Expression of apoptotic and cell proliferation regulatory proteins in keratoacanthomas and squamous cell carcinomas of the skin

The aim of this study was to investigate the expression of p53, caspase-3, bcl-2, MIB-1, and PCNA to validate more objective methods to differentiate squamous cell carcinoma and keratoacanthoma, as well as to understand their pathogenesis with accuracy. A total of 52 cases of histopathologically diagnosed keratoacanthoma in the proliferative stage and 56 cases of well-differentiated squamous cell carcinoma were selected in this study. The expression was evaluated by means of immunohistochemistry. Bcl-2 immunoreactivity was weak or absent in the majority of cases, either in squamous cell carcinoma or in keratoacanthoma. PCNA-positive cells did not show differences between two lesions evaluated. on the other hand, MIB-1 was statistically significant (p<0.05) between squamous cell carcinomas and keratoacanthomas. Moreover, p53 and caspase-3 were overexpressed in squamous cell carcinomas. Together, these results suggest that the biological behavior of the well-differentiated squanous cell carcinomas of the skin may be associated with cellular proliferation and/or deregulation of cell death, indicated by increased expression of the MIB-1 and apoptotic proteins p53 and caspase-3, respectively. (C) 2007 Elsevier GrnbH. All rights reserved.

Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells

Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500 ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500 ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500 ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500 ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation. (C) 2011 Elsevier B.V. All rights reserved.

The role of interleukin-10 in the differential expression of interleukin-12p70 and its beta 2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects

Paracoccidioidomycosis patients present an antigen-specific Th1 immunosuppression. To better understand this phenomenon, we evaluated the interleukin (IL)-12 pathway by measuring IL-12p70 production and CD3(+) T cell expression of the IL-12 receptor (IL-12R)beta1/beta2 chains, induced with the main fungus antigen (gp43) and a control antigen, from Candida albicans (CMA). We showed that gp43-induced IL-12p70 production and IL-12Rbeta2 expression were significantly decreased in acute and chronic patients as compared to healthy subjects cured from PCM or healthy infected subjects from endemic areas. Interestingly, the healthy infected Subjects had higher gp43-induced IL12p70 production and beta2 expression than the cured subjects. The addition of a neutralizing anti-IL-10 antibody to the cultures increased IL12p70 levels and beta2 expression in acute and chronic patients to levels observed in Cured subjects. Conversely, addition of the cytokine IL-10 strongly inhibited both parameters in the latter group. In conclusion, we have shown that paracoccidioidomycosis-related Th1 immunosuppression is associated with down-modulation of the IL-12 pathway, that IL-10 may participate in this process, and that patients cured from paracoccidioidomycosis may not fully recover their immune responsiveness. (C) 2004 Elsevier B.V. All rights reserved.

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.

Effects of β-glucan extracted from Agaricus blazei on the expression of ERCC5, CASP9, and CYP1A1 genes and metabolic profile in HepG2 cells

The polysaccharide β-glucan has biological properties that stimulate the immune system and can prevent chronic pathologies, including cancer. It has been shown to prevent damage to DNA caused by the chemical and physical agents to which humans are exposed. However, the mechanism of β-glucan remains poorly understood. The objective of the present study was to verify the protective effect of β-glucan on the expression of the genes ERCC5 (involved in excision repair of DNA damage), CASP9 (involved in apoptosis), and CYP1A1 (involved in the metabolism of xenobiotics) using real-time polymerase chain reaction and perform metabolic profile measurements on the HepG2 cells. Cells were exposed to only benzo[a]pyrene (B[a]P), β-glucan, or a combination of B[a]P with β-glucan. The results demonstrated that 50 μg/mL β-glucan significantly repressed the expression of the ERCC5 gene when compared with the untreated control cells in these conditions. No change was found in the CASP9 transcript level. However, the CYP1A1 gene expression was also induced by HepG2 cells exposed to B[a]P only or in association with β-glucan, showing its effective protector against damage caused by B[a]P, while HepG2 cells exposed to only β-glucan did not show CYP1A1 modulation. The metabolic profiles showed moderate bioenergetic metabolism with an increase in the metabolites involved in bioenergetic metabolism (alanine, glutamate, creatine and phosphocholine) in cells treated with β-glucan and to a lesser extent treated with B[a]P. Thus, these results demonstrate that the chemopreventive activity of β-glucan may modulate bioenergetic metabolism and gene expression. © 2013 The Author(s).

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells. © CSIRO 2013.

Compartmentalized gene expression of toll-like receptors 2, 4 and 9 in the brain and peripheral lymphoid organs during canine visceral leishmaniasis

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

Expression of MyoD, myogenin, myostatin and Hsp70 transcripts in chicken embryos submitted to mild cold or heat

The expression of the MyoD, myogenin, myostatin and Hsp70 genes was estimated in chicken embryos submitted to mild cold (36 +/- 0.5degreesC) or heat (44 +/- 0.5degreesC) for 1 h. 2. Marked decreases in MyoD, myogenin and myostatin transcript levels were observed in embryos exposed to high temperature, contrasting to the higher expression of the Hsp70 mRNA detected in heat-stressed embryos. 3. The exposure of chicken embryos to low temperature significantly affected only the abundance of myogenin mRNA. 4. These findings suggest that myogenic proliferation and differentiation events are compromised by variations in environmental temperature during avian embryogenesis. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Molecular mapping of the viral determinants of systemic wilting induced by a Lettuce mosaic virus (LMV) isolate in some lettuce cultivars

The isolate AF199 of Lettuce mosaic virus (LMV, genus Potyvirus) causes local lesions followed by systemic wilting and plant death in the lettuce cultivars Ithaca and Vanguard 75. Analysis of the phenotype of virus chimeras revealed that a region within the PI protein coding region (nucleotides 112-386 in the viral genome) and/or another one within the CI protein coding region (nucleoticles 5496-5855) are sufficient together to cause the lethal wilting in Ithaca, but not in Vanguard 75. This indicates that the determinants of this particular symptom are different in these two lettuce cultivars. The wilting phenotype was not directly correlated with differences in the deduced amino acid sequence of these two regions. Furthermore, transient expression of the LMV-AF 199 proteins, separately or in combination, did not induce local necrosis or any other visible reaction in the plants. Together, these results Suggest that the systemic wilting reaction might be Clue to RNA rather than protein sequences. (c) 2004 Elsevier B.V. All rights reserved.

Expression of nonclassical molecule human leukocyte antigen-G in oral lesions

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Presence and morphology of the molar tubercle according to dentition, hemi-arch and sex

The purpose of this study was to assess the presence and the degree of expression of the molar tubercle according to sex, dentition and hemi-arches. Study casts of 126 patients were assessed, and those were under orthodontic treatment at the University of Franca, UNIFRAN; they were from both sexs, from 4 to 13 years old. The upper second primary molars and the upper first permanent molars, from both sides, were evaluated regarding the presence and the degree of expression of the molar tubercle. For an association study, the qui-square test was utilized. The concordance about the presence or absence of the molar tubercle according to dentition, hemi-arch and sex, was estimated by the Kappa Statistics. There was a sexual dimorphism concerning the presence/absence of the molar tubercle (p=0.009), however there was no significant association between the degree of expression of the tubercle and the sex (p=0.791). The molar tubercle was more frequently observed in the male sex, in upper second primary molars and in the form of depression. There was a significant and "moderate" concordance between the left and right sides in primary dentition (k=0.596), there was a "good" concordance in permanent dentition (k=0.708) and a "weak" and significant concordance between the presence of the molar tubercle and dentition (k=0.207).

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

On the Apterous Line of the Termite Velocitermes heteropterus (Isoptera: Termitidae): Developmental Pathways and Cellulose Digestion

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

Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA

Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. Through a PCR methodology, the gsn cDNA coding for the N. crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced. The product of the cDNA seems to be the only glycogen synthase present in N. crassa. Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases. Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level. Expression of the gsn is highly regulated at the transcriptional level. Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. of these,14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.