Cloning of glucocorticoid-regulated genes in C6/ST1 rat glioma phenotypic reversion

The C6 rat glioma cell line is responsive to glucocorticoid hormones. C6 variants that are hyper-responsive (ST1) and resistant (P7) to hormone treatment have been derived previously. Glucocorticoid treatment of ST1 cells leads to complete reversion of the transformed phenotype and loss of tumorigenic potential. Production of C type retrovirus particles is also induced by glucocorticoids in ST1 cells. Cloning of the genes regulated by glucocorticoids in this cell system was used here as a strategy to uncover the gene products involved in the transformed-to-normal phenotypic change. Construction of a cDNA library from glucocorticoid-treated ST1 cells and screening by differential hybridization resulted in the isolation of three cellular sequences that code for rat metallothioneins (C27 and C41) and α1-acid glycoprotein (C36). Northern blot analysis revealed that expression of these genes was dramatically induced by hydrocortisone in ST1 but not in P7 cells. Viral genomic RNA was used to isolate and characterize retrovirus-related sequences that could also be responsible for the phenotypic reversion phenomenon.

Polishable and Renewable DNA Hybridization Biosensors

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.

Influence of the diet on in situ disappearance of dry matter, organic matter, and neutral detergent fiber of hydrolysed sugar cane bagasse

The effects of two diets based on hydrolysed sugarcane bagasse (HSB) and whole cottonseed (WCS), with or without oat hay, were analyzed for the in situ disappearance of dry matter (DM), organic matter (OM) and neutral detergent fiber (NDF) of HSB. Six mature castrated rams with a permanent T ruminai cannula were used in a complete randomized split plot design. The incubation times were 3, 6, 9, 12, 24, 48 and 72h. The diet with oat hay showed higher disappearance indexes for the NDF fraction. Furthermore, the maximum degradation of HSB constituents was reached around 48h of incubation. The diets were T1=64% hydrolyzed sugarcane + 36% whole cottonseed and T2=14% hydrolyzed sugarcane bagasse + 36% cottonseed + 50% oat hay.

Intrachromosomal distribution of telomeric repeats in Eumops glaucinus and eumops perotis (Molossidae, Chiroptera)

The location of chromosomal telomeric repeats (TTAGGG)(n) was investigated in two species of the Molossidae family, Eumops glaucinus and Eumops perotis. The diploid chromosome number (2n) is 40 in E. glaucinus and 48 in E. perotis and the fundamental numbers (FN) are 64 and 58, respectively. It has been suggested that the E. glaucinus karyotype has evolved from the E. perotis karyotype through Robertsonian fusion events. In the present study, the telomeric sequences were detected at the termini of chromosomes in both species. In addition, E. glaucinus also displayed telomeric repeats in centromeric and pericentromeric regions in almost all biarmed chromosomes. Conversely, in E. perotis pericentromeric signals were only observed in two biarmed chromosomes. In both E. glaucinus and E. perotis, such telomeric sequences were observed as part of the heterochromatin. The interstitial sites of telomeric sequences suggest that they are remnants of telomeres of ancestral chromosomes that participated in the fusion event.

Molecular and cytogenetic analysis of the telomeric (TTAGGG)n repetitive sequences in the Nile tilapia, Oreochromis niloticus (Teleostei: Cichlidae)

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.

Hodgkin disease in adult and juvenile groups from two different geographic regions in Brazil: Characterization of clinicopathologic aspects and relationship with Epstein-Barr virus infection

We analyzed clinicopathologic data, immunophenotype, and Epstein-Barr virus (EBV) status in 96 cases of Hodgkin disease (HD) in juveniles (younger than 20 years) and adults (20 years or older) from 2 distinctive states in Brazil. We studied 34 juvenile (group 1) and 16 adult (group 2) cases from Ceara and 31 juvenile (group 3) and 15 adult (group 4) cases from São Paulo. Ceara has a socioeconomic profile similar to a developing country; São Paulo is in better economic condition. Mixed cellularity (MC) was the major histologic subtype among groups 1 (22 [65%]), 3 (21 [68%]), and 4 (7 [47%]); nodular sclerosis (NS) was more frequent in group 2 (8 [50%]). EBV infection was observed in 61 cases (64%), including the following (among others): group 1, MC, 22 (65%) and NS, 4 (12%); group 2, NS, 3 (19%) and MC, 2 (12%); group 3, MC, 16 (52%) and NS, 1 (3%); and group 4, MC, 7 (47%). There was predominance of EBV+ HD cases in group 1 compared with group 3. HD in Brazilian patients is highly associated with EBV infection, but geographic differences reflect histologic subtypes and age distribution.

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P=.017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P=.037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces, Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 metasubmetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.

p16INK4A immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16 INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.

Chromosome localization of the ribosomal genes 18S and 5S in four stocks of rainbow trout (Oncorhynchus mykiss) from cultivated and naturalized stocks in Brazil

In the present study, fluorescence in situ hybridization (FISH) was employed to determine the chromosomal location of genes 18S rDNA and 5S rDNA in four rainbow trout stocks. In specimens from the stocks of Núcleo Experimental de Salmonicultura de Campos do Jordão and Gavião river, 18S genes were located at a subterminal position in the long arms of two submetacentric chromosomes, whereas in specimens from stocks of Mount Shasta and Teresópolis they were found in the short arms. In all analyzed stocks, 5S genes were located in two chromosome pairs. In a subtelocentric pair, 5S genes were present in the short arms and, in the other submetacentric pair, 5S genes were at an interstitial position. In the latter, 18S and 5S genes were contiguous. Taking into account that both 18S and 5S rDNA genes have been localized in the short arm of a submetacentric chromosome in almost all rainbow trout samples so far studied, the presence of such genes in the long arm, as seen in the samples from Núcleo Experimental de Salmonicultura de Campos do Jordão and Gavião river, supports the hypothesis of a pericentric inversion involving this chromosome segment in the ancestor line of these stocks. The observed polymorphism allowed the identification of a very useful genomic marker, and may therefore constitute an important tool in the genetic management of rainbow trout stocks.

An ellipsometric study of manganese oxide films: In situ characterization of the deposition and electroreduction of MnO2

The electrodeposition of manganese oxide films onto a platinum substrate was investigated by means of in situ ellipsometry. In the thickness range from 0 to 150 nm, the anodic oxide behaves as an Isotropic single layer with optical constants that are independent of thickness. Deviations at higher thickness are explained in terms of anisotropic properties of the film. The electroreduction of thin films (up to ca. 150 nm) in an alkaline electrolyte leads to a decrease in both the refractive index and the extinction coefficient and is accompanied by a thickness increase of ca. 10%. The Mn(IV) to Mn(III) conversion takes place from the oxide/electrolyte interface inwards. © 2004 The Electrochemical Society. All rights reserved.

Localization of rDNA sites in holocentric chromosomes of three species of triatomines (Heteroptera, Triatominae).

Chromatin organization in the holocentric chromosomes of three triatomines species was cytologically studied by fluorescent in situ hybridization with a 45S rDNA probe of Drosophila melanogaster to localize ribosomal genes. In Triatoma tibiamaculata, metaphases I showed telomeric highlights in a single, larger bivalent. In T. protacta, hybridization was detected in one of the telomeres of an autosomal chromosome. In T. platensis, there were highlights in a single, smaller chromosome (X chromosome). The results obtained did not agree with the expected localization of rDNA genes in the sex chromosomes of triatomines, as demonstrated by silver impregnation, and suggest that the chromosome reorganization that occurred in this group during evolution may be a more important mechanism involved in rDNA distribution.

Genetic alterations in benign lesions: Chronic gastritis and gastric ulcer

Aim: To investigate the occurrence of chromosome 3, 7, 8, 9, and 17 aneuploidies, TP53 gene deletion and p53 protein expression in chronic gastritis, atrophic gastritis and gastric ulcer, and their association with H pylori infection. Methods: Gastric biopsies from normal mucosa (NM, n = 10), chronic gastritis (CG, n = 38), atrophic gastritis (CAG, n = 13) and gastric ulcer (GU, n = 21) were studied using fluorescence in situ hybridization (FISH) and immunohistochemical assay. A modified Giemsa staining technique and PCR were used to detect H pylori. An association of the gastric pathologies and aneuploidies with H pylori infection was assessed. Results: Aneuploidies were increasingly found from CG (21%) to CAG (31%) and to GU (62%), involving mainly monosomy and trisomy 7, trisomies 7 and 8, and trisomies 7, 8 and 17, respectively. A significant association was found between H pylori infection and aneuploidies in CAG (P = 0.0143) and GU (P = 0.0498). No TP53 deletion was found in these gastric lesions, but p53-positive immunoreactivity was detected in 45% (5/11) and 12% (2/17) of CG and GU cases, respectively. However, there was no significant association between p53 expression and H pylori infection. Conclusion: The occurrence of aneuploidies in benign lesions evidences chromosomal instability in early stages of gastric carcinogenesis associated with H pylori infection, which may confer proliferative advantage. The increase of p53 protein expression in CG and GU may be due to overproduction of the wild-type protein related to an inflammatory response in mucosa. © 2006 The WJG Press. All rights reserved.

In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.