isolated and established primary cell cultures and observed that these cells are sensitive (proliferate in response to insulin). These results suggest that the proliferative cells stimulated by host biomolecules like hormones would be responsible for the abnormal growth of racemose larvae. Further studies are needed to identify all genes and pathways altered in the racemose form. The primary cell cultures will be used as a therapeutic target and allow us in future works to understand the developmental biology of the parasite.

Non-muscle actins have been studied for many decades, however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either &beta, actin coded by ACTB or &gamma, actin coded by ACTG1. First, we observed different distributions of &beta, and &gamma, actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that &gamma, actin knockout had more severe consequences on cell migration and invasion than &beta, actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones, however, a more detrimental effect was observed for &gamma, actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells&rsquo, FA formation and motility.

SUMMARY: Tyrosine phosphorylated proteins have been localized and identified in male reproductive tissues such as testis and capacitated/ acrosome reacted sperm except epididymis. The changes of such proteins are associated with decreased sperm quality of valproic acid treatment. This study aimed to investigate the presence and alterations of protein phosphorylation in epididymal epithelium and fluid of rats treated VPA. Sixteen adult male rats were divided into control and VPA-treated groups (n=8/ each). Treated rats were injected with VPA (500 mg/ kgBW, intraperitoneally) for 10 consecutive days. At the end of experiment, the monoclonal antiphosphotyrosine (clone 4G10) was used for immunohistochemistry to probe tyrosine phosphorylated proteins and also to examine the expression of such proteins using immuno-Western blotting in epididymal tissue and fluid. The result showed that positive reactivity of phosphorylated proteins was clearly observed in cytoplasmic principle cells, nuclei of apical & basal cells and sperm mass surrounded with epididymal fluids. The profiles of phosphorylated proteins in epididymal fluid were 182, 127, 80, 70, 57, 45, 34, and 31 kDas, respectively. Interestingly, VPA affected the changes of phosphorylated proteins and β actin in head, body, and tail epididymal fluids. We conclude that tyrosine phosphorylated proteins were detected in epididymal epithelium and fluid. The expressions of those proteins and actin were altered under VPA treating.

BACKGROUND: The insulin-like growth factor binding protein5 (IGFBP5) is often dysregulated in human cancers and considered neither a tumor suppressor nor an oncogene. OBJECTIVE: We aim to examine the reason of the changeable gene regulation of IGFBP5 in the case of methylation in breast cancer. METHODS: We used methyl-specific polymerase (MSP) chain reaction to detect CpG methylation of IGFBP5 promoter and exon-I in breast cancer and adjacent tissues. Gene expression is evaluated by quantative polymerase chain reaction (qPCR). RESULTS: IGFBP5 methylation was detected in 24 of 58 (41%) and 54 of 56 (96.5%) promoter and exon-I site respectively in tumor tissues. In adjacent tissues 17 of 58 (29%) and 53 of 56 (96.5%) was methylated. IGFBP5 expression was higher estrogene receptor (ER)(+) than ER(-) patients (p = 0:0549). Beside, we found a positive correlation between the expression of IGFBP5 and G2 tumor grade (p = 0:0131). However, no correlation was observed between IGFBP5 expression and age, menopause or the presence of lymph node metastasis (p > 0:05). CONCLUSIONS: In summary, our results showed that IGFBP5 promoter and exon-I methylation did not have any differences between tumor and adjacent tissues so that IGFBP5 methylation did not change IGFBP5 gene regulation in breast cancer. This is the first study investigating the IGFBP5 gene methylation in breast cancer. © 2016 - IOS Press and the authors.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J. M., Padr, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

WOS: 000442483900002 PubMed ID: 30132448 Background: We aimed to determine the effects of exercise followed by detraining on systolic blood pressure (SBP), heme oxygenase 2 (HO-2) expression, and carboxyhemoglobin (COHb) concentration in spontaneously hypertensive rats (SHR) to explain the role of carbon monoxide (CO) in this process. Material/Methods: Animals were randomized into exercised and detrained groups. Corresponding sedentary rats were grouped as Time 1-2. Swimming of 60 min/5 days/week for 10 weeks was applied. Detraining rats discontinued training for an additional 5 weeks. Gene and protein expressions were determined by real-time PCR and immunohistochemistry. Results: Aorta HO-2 histological scores (HSCORE) of hypertensive rats were lower, while SBP was higher. Swimming caused enhancement of HO-2 immunostaining in aorta endothelium and adventitia of SHR. Exercise induced elevation of blood COHb index in SHR. Synchronous BP lowering effect of exercise was observed. HO-2 mRNA expression, HSCORE, and blood COHb index were unaltered during detraining, while SBP was still low in SHR. Conclusions: CO synthesized by HO-2 at least partly plays a role in SBP regulation in the SHR-and BP-lowering effect of exercise. Regular exercise with short-term pauses may be advised to both hypertensives and individuals who are at risk. Pamukkale University Scientific Research Projects Coordination UnitPamukkale University [2012ARS002, 2013SBE002, 2016HZLDP018, 2018KRM002-201] This study is part of a PhD thesis and was supported by Pamukkale University Scientific Research Projects Coordination Unit through project numbers 2012ARS002, 2013SBE002, 2016HZLDP018 and 2018KRM002-201

The objective of this study was to compare effects of Western diet (WD) with low carbohydrate high protein (LCHP) diet on lipid metabolism, liver and kidney function in rats. Eighteen rats were randomly assigned to three experimental groups and fed for the next 2 months. The experimental diets were: Control (7% of soybean oil, 20% protein), WD (21% of butter, 20% protein), and LCHP (21% of butter and 52.4% protein) diet. The LCHP diet significantly decreased the body weight of the rats. Diet consumption was differentiated among groups, however significant changes were observed since third week of the experiment duration. Rats fed LCHP diet ate significantly less (25.2 g/animal/day) than those from Control (30.2 g/animal/day) and WD (27.8 g/animal/day) groups. Additionally, food efficiency ratio (FER) tended to decrease in LCHP fed rats. Serum homocysteine concentration significantly decreased in rats fed WD and LCHP diets. Liver weights were significantly higher in rats fed WD and LCHP diets. At the end of the experiment (2 months) the triacylglycerol (TAG) was significantly decreased in animals fed LCHP compared to WD. qRT-PCR showed that SCD-1 and FAS were decreased in LCHP fed rats, but WD diet increased expression of lipid metabolism genes. Rats receiving LCHP diet had two fold higher kidney weight and 54.5% higher creatinin level compared to Control and WD diets. In conclusion, LCHP diet decreased animal's body weight and decreased TAG in rat's serum. However, kidney damage in LCHP rats was observed.

Chagas disease is a complex zoonotic pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite presents remarkable genetic variability and has been grouped into six discrete typing units (DTUs). The association between the DTUs and clinical outcome remains unknown. Chagas disease and co-infection with HIV/AIDS has been reported widely in Brazil and Argentina. Herein, we present the molecular analyses from a Chagas disease patient with HIV/AIDS co-infection in Colombia who presented severe cardiomyopathy, pleural effusion, and central nervous system involvement. A mixed infection by T. cruzi genotypes was detected. We suggest including T. cruzi in the list of opportunistic pathogens for the management of HIV patients in Colombia. The epidemiological implications of this finding are discussed. © 2014 The Authors.

Background/Aims: Bile flow consists of bile salt-dependent bile flow (BSDF), generated by canalicular secretion of bile salts, and bile salt-independent flow (BSIF), probably of combined canalicular and ductular origin. Bile salt transport proteins have been identified in cholangiocytes, suggesting a role in control of BSDF and/or in control of bile salt synthesis through cholehepatic shunting. Methods: We studied effects of bile duct proliferation under non-cholestatic conditions in multidrug resistance-2 P-glycoprotein (Abcb4)-deficient multidrug resistance gene-2 (Mdr2(-/-)) mice. BSDF and BSIF were determined in wild-type and Mdr2(-/-) mice during infusion of step-wise increasing dosages of tauroursodeoxycholate (TUDC). Cholate synthesis rate was determined by 2H4-cholate dilution. Results were related to expression of transport proteins in liver and intestine. Results: During TUDC infusion, BSDF was increased by ∼ 50% and BSIF by ∼ 100% in Mdr2(-/-) mice compared with controls. Cholate synthesis rate was unaffected in Mdr2(-/-) mice. Hepatic expression of the apical sodium-dependent bile salt transporter (Asbt), its truncated form (tAsbt) and the multidrug resistance-related protein 3 were upregulated in Mdr2(-/-) mice. Conclusions: Bile duct proliferation in Mdr2(-/-) mice enhances cholehepatic shunting of bile salts, which is associated with a disproportionally high bile flow but does not affect bile salt synthesis. © Blackwell Munksgaard 2005. Chemicals / CAS: carrier protein, 80700-39-6; chenodeoxycholic acid, 474-25-9; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; lithocholic acid, 434-13-9; multidrug resistance protein 2, 256503-65-8; multidrug resistance protein 3, 231947-64-1; muricholic acid, 39016-49-4; tauroursodeoxycholic acid, 14605-22-2; bile acid binding proteins; Bile Acids and Salts; Carrier Proteins; Cholates; Deuterium, 7782-39-0; Membrane Glycoproteins; Organic Anion Transporters, Sodium-Dependent; P-glycoprotein 2; P-Glycoproteins; Phospholipids; RNA, Messenger; sodium-bile acid cotransporter, 145420-23-1; Symporters

HPV-transformed cells exhibit activation of NF-?B and STAT3 (mediators of inflammation), but very little is known about their regulation under inflammatory conditions before HPV integration. This study reports that cervical tissues with stromal inflammation and intact HPV16 E2 gene show increased expression of target genes of NF-?B and/or STAT3 which can regulate cell survival (cyclin D1, c-Myc, survivin and Bcl2) and inflammatory responses (TNF-?, IL-1?, IL-6, IL-8 and CCR2). Increased expression of RelA, p-I?B?, STAT3, p-STAT3 (Ser727), Pin1 (peptidyl-prolyl cis/trans isomerase) and MCM2 in the squamous epithelia of cervices with stromal inflammation supports early activation of NF-?B-STAT3. Furthermore, HPV16 E2 potentiated NF-?B activation induced by inflammatory mediators, IL-1? and SDF-1?, in HEK293 cells. These results reveal a novel role for E2 in regulating the activities of NF-?B and STAT3 that may have implications in carcinogenic progression of HPV16-infected cells under conditions of stromal inflammation. � 2014 Elsevier Inc.

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis. © 2013 Macmillan Publishers Limited. All rights reserved.

Oxidative stress and neuroinflammation play important roles in Alzheimer's disease (AD). ABCG2 is a transporter protein expressed in the brain and involved in GSH transport. To study the roles of Abcg2 in oxidative stress and AD, we cross-bred Tg-SwDI and Abcg2-KO mice and generated Tg-SwDI/Abcg2-KO (double-tg) mice. Brain tissues from double-tg, Tg-SwDI, wild-type, and Abcg2-KO mice at various ages were analyzed. Aβ40 and Aβ42 were detected in Tg-SwDI and double-tg mice. Total brain GSH was decreased and levels of lipid/DNA oxidation were increased in 3-month double-tg compared to Tg-SwDI mice. Low brain GSH was still detected in 9-month double-tg mice. Increased HMOX-1 and MCP-5 expression was observed in 9-month double-tg mice but not in Tg-SwDI mice compared to WT and Abcg2-KO mice. Increased HMOX-1 and decreased ICAM-1 expression were observed in 12-month double-tg mice compared to Tg-SwDI mice. The levels of Nrf-2 expression and activity were decreased in 6-month double-tg mice. Behavioral tests show impaired cognitive/memory performance of 9-month double-tg compared to Tg-SwDI mice as well as WT and Abcg2-KO mice. These results suggest that Abcg2 deficiency increases oxidative stress and alters inflammatory response in the brain and exacerbates cognitive/memory deficit in double-tg mice at different developmental stages. © 2012 National Research Council Canada & The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Purpose: Loss of sensitivity to transforming growth factor ? (TGF-?) signaling typically occurs in human ovarian cancer cells, but there is paucity of information regarding this in human ovarian tumors. Thus the association of inactivating mutations and/or variations in expression levels of TGF-? signaling components with human ovarian tumors was evaluated. Methods: Forty human ovarian tissue samples were analyzed for mutations and/or variations in the expression of transforming growth factor ? signaling components. Mutation studies were done through reverse transcription (RT) PCR, single strand conformation polymorphism analysis and automated DNA sequencing. Expression studies were carried out by semi quantitative RT PCR and western blotting. DNA binding ability of Smad complexes and expression of downstream targets were also analyzed. Results: The six alanine repeat containing variant of TGF-? RI was seen in 27% of the tumor cases studied, in addition to the 45 bp nucleotide deletions in exon 1 of the receptor in two ovarian tumor samples. A deletion in the polyadenine tract of exon 3 of TGF-? RII was seen in 22% of the tumor samples. We also report a loss or decrease in the expression of Smad 4 protein in tumor samples with a concurrent loss or reduced DNA binding ability of the Smad complex and deregulated expression of p21 and c-Myc. Conclusions: Our results suggest that mutations and/or alterations in expression of TGF-? receptors and loss of Smad 4 are frequent in human ovarian cancers and may potentially explain the frequent loss of TGF-? responsiveness that typically occurs in human ovarian cancer. � 2009 Springer-Verlag.

Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TTC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, oil average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most Suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor-normal paired breast cancer qRT-PCR studies.

In this study, we demonstrated that bcl-2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis-related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl-2 overexpressing derivatives, we found that bcl-2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP-2, MMP-7, MT1-MMP, and tissue inhibitors of metalloproteases-1 and -2. Moreover, bcl-2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl-2 transfectants tumors is significantly associated to an increase in MMP-2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl-2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression.

This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org. Copyright © by the Society of Toxicologic Pathology.

Background/Aims: Bile flow consists of bile salt-dependent bile flow (BSDF), generated by canalicular secretion of bile salts, and bile salt-independent flow (BSIF), probably of combined canalicular and ductular origin. Bile salt transport proteins have been identified in cholangiocytes, suggesting a role in control of BSDF and/or in control of bile salt synthesis through cholehepatic shunting. Methods: We studied effects of bile duct proliferation under non-cholestatic conditions in multidrug resistance-2 P-glycoprotein (Abcb4)-deficient multidrug resistance gene-2 (Mdr2(-/-)) mice. BSDF and BSIF were determined in wild-type and Mdr2(-/-) mice during infusion of step-wise increasing dosages of tauroursodeoxycholate (TUDC). Cholate synthesis rate was determined by 2H4-cholate dilution. Results were related to expression of transport proteins in liver and intestine. Results: During TUDC infusion, BSDF was increased by ∼ 50% and BSIF by ∼ 100% in Mdr2(-/-) mice compared with controls. Cholate synthesis rate was unaffected in Mdr2(-/-) mice. Hepatic expression of the apical sodium-dependent bile salt transporter (Asbt), its truncated form (tAsbt) and the multidrug resistance-related protein 3 were upregulated in Mdr2(-/-) mice. Conclusions: Bile duct proliferation in Mdr2(-/-) mice enhances cholehepatic shunting of bile salts, which is associated with a disproportionally high bile flow but does not affect bile salt synthesis. © Blackwell Munksgaard 2005. Chemicals / CAS: carrier protein, 80700-39-6; chenodeoxycholic acid, 474-25-9; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; lithocholic acid, 434-13-9; multidrug resistance protein 2, 256503-65-8; multidrug resistance protein 3, 231947-64-1; muricholic acid, 39016-49-4; tauroursodeoxycholic acid, 14605-22-2; bile acid binding proteins; Bile Acids and Salts; Carrier Proteins; Cholates; Deuterium, 7782-39-0; Membrane Glycoproteins; Organic Anion Transporters, Sodium-Dependent; P-glycoprotein 2; P-Glycoproteins; Phospholipids; RNA, Messenger; sodium-bile acid cotransporter, 145420-23-1; Symporters

This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org. Copyright © by the Society of Toxicologic Pathology.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins αβ-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of β-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of αβ-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock. Chemicals/CAS: Chaperonin 60; Crystallins; DNA, Complementary; Heat-Shock Proteins; HSPB1 protein, human; Neoplasm Proteins; Reagent Kits, Diagnostic; RNA, Messenger; RNA, Neoplasm

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins αβ-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of β-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of αβ-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock. Chemicals/CAS: Chaperonin 60; Crystallins; DNA, Complementary; Heat-Shock Proteins; HSPB1 protein, human; Neoplasm Proteins; Reagent Kits, Diagnostic; RNA, Messenger; RNA, Neoplasm

The testis is a double gland comprised of the sperm-producing seminiferous tubules and a complex endocrine interstice. The former structures generate and release whole cells (exocrine aspect of the gland) while the latter synthesize and release androgens and related hormones. The testis also has poorly understood paracrine connexions. These connexions play an important role during spermatogenesis. A key molecule within these structures is β-actin. Therefore, the present study aims at examining the expression pattern of β-actin during the various stages of the spermatic cycle in the rat. To achieve this goal, we used a combination of trans-illumination assisted microdissection of seminiferous tubules, confocal immunofluorescence and Western blot analysis. Our experiments revealed that the levels of β-actin fluctuate significantly during spermatogenesis, peaking immediately after spermiation. Fil: Acosta, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Foscolo, Mabel Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Cavicchia, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina