Total RNA Expression Research Articles

Genotoxic effects and serum abnormalities in residents of regions proximal to e-waste disposal facilities in Jinghai, China

Electronic waste (e-waste) disposal is a growing problem in China, and its effects on human health are a concern. To determine the concentrations of pollutants in peripheral blood and genetic aberrations near an e-waste disposal area in Jinghai, China, blood samples were collected from 30 (age: 41±11.01 years) and 28 (age: 33±2.14 years) individuals residing within 5 and 40km of e-waste disposal facilities in Jinghai (China), respectively, during the week of October 21–28, 2011. Levels of inorganic pollutants (calcium, copper, iron, lead, magnesium, selenium, and zinc) and malondialdehyde (MDA), identities of persistent organic pollutants (POPs), micronucleus rates, and lymphocyte subsets were analyzed in individuals. Total RNA expression profiles were analyzed by group and gender. The population group living in proximity to the e-waste site displayed significantly higher mean levels of copper, zinc, lead, MDAs, POPs (B4–6DE, B7–9DE, total polychlorinated biphenyls, and BB-153). In addition, micronucleus rates of close-proximity group were higher compared with the remote group (18.27% vs. 7.32%). RNA expression of genes involved in metal ion binding and transport, oxidation/reduction, immune defense, and tumorigenesis varied between groups, with men most detrimentally affected (p<0.05). CD4+/CD8+T cell ratios, CD4+CD25nt/hiCD127loregulatory T cell percentages, and CD95 expression were greater in the e-waste group (p<0.05). Residing in close proximity to e-waste disposal facilities (≤5km) may be associated with the accumulation of potentially harmful inorganic/organic compounds and gender-preferential genetic aberrations.

Changes of gene expressions in zinc deficiency and diet restriction in rats (1043.8)

We previously reported that both zinc deficiency and diet restriction affect total RNA concentration and expression of Gapdh and Actb in kidney. In this study, we have examined whether zinc deficiency and diet restriction affect Crebbp and Taf5l in kidney. Thirty 3‐week‐old male Sprague‐Dawley rats were assigned into 3 dietary treatment groups of 10 for the four‐week study: Control group (AIN‐93G), Zn‐deficient group (4.5 mg Zn/kg) and pair‐fed group (AIN‐93G). Kidney was separated into cortex and medulla for RNA isolation. Quantitative real time RT‐PCR using TaqMan proves was performed for mRNAs of Crebbp and Taf5l. Difference between groups was statistically analyzed by Fisher's PLSD. P value less than 0.05 was considered significant. Kidney cortex Crebbp per total RNA was not changed by the treatments. Kidney medulla Crebbp per total RNA was significantly increased by zinc deficiency and diet restriction, respectively. Kidney cortex Taf5l per total RNA was significantly increased by zinc deficiency and diet restriction, respectively. Kidney medulla Taf5l per gram tissue was significantly decreased by zinc deficiency and diet restriction, respectively. These results suggest that zinc deficiency and diet restriction differently affect expression of Crebbp and Taf5l in kidney cortex and medulla. This work was supported by KAKENHI grant‐in‐aid for Young Scientists (B) (23700928).

Abstract B121: AIB1 is a transcriptional and translational mediator of breast cancer cell growth and migration targets

Abstract The nuclear p160 steroid receptor co-activator, AIB1 is a downstream regulator of the insulin-like growth factor (IGF-I) tyrosine kinase signaling axis [1]. Since AIB1 is over-expressed in 31-64% of breast cancers cell several mechanisms have been described to explain its function in breast cancer [2]. AIB1 transcriptionally regulates genes involved in carcinogenesis via its co-partnership with estrogen receptor alpha (ERα) and E2F1 [3]. Interestingly the transcriptional mediators, c-myc and E2F1 induce mRNA translation potentially via the transcriptional regulation of ribosome biogenesis factors [4]. However, whether AIB1 acts in similarly is not well known [3]. Furthermore, since IGF-I signaling stimulates mRNA cap-dependent translation and also affects AIB1, we hypothesize that AIB1 acts as both a transcriptional and translational regulator of breast cancer cell growth and migration in an IGF-dependent manner. ER positive (MCF7L) wild-type, stably expressing AIB1 and scrambled control short hairpin RNA (shAIB1 and shCON) cells were created. Cells were treated for 24h in serum free media plus and minus IGF-I (5nM). Polyribosomal mRNA was stratified using sucrose gradients and fractionated into ten individual fractions and total-RNA samples were obtained. qRT-PCR was performed to determine IGF-I and AIB1 mediated transcriptional and translational effects on AIB1, markers of proliferation (i.e. KI67 and PCNA), cell cycle (i.e. p21), survival (i.e. BCL2), migration (i.e. VEGFA) and ribosome biogenesis factors (i.e. eIF3A and eIF5) in MCF7L shCON and shAIB1 cells. Statistical significance was determined by one-way ANOVA (p&amp;lt;0.05). Total-RNA expression of VEGFA, Ki67, PCNA and eIF5 was induced by IGF-I in the shCON and shAIB1 cells (p&amp;lt;0.05), apart from eIF5 where no induction by IGF-I was detected in the shAIB1 cells. No change in total-RNA expression of AIB1, BCL2, p21 or eIF3A was detected by IGF-I treatment. However, AIB1 and p21 total-RNA expression was reduced under both basal and IGF-I treated conditions in the shAIB1 vs. shCON cells (p&amp;lt;0.05). The percentage of ribosome-bound Ki67, PCNA, p21, VEGFA, and eIF3A mRNA was increased by IGF-I in all ten pooled fractionated RNA samples combined (range: 11% to 340%, mean: 110%), compared to control cells. This effect was either reversed (i.e. Ki67, PCNA and eIF3A), slightly increased (i.e. p21) or unaffected (i.e. VEGFA) in the shAIB1 IGF-I treated vs. control cells. IGF-I treatment reduced ribosome-bound BCL2 (23%) and eIF5 (20%) mRNA in the shCON cells, which was further reduced to 68% and 46% respectively in the shAIB1 cells. We observed a basal reduction of ribosome-bound mRNA for AIB1 (63%), VEGFA (24%), p21 (43%) and eIF3A (37%) and an increase for Ki67 (26%), PCNA (12%), eIF5 (32%) and BCL2 (59%) in the shAIB1 vs. shCON cells. Moreover, all genes had reduced ribosome-bound mRNA following IGF-I treatment in the shAIB1 vs. shCON fractions, except for eIF5 which increased. Our studies demonstrate that IGF-I transcriptionally induces Ki67, PCNA, VEGFA and eIF5 in an AIB1-dependent and independent manner and p21 was only transcriptionally regulated by AIB1. Importantly, IGF-I regulated mRNA ribosome recruitment was predominantly reduced in the AIB1 knock-down cells, indicative of a reduced malignant potential. These findings give preliminary evidence of AIB1 acting as a novel mediator of ribosomal recruitment, which may lead to novel combinatorial therapeutic approaches using AIB1 and translational targeted inhibitors in AIB1 over-expressing breast cancers. 1. Oh, A., et al. Cancer Res, 2004. 15 (64): p.8299-308 2. Lahusen, T., et al. Breast Cancer Res Treat, 2009: 116(2): p.225-37 3. Louie, MC., et al. Mol Cell Biol, 2004: 24(12): p. 5157-71 4. Cole, MD and Cowling VH. Oncogene, 2009: 28: p 1169-1175 Citation Format: Aleksandra M. Ochnik, Mark S. Peterson, Svetlana V. Avdulov, Douglas Yee. AIB1 is a transcriptional and translational mediator of breast cancer cell growth and migration targets. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B121.

Granulocyte colony stimulating factor (GCSF) improves memory and neurobehavior in an amyloid-β induced experimental model of Alzheimer's disease

GCSF is an endogenous neuronal hematopoietic factor that displays robust in vitro and in vivo neuroprotective activity. The present study aimed to evaluate the effect of GCSF on Aβ-induced memory loss in an Alzheimer's disease model of rats. A total of 42 male adult Wistar rats weighing 200–250g were used in the study and were divided into 7 experimental groups. Animals were subjected to intracerebroventricular (ICV) injection stereotaxically at day 0 to instill amyloid-β1–42 (Aβ1–42) or PBS (sham operated group) at 10μl (5μl bilaterally). GCSF treatment was given from day 7 to 12 of Aβ injection. On day 21, behavioral tests (short term memory, exploratory behavior and motor coordination) in all groups were evaluated. Biochemical parameters and RNA expression were measured to ensure the efficacy of GCSF. GCSF (35 and 70μg/kg, s.c.) showed statistically significant improvement in memory as compared to control and sham operated groups (p<0.05). Mean time spent in the platform placed quadrant was found to be significantly increased in the GCSF (70μg/kg, s.c.) as compared to GCSF (35μg/kg, s.c.) and GCSF (10μg/kg, s.c.) groups (p<0.001). GCSF (35 and 70μg/kg, s.c.) also improved motor coordination and exploratory behavior significantly as compared to naïve sham operated and GCSF (10μg/kg, s.c.) groups (p<0.05). Improvement in memory by GCSF (35 and 70μg/kg, s.c.) was coupled with marked reduction of lipid peroxidation, acetylcholinesterase levels and a significant increase in antioxidant enzymes as well as total RNA expression in the brain. Additionally, GCSF (35 and 70μg/kg, s.c.) significantly increased progenitor cells (iPSCs) and surface marker CD34+ in the brain and hence induced neurogenesis. The present findings demonstrate an improvement of memory and neurobehavioral function with GCSF in Aβ-induced Alzheimer's disease model in rats.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

Both zinc deficiency and diet restriction decreased kidney total RNA concentration in rats

Total RNA concentration in liver is known to be decreased by dietary zinc deficiency. In this study, we have examined whethre zinc deficiency and diet restriction affect total RNA concentration and expression of Actb and Gapdh in kidney. Thirty 3‐week‐old male SD rats were assigned into 3 dietary treatment groups of 10 for the four‐week study: Control group (AIN‐93G), Zn‐deficient group (4.5 mg Zn/kg; ZD) and pair‐fed group (AIN‐93G; PF). Kidney was separated into cortex and medulla for RNA isolation. Real‐time PCR analysis was performed. Difference between groups was statistically analyzed by Fisher's PLSD. P value less than 0.05 was considered significant. Total RNA concentrations in the medulla of ZD and PF were significantly decreased compared with Control. Total RNA concentrations in the cortex of PF was significantly decreased compared with Control. The Gapdh/total RNA ratio in the cortex were increased in PF compared with Control. The Actb/total RNA ratio in the cortex of PF was significantly increased compared with Control, and the Actb/total RNA ratio in the medulla of ZD and PF were significantly increased compared with Control. These results suggest that both zinc deficiency and diet restriction affect total RNA concentration and expression of Gapdh and Actb in kidney. This work was supported by KAKENHI grant‐in‐aid for Research Activity Start‐up (21800073) and Young Scientists (B) (23700928).

Effects of leptin on the expression of fatty acid-binding proteins in human placental cell cultures

The present study aimed to evaluate the protein expression of the transmembrane translocase FAT/CD36 and cytoplasmic H-FABP and L-FABP in human trophoblast tissue and evaluate the effects of exogenous leptin upon differential expression of each biomolecule; consequently, it aimed to derive information regarding the effects of leptin upon the expression of proteins implicated in fatty acid metabolism. Protein and total RNA were isolated from 72 samples of trophoblast tissue obtained from chorionic villous sampling. Of these, 36 samples were evaluated for protein (supernatant and pellet fraction separated) and the other 36 for total RNA expression. For each subgroup of samples, 12 were treated immediately and 24 were cultured. Half of the cultured samples were treated with 10 ng/-ml exogenously added leptin and the other half were untreated. Western blotting and PCR techniques were used for the evaluation of biomolecule expression. Our results were obtained from samples at a mean gestational week of 12+5 (n=72; min, 11+0; max, 14+1 gw; SD,0.89). In promptly treated samples we observed the presence of FAT/‌CD36 protein and absence of cytoplasmic FABPs. In the latter, only mRNA transcription of H-FABP was noted. A cytoplasmic pool of FAT/CD36 was also noted in the supernatant fraction of proteins. For cultured samples, when leptin was added, a statistically significant increase in FAT/CD36 protein expression was observed (n=24, p<0.001; mean difference, 0.219; SD, 0.0315; CI, -0.284 to -0.154). In our study we demonstrated the protein and mRNA expression of biomolecules implicated in fatty acid metabolism in human placenta. A cytoplasmic pool of the transmembrane protein FAT/CD36 was noted. Leptin caused the increase in FAT/CD36 protein expression in the cultured samples. Therefore, we conclude that leptin has an immediate effect and plays a role in lipid metabolism in human placenta.

Identification of ATPAF1 as a novel candidate gene for asthma in children

Asthma is a common disease of children with a complex genetic origin. Understanding the genetic basis of asthma susceptibility will allow disease prediction and risk stratification. We sought to identify asthma susceptibility genes in children. A nested case-control genetic association study of children of Caucasian European ancestry from a birth cohort was conducted. Single nucleotide polymorphisms (SNPs, n= 116,024) were genotyped in pools of DNA samples from cohort children with physician-diagnosed asthma (n= 112) and normal controls (n= 165). Agenomic region containing the ATPAF1 gene was found to be significantly associated with asthma. Additional SNPs within this region were genotyped in individual samples from the same children and in 8 independent study populations of Caucasian, African American, Hispanic, or other ancestries. SNPs were also genotyped or imputed in 2 consortia control populations. ATPAF1 expression was measured in bronchial biopsies from asthmatic patients and controls. Asthma was found to be associated with a cluster of SNPs and SNP haplotypes containing the ATPAF1 gene, with 2 SNPs achieving significance at a genome-wide level (P= 2.26 × 10(-5) to 2.2 × 10(-8)). Asthma severity was also found to be associated with SNPs and SNP haplotypes in the primary population. SNP and/or gene-level associations were confirmed in the 4 non-Hispanic populations. Haplotype associations were also confirmed in the non-Hispanic populations (P= .045-.0009). ATPAF1 total RNA expression was significantly (P< .01) higher in bronchial biopsies from asthmatic patients than from controls. Genetic variation in the ATPAF1 gene predisposes children of different ancestries to asthma.

Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is characterized by a multi-cause, multi-stage and multi-focus process of tumor progression. Its prognosis is poor and early diagnosis is of utmost importance. This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC. A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression. Total RNA was purified and transcribed into cDNA by reverse transcription. Fragments of the GPC-3 gene were amplified by nested RT-PCR, and confirmed by sequencing. GPC-3 was analyzed by immunohistochemistry, Western blotting or ELISA. Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm. Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver total RNA and GGT expression. The incidence of liver GPC-3 mRNA and GPC-3, and serum GPC-3 was 100%, 100% and 77.8% in the HCC group, 100%, 100%, and 66.7% in the precancerous group, 83.3%, 83.3%, and 38.9% in the degeneration group, and no expression in the liver or blood of the control group, respectively. There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

Homeostatic regulation of electrical excitability in physiological cardiac hypertrophy

Pathological biomechanical stresses cause cardiac hypertrophy, which is associated with QT prolongation and arrhythmias. Previous studies have demonstrated that repolarizing K(+) current densities are decreased in pressure overload-induced left ventricular hypertrophy, resulting in action potential and QT prolongation. Cardiac hypertrophy also occurs with exercise training, but this physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk, suggesting that repolarizing K(+) currents are upregulated, in parallel with the increase in myocyte size, to maintain normal cardiac function. To explore this hypothesis directly, electrophysiological recordings were obtained from ventricular myocytes isolated from two mouse models of physiological hypertrophy, one produced by swim-training of wild-type mice and the other by cardiac-specific expression of constitutively active phosphoinositide-3-kinase-p110α (caPI3Kα). Whole-cell voltage-clamp recordings revealed that repolarizing K(+) current amplitudes were higher in ventricular myocytes isolated from swim-trained and caPI3Kα, compared with wild-type, animals. The increases in K(+) current amplitudes paralleled the observed cellular hypertrophy, resulting in normalized or increased K(+) current densities. Electrocardiographic parameters, including QT intervals, as well as ventricular action potential waveforms in swim-trained animals/myocytes were indistinguishable from controls, demonstrating preserved electrical function. Additional experiments revealed that inward Ca(2+) current amplitudes/densities were also increased in caPI3Kα, compared with WT, left ventricular myocytes. The expression of transcripts encoding K(+), Ca(2+) and other ion channel subunits was increased in swim-trained and caPI3Kα ventricles, in parallel with the increase in myocyte size and with the global increases in total cellular RNA expression. In contrast to pathological hypertrophy, therefore, the functional expression of repolarizing K(+) (and depolarizing Ca(2+)) channels is increased with physiological hypertrophy, reflecting upregulation of the underlying ion channel subunit transcripts and resulting in increased current amplitudes and the normalization of current densities and action potential waveforms. Taken together, these results suggest that activation of PI3Kα signalling preserves normal myocardial electrical functioning and could be protective against the increased risk of arrhythmias and sudden death that are prevalent in pathological cardiac hypertrophy.

Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth.

Changes of total RNA and mRNA differential expressions in leaves of Pinellia ternata under high temperature stress

To study the total RNA and mRNA differential expression in leaves of Pinellia ternata under high temperature, provide more information of the molecular mechanism of the sprout tumble. The total RNA and mRNA differential expression in leaves of P. ternata at different stress time was analyzed. The results showed that the trend of total RNA content was divided into three descending stages and two ascending stages, the total RNA content was the highest at 0, 6 h, but it was the lowest at and 42 h, as well as when the sprout tumbled. The differential display showed that the polymorphism and type of bands of the sample at 6 h were similar to those at 0 h. But the bands numbers at other time were far less than those at 0, 6 h. And there were some different mRNA differential expression bands between the different samples. In the process of the sprout tumble caused by high temperature stress, the RNA and mRNA differential expression in leaves of P. ternata changed.

Abstract 4087: Large intergenic noncoding RNAs associated with Ewing sarcoma family of tumors

Abstract BACKGROUND: Ewing sarcoma family of tumors (EFT) represents the second most common primary malignant bone tumor in children and adolescents. The majority of EFTs harbor a translocation (t11;22)(q24;q12) resulting in the expression of the EWS-FLI1 chimeric oncoprotein. We hypothesized that microarray gene expression profiling, in combination with next-generation sequencing technology, could be used to identify EFT-specific genes and transcripts including large intergenic noncoding (linc)RNAs. Next-generation sequencing, coupled with target enrichment for validation, can also identify driver mutations of metastasis and treatment resistance in high-risk cases. RESULTS: Genome-wide expression profiles of childhood sarcoma and normal tissues were analyzed using both Partek Genomics Suite as well as our own customized software, Genetrix. These are compared with Helicos single molecule sequencing data. The results show that both known genes and candidate lincRNAs strongly associate with EFT and can be used to distinguish EFT from other childhood tumors. Moreover, many lincRNAs are seen on both the poly-A selected track and on the random hexamer primed total RNA expression profile, indicating that the transcripts are multi-exonic and poly-adenylated. Importantly, regions of EFT-associated lincRNA expression may indicate regions that are deregulated by EWS-FLI1 in these tumors. We also focused on lincRNAs with differential expression in an EFT cell line pair (CHLA-9 and −10) derived, respectively, from patient-matched primary and metastatic tumors. RNA-seq determined ∼160,000 regions representing ∼16MB of genomic sequence as being at least 3-fold differentially expressed between the two cell lines. A higher threshold of at least 10-fold expression difference still revealed ∼20,000 regions representing ∼2MB of genomic sequence. In addition, the proportion of differentially-expressed intergenic transcripts was higher in CHLA-9 (18.1%) versus CHLA-10 (11.2%). Annotated transcripts with at least 10-fold expression in CHLA-10 are enriched in plasma membrane and adhesion-related functions, and this is consistent with metastatic behavior. Genes most highly expressed in CHLA-10 include ANXA5, FABP3, and HIST1H1A, whereas genes with much lower expression include CXCL14, MCTP2, and TRIM22. These genes, along with lincRNAs, may play important roles in disease progression and drug resistance. CONCLUSIONS: We have identified several large intergenic noncoding (linc)RNAs that are highly and differentially expressed by EFT. We hypothesize that these lincRNAs may be novel therapeutic targets in EFT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4087.

225 FOLLICULAR DIAMETER, OVULATION RATE, AND LH RECEPTOR GENE EXPRESSION IN NELLORE COWS

The aim of the present experiment was to verify the relationship among follicular diameter, ovulation rate, and gene expression of LH receptor (LHR) isoforms in order to know whether these aspects could or could not influence ovulation rates in Nellore cows. In Experiment 1, at a random stage of the estrous cycle (Day 0), Nellore cows (n = 53) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, São Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB; i.m. Estrogin®; Farmavet, São Paulo, Brazil). On Day 8, PGF2 (150 μg d-cloprostenol; Prolise® ARSA S.R.L., Buenos Aires, Argentina) was administered i.m. and the device was removed. Twenty-four hours after device removal, cows were treated i.m. with EB (1.0 mg) and, 48 h afterwards, ovulation was determined by ultrasonography (US; Aloka 900, Tokyo, Japan). Three days after ovulation, follicular growth was observed daily by US and cows were randomly allocated into 3 groups according to follicular diameter (mm) [G1 (7.0-8.0), G2 (8.1-9.0), and G3 (9.1-10.0)] to receive 6.25 mg of LH (i.m. Lutropin®-V, Bioniche, Belleville, Ontario, Canada), which corresponds to twice the minimum ovulatory dose (3.12 mg) as determined in a preliminary experiment. The results were analyzed by logistic regression (PROC GEN MOD, SAS Institute, Cary, NC). The ovulation rates were 9 (2/21), 36 (8/22), and 90% (9/10) for G1, G2, and G3, respectively. There were significant differences when comparing G1 v. G3 (P &lt; 0.01), G2 v. G3 (P &lt; 0.02), and G1 v. G2 (P &lt; 0.03). In Experiment 2, granulosa and theca cells from Nellore cows were recovered from follicles obtained in a local abattoir and submitted to total RNA extraction and expression of LHR isoforms (LHR-B3, LHR-B4, LHR-B5, and LHR-B6) by semiquantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPD) as the internal control. Follicles were dissected, measured with a paquimeter, and allocated in 3 groups according to follicular diameter (mm): A (8.0-9.0), B (9.1-10.0), and C (10.1-11.0). Considering that follicles measured with paquimeter are on average 1.0 mm larger than those measured by US, Groups A, B, and C correspond to Groups G1, G2, and G3 (Experiment 1). In order to select only nonatretic (healthy) follicles, the E2/P4 &gt;1.0 ratio was used. Therefore, from a total of 400 ovaries, only 5, 4, and 4 granulosa (n = 13) and 7, 8, and 8 theca samples (n = 23) from Groups A, B, and C, respectively, were obtained. The data were analyzed by ANOVA and Pearson’s correlation. There were no significant differences in total LHR expression (LHR-B3 + LHR-B4 + LHR-B5 + LHR-B6) in theca cells from Groups A, B, and C. However, in granulosa cells, follicles from Group A had lower LHR expression (16.5; mRNA LHR/mRNA GAPD) compared with Group C (37.6; P &lt; 0.05). There was a positive correlation between expression of LHR-B5 and LHR-B6 isoforms and an increase in follicular diameter. In conclusion, these preliminary results indicate that ovulatory capacity in Nellore cattle is related to an increase in follicular diameter and LHR expression in granulosa cells. R. A. L. Simões, R. A. Satrapa, and A. C. S. Castilho are recipients of fellowship and funding from FAPESP (São Paulo, Brazil).

Neuronal in vitro models for the estimation of acute systemic toxicity

The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA A receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro–in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Vascular Endothelial Growth Factor Isoform and Receptor Expression During Compensatory Lung Growth

Vascular endothelial growth factor (VEGF) is required for blood vessel formation during lung growth and repair. Alteration of VEGF isoform expression has been demonstrated in response to fetal tracheal occlusion and in models of lung injury. The purpose of this study was to investigate VEGF expression during compensatory lung growth in the mouse. Under general anesthesia, adult mice underwent left thoracotomy with (n = 5) or without (sham, n = 5) pneumonectomy. The right lungs were harvested at 1, 3, and 7 d after the operation. Lung-to-body weight ratio as well as total DNA and protein content were measured. VEGF protein expression was analyzed by Western blot and ELISA. VEGF isoform expression was evaluated using semi-quantitative PCR followed by Imagequant optical densitometry. Values were compared by Student's t-test and ANOVA using Fisher's protected least significant difference post-hoc test where appropriate. Compensatory lung growth was observed as measured by increases in right lung-to-body weight ratio and in DNA and protein content. Total VEGF RNA and protein expression did not change after pneumonectomy. However, on post-operative day 1, there was a decrease in the relative percentage of VEGF188 mRNA (P < 0.01), and an increase in the relative percentage of VEGF164 mRNA (P = 0.05). At 3 d postpneumonectomy, low relative VEGF188 expression persisted (P < 0.05), VEGF164 expression normalized, and relative VEGF120 expression increased (P < 0.01). Isoform expression in the pneumonectomy animals was identical to sham animals by the seventh d. There were no differences observed in VEGF receptor expression. During compensatory lung growth, we have observed an early postoperative reversion of VEGF isoform expression to the pattern seen during fetal lung development and in lung injury models.

The Effects of Long-term Smoking on Endothelial Nitric Oxide Synthase mRNA Expression in Human Platelets as Detected With Real-time Quantitative RT-PCR

Endothelium-derived nitric oxide (NO) plays an important role in the prevention of platelet aggregation and adhesion to the vascular wall. Endothelial nitric oxide synthase (eNOS) and L-arginine/NO pathway are both present in human platelets. Platelet-derived NO inhibits excessive activation and aggregation of platelets. However, the expression level of the eNOS gene in human platelets has yet to be elucidated. The current study investigates the individual expression level of platelet eNOS mRNA using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection method. eNOS mRNA expression was examined in platelets isolated from 50 subjects: 11 male smokers, 15 male nonsmokers, and 24 female non-smokers. After extraction of platelet total RNA, eNOS (target) and GAPDH (internal control) mRNA expression levels were quantitated using real-time RT-PCR. The expression levels of eNOS mRNA (relative copy numbers) were significantly lower in male smokers (59+/-17) than in male nonsmokers (195+/-71, P < .03), and higher in female nonsmokers (285+/-60) than in the male nonsmokers (195+/-71, P < .03). By multiple linear regression analysis, cigarette smoking (P = .008) and diabetes mellitus (P = .047) were found to be significantly negative predictors, and antioxidant (vitamin E) treatment (P = .01) was a significantly positive predictor of platelet eNOS mRNA expression. Age, other medications, and other risk factors for coronary artery disease were not significant. Using this method, eNOS mRNA abundance in human platelets was detected and quantitated in real-time. The intraplatelet eNOS mRNA expression levels were significantly decreased in cigarette smokers. Low platelet NO synthesis in smokers may result in the augmentation of platelet aggregation and thrombus formation, developing into acute coronary syndromes.

Identification of an Aromatase Haplotype That Is Associated with Gene Expression and Postmenopausal Osteoporosis

Osteoporosis has a significant genetic component. The aromatase-dependent conversion of androgenic precursors is the main source of estrogens in postmenopausal women. The objective of the investigation was to study the relationship of a set of single nucleotide polymorphisms (SNPs) of the aromatase gene with osteoporosis and determine their functional influence on gene transcription. This was a case-control study including 135 women with vertebral fractures due to postmenopausal osteoporosis and 312 controls. Alleles at four SNPs situated between exons I.2 and 3 were determined by Taqman assays. Total aromatase RNA and differential allelic-specific expression were studied by RT-real time PCR in adipose tissue samples taken from 50 individuals. The SNPs studied were in strong linkage disequilibrium. A common haplotype, present in about half of the population, was identified as being associated with an increased risk of fractures (odds ratio 1.8, 95% confidence interval 1.2-2.8, P = 0.006). There was evidence of differential allelic expression. In heterozygous individuals, transcripts bearing T alleles at rs700518 SNP (which were included in the risk haplotype) were less abundant than those with the alternative C alleles (P < 0.001). Total aromatase expression was four times lower in fat samples from individuals who were homozygotes for the unfavorable alleles than in the opposite homozygotes (P = 0.007). A common haplotype of aromatase associated with gene expression is also associated with the risk of osteoporotic vertebral fractures in postmenopausal women. These data are in line with the hypothesis that the aromatase-dependent synthesis of estrogens plays an important role in bone homeostasis in postmenopausal women.

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3'UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.

Evaluation of gene expression analysis using RNA-targeted partial genome arrays

Highly parallel cDNA targeting microarrays have been established over the last years as the quasi-standard for genome wide expression profiling in pro- and eukaryotes. Protocols for the direct detection of RNA or aRNA (amplified RNA) are currently emerging. This allows to circumvent the bias introduced by enzymatic target molecule preparation. To systematically evaluate the extent of non-specific target binding on oligonucleotide microarrays designed for total RNA expression profiling, a model system of 70-mer probes targeting genes involved in magnetosome formation ( mam genes) of the bacterium Magnetospirillum gryphiswaldense was established utilizing wild-type strain MSR-1 and an isogenic deletion mutant MSR-1B that lacks all mam genes. An optimized protocol for the direct chemical labelling of total cellular RNAs was used. A linear correlation between the amount of applied RNA and the mean global background intensity was found which enables a simple and unbiased way of normalizing the data. The results obtained with the mam deletion mutant MSR-1B revealed a significant number of false positive signals, even under optimal hybridization conditions. This indicates a high degree of non-specific binding in microarray experiments when using longer oligo- or polynucleotides and RNA as target molecule. Comparative microarray analysis of an MSR-1B culture and two MSR-1 wild-type cultures grown under different conditions was done via a three-colour hybridization assay. The additional information provided by the MSR-1B transcriptome revealed differential gene expression in the two MSR-1 cultures, which was otherwise undetectable.