Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression in placentation of farm animals: An overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish - a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] - for humans, and tissue-specific genes database (TiSGeD) - for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta. (C) 2011 Elsevier B.V. All rights reserved.

Transference of a crystal protein gene from bacillus thuringiensis and its expression in bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a δ-endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5α

Acute and short-term insulin-induced molecular adaptations of GLUT2 gene expression in the renal cortex of diabetic rats

Increased GLUT2 gene expression in the renal proximal tubule of diabetic rats is an adaptive condition, which may be important in the diabetic nephropathy development. We investigated the effects of insulin treatment upon the renal GLUT2 overexpression of diabetic rats. Acute treatment, surprisingly, induced a rapid further increase in GLUT2 mRNA content. Twelve hours after insulin injection, GLUT2 mRNA was twice the value of saline-injected rats (P < 0.001), when GLUT2 protein remained unchanged. In response to short-term treatment, both GLUT2 mRNA and protein were increased in 1-day treated rats (P < 0.05 versus saline-injected), decreasing after that, and reaching, within 6 days, values close to those of non-diabetic rats. Concluding, insulin treatment induced: initially, an additional upregulation of GLUT2 gene expression, involving posttranscriptional modulation; thereafter, downregulation of GLUT2 expression, which returns to non-diabetic levels. The former may be related to increased insulin concentration, the latter may be due to glycemic control. © 2005 Elsevier B.V. All rights reserved.

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1null mice, the peritonitis response was exaggerated (∼40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased ai both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1null mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection. Copyright © 2006 by The American Association of Immunologists, Inc.

Tamoxifen inhibits transforming growth factor-α gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T3), 17β-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-α gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-α mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-α mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-α mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-α mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-α upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-α, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER α and β; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2. ©2008, Editrice Kurtis.

Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR

Background: Rust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions. Findings. We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses. Conclusions. For tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments. © 2010 Laia et al; licensee BioMed Central Ltd.

Expression of pro-and-anti-apoptotic antigens in the cerebellum of dogs naturally infected with canine distemper virus

Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The present work investigated apoptosis in white and gray matter (granular layer) in the cerebellum of naturally infected dogs by the analysis of the expression of the pro-apoptotic antigens caspase - 2 and - 3, b(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL-staining) positivity, annexin-V immunodetection, and the presence of the anti-apoptotic antigens, BCl-2 and p53. Cerebellum specimens were obtained from the Laboratory of Animal Pathology, from 1995 to 2009, and the 5-μm thick fragments were stained both with hematoxylin-eosin and Shorr. All samples were diagnosed as positive for CDV genome by reverse transcriptase polymerase chain reaction targeting the nucleocapsid gene. The anti-apoptotic pathways evidenced in this study were BCl-2 and p53 proteins that were intensively detected in cerebellum of CDV positive slides (40-80% of labeled cells/mm2). In addition, the apoptosis markers annexin-V and TUNEL are directly correlated among the same samples (80 and 40% of labeled cells, respectively). This is the first description of p53 and annexin-V expression, characterized as anti-apoptotic and apoptotic proteins, involvement in canine natural cases of CDV infections.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Experimental hyperthyroidism decreases gene expression and serum levels of adipokines in obesity

Aims. To analyze the influence of hyperthyroidism on the gene expression and serum concentration of leptin, resistin, and adiponectin in obese animals. Main Methods. Male Wistar rats were randomly divided into two groups: control (C)fed with commercial chow ad libitumand obese (OB)fed with a hypercaloric diet. After group characterization, the OB rats continued receiving a hypercaloric diet and were randomized into two groups: obese animals (OB) and obese with 25g triiodothyronine (T3)/100BW (OT). The T3 dose was administered every day for the last 2 weeks of the study. After 30 weeks the animals were euthanized. Samples of blood and adipose tissue were collected for biochemical and hormonal analyses as well as gene expression of leptin, resistin, and adiponectin. Results. T3 treatment was effective, increasing fT3 levels and decreasing fT4 and TSH serum concentration. Administration of T3 promotes weight loss, decreases all fat deposits, and diminishes serum levels of leptin, resistin, and adiponectin by reducing their gene expression. Conclusions. Our results suggest that T3 modulate serum and gene expression levels of leptin, resistin, and adiponectin in experimental model of obesity, providing new insights regarding the relationship between T3 and adipokines in obesity. Copyright © 2012 Renata de Azevedo Melo Luvizotto et al.

The effect of laminin-1-doped nanoroughened implant surfaces: Gene expression and morphological evaluation

Aim. This study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model. Materials and Methods. Nanostructured implants were coated with laminin-1 (test; dilution, 100 g/mL) and inserted into the rabbit tibiae. Noncoated implants were used as controls. After 2 weeks of healing, the implants were removed and subjected to morphological analysis using scanning electron microscopy (SEM) and gene expression analysis using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. SEM revealed bony tissue attachment for both control and test implants. Real-time RT-PCR analysis showed that the expression of osteoblast markers RUNX-2, osteocalcin, alkaline phosphatase, and collagen I was higher (1.62-fold, 1.53-fold, 1.97-fold, and 1.04-fold, resp.) for the implants modified by laminin-1 relative to the control. All osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows: tartrate-resistant acid phosphatase (1.67-fold), calcitonin receptor (1.35-fold), and ATPase (1.25-fold). The test implants demonstrated higher expression of inflammatory markers interleukin-10 (1.53-fold) and tumour necrosis factor-α (1.61-fold) relative to controls. Conclusion. The protein-doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface. © 2012 Humberto Osvaldo Schwartz-Filho et al.

Morphology, sex ratio and gene expression of Day 14 in vivo and in vitro bovine embryos

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo-and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down-and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality. © 2013 CSIRO.

Detecting Loci under Recent Positive Selection in Dairy and Beef Cattle by Combining Different Genome-Wide Scan Methods

As the methodologies available for the detection of positive selection from genomic data vary in terms of assumptions and execution, weak correlations are expected among them. However, if there is any given signal that is consistently supported across different methodologies, it is strong evidence that the locus has been under past selection. In this paper, a straightforward frequentist approach based on the Stouffer Method to combine P-values across different tests for evidence of recent positive selection in common variations, as well as strategies for extracting biological information from the detected signals, were described and applied to high density single nucleotide polymorphism (SNP) data generated from dairy and beef cattle (taurine and indicine). The ancestral Bovinae allele state of over 440,000 SNP is also reported. Using this combination of methods, highly significant (P<3.17×10-7) population-specific sweeps pointing out to candidate genes and pathways that may be involved in beef and dairy production were identified. The most significant signal was found in the Cornichon homolog 3 gene (CNIH3) in Brown Swiss (P = 3.82×10-12), and may be involved in the regulation of pre-ovulatory luteinizing hormone surge. Other putative pathways under selection are the glucolysis/gluconeogenesis, transcription machinery and chemokine/cytokine activity in Angus; calpain-calpastatin system and ribosome biogenesis in Brown Swiss; and gangliosides deposition in milk fat globules in Gyr. The composite method, combined with the strategies applied to retrieve functional information, may be a useful tool for surveying genome-wide selective sweeps and providing insights in to the source of selection.