AgNOR Staining Research Articles

Diagnostic and Prognostic Significance of AgNOR Counts in Before and After Radiotherapy-Treated Cervical Carcinogenesis is Compared with Cervical Cytology and Histology

Nucleolar organizer region-associated proteins are argyrophilic and visualized by silver stains. There are two types of dots are present. One is the presence of single dots, and another is pleomorphic dots. Pleomorphic dots are few, increase with severity of cervical lesions and hence indicate a more severe prognosis. The present study aimed at investigating the diagnostic value of both pleomorphic and single AgNOR dots in cervical carcinogenesis in different cytological stages of cervix before and after radiotherapy. The results were correlated with cervical cytology and histopathology. The study was conducted on the samples of 37 untreated cases of patients coming to the outpatient door of AHRCC. In two slides, samples were taken for PAP smear cytology and AgNOR staining. From histopathological detection, various grades of squamous cell carcinoma of the cervix were determined. Samples were divided into cytological/histological normal/controls (N = 10), inflammation (N = 7), LSIL (N = 8), HSIL (N = 7) and invasive carcinoma (N = 5). AgNOR staining was performed slides containing cervical smears. Samples from same patients [LSIL (N = 8), HSIL (N = 7) and invasive carcinoma (N = 5)] that were subjected to radiotherapy were assessed again for AgNOR scoring. The rate of increase in cell counts of pleomorphic dots was higher than the rate of increase in cell counts of single dots, and there is a positive and significant correlation of cell counts of both pleomorphic and single dots with disease severity. The results were correlated with PAP stained cervical cytology and histopathology to find out the diagnostic and prognostic significance. In squamous cells of the normal/control group, the AgNORs were fine and centrally placed in the nucleoli. AgNORs in the case of LSIL, HSIL and invasive carcinoma of the cervix before radiotherapy were large and variable in size and shape. The cases studied for AgNORs count after 6 month of radiotherapy showed less number of AgNORs dots which were single and fewer in numbers as compared to the pre-radiation group. AgNOR pleomorphic dot counts increase with severity of cervical lesions and may be used as an effective tool along with cervical cytology and histopathology for detection of cervical carcinogenesis.

The Karyotype of Salvator merianae (Squamata, Teiidae): Analyses by Classical and Molecular Cytogenetic Techniques

In this study, we analyzed the karyotype of Salvator merianae (Teiidae) from the Brazilian semiarid region using different cytogenetic markers. Chromosomes were examined by classical (Giemsa and AgNOR staining) and molecular (FISH with ribosomal, telomeric, and microsatellite probes) cytogenetic approaches. S. merianae showed a diploid chromosome number of 2n = 38 (10 biarmed macrochromosomes + 28 microchromosomes). No sex-linked chromosome heteromorphisms were observed. Clusters of 18S/28S rDNA were localized in the terminal region of the long arm of pair 2. In addition to the typical telomeric signals, (TTAGGG)_n repeats were detected in the pericentromeric region of some macrochromosome pairs, which might indicate the occurrence of chromosomal rearrangements via chromosome fusions. Hybridization signals of the microsatellite probes (GA)_n, (GAA)_n, and (GAG)_n were uniformly distributed across all chromosomes, while (CA)_n, (CAA)_n, and (CAC)_n produced brighter signals in the telomeric and pericentromeric regions of specific chromosome pairs. The comparison with previous studies demonstrates that, despite the wide distribution of S. merianae, the macrostructure organization of the karyotype remained unchanged, showing stability in diploid number and chromosome morphology.

Effect of MGHPPGP Peptide Administered during the Post-Traumatic Period on Morphometric Parameters of the Neocortex and Hippocampus, Hepatocytes, and Epithelium of the Tongue and Small Intestine in Rats.

The effect of glyproline-containing peptide MGHPPGP (Met-Glu-His-Phe-Pro-Gly-Pro) was studied in experiments on male Wistar rats with modeled traumatic brain injury. The peptide was administered intraperitoneally in a dose of 1 mg/kg in 3 h and on days 2, 3, 4, 5 after injury. We evaluated morphometric parameters of the epithelial cells of the tongue, small intestine, and liver cells (AgNOR staining), neuronal layers II and V of the neocortex of the parietal lobe and hippocampal CA1 field (staining with gallocyanin) were evaluated in the post-traumatic period. Traumatic brain injury (TBI) was induced in rats by using the impact model (WDM; weight drop method). MGHPPGP peptide corrected the activity indicators of the nuclear organizer regions in epitheliocytes of the tongue.

Comparative cytogenetics of the ground frogs Eupsophus emiliopugini Formas, 1989 and E. vertebralis Grandison, 1961 (Alsodidae) with comments on their inter- and intraspecific chromosome differentiation.

South American frogs of the genus Eupsophus Fitzinger, 1843 comprise 10 species. Two of them, Eupsophus vertebralis Grandison, 1961 and E. emiliopugini Formas, 1989 belong to the Eupsophus vertebralis group, exhibiting 2n = 28. Fundamental number differences between these species have been described using conventional chromosome staining of few specimens from only two localities. Here, classical techniques (Giemsa, C-banding, CMA3/DAPI banding, and Ag-NOR staining), and fluorescence in situ hybridization (FISH, with telomeric and 28S ribosomal probes), were applied on individuals of both species collected from 15 localities. We corroborate differences in fundamental numbers (FN) between E. vertebralis and E. emiliopugini through Giemsa staining and C-banding (FN = 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes and Ag-NOR staining confirmed the active nucleolus organizer regions signal on pair 5 for both species. Nevertheless, one E. emiliopugini individual from the Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. Chromosomal rearrangements, rRNA gene dosage control, mobile NORs elements, and/or species hybridization process could be involved in this interpopulation chromosomal variation.

Odontogenic keratocyst: Analysis of recurrence by AgNOR, p53 and MDM2 profiling.

Background:Odontogenic keratocyst (OKC) is a clinical entity with characteristic microscopic features, high growth potential and propensity to recur. Aggressive behavior and higher tendency for recurrence have been attributed to greater proliferative activity of epithelial lining. The incidence of recurrence in various reported series ranges from 2.5% to 62%.Objectives:The objective of the study was to investigate the clinical behavior of OKC by evaluating p53, MDM2 expression, AgNOR staining and to ascertain if the expression of these markers correlate with the clinical outcome and tendency for recurrence.Materials and Methods:All recurrent and nonrecurrent OKCs from the archives were included, and sections were subjected to AgNOR staining, p53 and MDM2 immunohistochemical staining.Results and Conclusion:There was a significant difference in the staining pattern of MDM2 and AgNOR in the recurrent group as compared to the nonrecurrent group. The higher expression of these markers in recurrent lesions may be important in order to consider additional surgical interventions to improve prognosis.

The investigation of the antitumoral effect of Cornus mas L in mice with ehrlich solid tumor.

Cornus mas L is commonly used due to its anti-inflammatory, anti-carcinogenic and anti-oxidant properties. In the study, the effects of C. mas L extract on a solid tumor were examined in the Ehrlich solid tumor model developed in Balb/C type mice. Ehrlich acid tumor (EAT) cells (1x106 EAT cell) from the stock animal were injected subcutaneously (s.c.) through the nape of the mice. Treatment groups of solid tumor-induced animals received 100 mg/kg and 200 mg/kg of C. mas L extract intraperitoneally (i.p.) for 14 days. Tumor volumes and animal weights were found to be statistically significant compared to the control group (p < 0.05). AgNOR staining was performed in tumor tissues. Statistically significant differences were observed between the groups in terms of TAA/NA ratio (p < 0.05). Immunohistochemical and biochemical parameters were also evaluated. An estimation of tumor proliferation of the lung, liver, brain, kidney, testis and tumor antioxidant parameters viz. lipid peroxidation, reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) was made. Our study showed that the anti-tumor effect of C. mas L in assisted tumor development with EAT cells, was mediated by the enhancement of oxidative stress with multiple mechanisms (Tab. 6, Fig. 12, Ref. 38).

Cytogenetic analysis of Rhinella jimi (Stevaux, 2002) (Anura, Bufonidae) from northeastern Brazil

In this work we analyzed the karyotype of Rhinella jimi (Stevaux, 2002) (Anura, Bufonidae)from Picos (Piaui) in Northeastern Brazil. The chromosomes were examined using classicalcytogenetic approaches (Giemsa, C-banding, and Ag-NOR staining). This species has 2n = 22chromosomes, all metacentric or submetacentric. Heterochromatic segments were visualized atthe centromeric region and the nucleolus organizer regions (NOR) were restricted to terminalregions of the short arms in pair 7. There was no evidence of heteromorphic sex chromosomes.The chromosomal analysis of R. jimi allowed us to identify a karyotype that is similar to manyother species of Rhinella, in which the diploid number remains unchanged and without evidencesof structural rearrangements.

Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue

BackgroundVarious stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as ‘directed staining technologies’. New methodsThe argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature (‘unravel’) the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. Comparison with old methodsPrior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. ConclusionsThis method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

Evidence of genotoxicity and cytotoxicity of X-rays in the oral mucosa epithelium of adults subjected to cone beam computed tomography.

Evidence of genotoxicity and cytotoxicity of X-rays in the oral mucosa epithelium of adults subjected to cone beam computed tomography.

Karyotype Evolution and Distinct Evolutionary History of the W Chromosomes in Swallows (Aves, Passeriformes)

As in many other bird groups, data on karyotype organization and distribution of repetitive sequences are also lacking in species belonging to the family Hirundinidae. Thus, in the present study, we analyzed the karyotypes of 3 swallow species (Progne tapera, Progne chalybea, and Pygochelidon cyanoleuca) by Giemsa and AgNOR staining, C-banding, and FISH with 11 microsatellite sequences. The diploid chromosome number was 2n = 76 in all 3 species, and NORs were observed in 2 chromosome pairs each. The microsatellite distribution pattern was similar in both Progne species, whereas P. cyanoleuca presented a distinct organization. These repetitive DNA sequences were found in the centromeric, pericentromeric, and telomeric regions of the macrochromosomes, as well as in 2 interstitial blocks in the W chromosome. Most microchromosomes had mainly telomeric signals. The Z chromosome displayed 1 hybridization signal in P. tapera but none in the other species. In contrast, the W chromosome showed an accumulation of different microsatellite sequences. The swallow W chromosome is larger than that of most Passeriformes. The observed enlargement in chromosome size might be explained by these high amounts of repetitive sequences. In sum, our data highlight the significant role that microsatellite sequences may play in sex chromosome differentiation.

Paracentric Inversions Differentiate the Conservative Karyotypes in Two Centropomus Species (Teleostei: Centropomidae)

Centropomus is the sole genus of the Centropomidae family (Teleostei), comprising 12 species widely distributed throughout the Western Atlantic and Eastern Pacific, with 6 of them occurring in the Western Atlantic in extensive sympatry. Their life history and phylogenetic relationships are well characterized; however, aspects of chromosomal evolution are still unknown. Here, cytogenetic analyses of 2 Centropomus species of great economic value (C. undecimalis and C. mexicanus) were performed using conventional (Giemsa, Ag-NOR, and fluorochrome staining, C- and replication banding) and molecular (chromosomal mapping of 18S and 5S rDNA, H2A-H2B and H3 hisDNA, and (TTAGGG)_n repeats) approaches. The karyotypes of both species were composed of 48 solely acrocentric chromosomes (2n = 48; FN = 48), but the single ribosomal site was located in varying positions in the long arms of the second largest chromosome pair. Replication bands were generally similar, although conspicuous differences were observed in some chromosome regions. In both species, the histone H3 genes were located on 3 apparently homeologous chromosome pairs, but the exact position of these clusters differed slightly. Interspecific hisDNA and rDNA site displacements can indicate the occurrence of multiple paracentric inversions during the evolutionary diversification of the Centropomus genomes. Although the karyotypes remained similar in both species, our data demonstrate an unsuspected microstructural reorganization between them, driven most likely by a series of paracentric inversions.

Anti-Estrogenic and Anti-Cell Proliferative Effect of Allyl Isothiocyanate in Chemoprevention of Chemically Induced Mammary Carcinogenesis in Rats.

The anti-estrogenic and anti-cell proliferative effect of allyl isothiocyanate (AITC) was carried out by analyzing the status of sex hormones and its receptors and cell proliferative markers in chemically induced mammary carcinogenesis in rats. Mammary tumor was induced by a single dose of DMBA (25mg/rat) and MNU (50mg/kg bw) injected subcutaneously near mammary gland. RT-PCR, western blotting and immunohistochemical analysis of mammary tissues show an upregulation of ER-α, PR, aromatase, PCNA, cyclin D1 and AgNORs staining and down regulation of p53 expression as well as plasma estradiol, prolactin and testosterone levels increased in DMBA and MNU-induced tumor bearing rats. Oral administration of AITC at a dose of 20mg/kg bw restored the levels of sex hormones and its receptors, aromatase, cell proliferative markers and AgNORs staining near to normal levels. Molecular docking studies also supported these findings. The results suggest that anti-estrogenic and anti-proliferative effect of AITC prevent the development of DMBA and MNU-induced mammary carcinogenesis in rat.

Unbalanced X;9 translocation in an infertile male with de novo duplication Xp22.31p22.33.

Male carriers of an X-autosome translocation are generally infertile, regardless of the position of the breakpoint on the X chromosome while the pathogenicity of Xp22.3 subtelomeric duplications is under debate. To shed light into this controversy, we present a rare case, of an azoospermic male with no other significant clinical findings, in whom classical cytogenetics revealed additional unbalanced chromosomal material, at the telomere of the long arm of one homolog of chromosome 9. In peripheral blood specimens of the index case and his parents, we performed GBanding, Inverted-DAPI Banding, AgNOR staining, Telomere specific Fluorescence in Situ Hybridization (FISH), Molecular karyotyping by Multi-color FISH, whole genome SNP microarrays, sub-telomeric MLPA, and transcription analysis of the expression of KAL1 gene by RT-PCR. Multi-color FISH revealed an unbalanced translocation involving the short arm of chromosome X. SNP microarray analysis combined to classical cytogenetics and MLPA demonstrated a de novo 8.796Mb duplication of Xp22.31-p22.33. Compared to three control specimens, the patient presented significantly elevated expression levels of KAL1 mRNA in peripheral blood, suggesting transcriptional functionality of the duplicated segment. The duplicated segment contains the pseudo-autosomal region PAR1 and more than 30 genes including SHOX, ARSE, STS, KAL1, and FAM9A and is not listed as polymorphic. Our data advocate that duplications of the Xp22.3 region may not be associated with a clinical consequence.

Evaluation of Cytomorphologic Changes of Oral Mucosa and Copper Level of Saliva in Occupationally Copper Exposed, Oral Squamous Cell Carcinoma and Normal Population

Introduction: Oral Squamous Cell Carcinoma (SCC) involves half of the oral cavity malignancies. It is very important to find the etiologic factors of oral SCC and its early diagnosis. Copper is one of the most useful metals that have been used by men since ancient time. The level of copper increases when the cancerous changes occur in the body, saliva analysis can be used to discover various illnesses because of its non-invasive methods. In addition to biopsy, an alternative method for examining oral lesions is exfoliative cytology. The aim of this study was to determine the salivary copper level and cytomorphologic changes of oral mucosa among three study groups.Materials and Methods: In this cross-sectional study, 15 individuals with oral SCC, 15 workers exposed to copper and 15 healthy individuals were included. Saliva samples were collected and analyzed by Atomic Absorption Spectrophotometer. The exfoliative smears were prepared by brush biopsy and stained by Papanicolaou and AgNOR staining methods. Data were analyzed by One-way ANOVA and Kruskal-Wallis test. P value <0.05 was considered significant.Result: The results showed a significant difference in mean salivary copper (P=0.008), cytomorphology of oral mucosa and AgNOR among the three groups (P<0.001).Conclusion: Occupational exposure to copper increases the salivary levels of this element and causes changes in mucosal cells. Since this increase in is very high salivary level of copper and nuclear activity are in oral SCC patients, exposure to copper should be considered an important risk factor for oral mucosal changes.

Poorly differentiated vulvar squamous cell carcinoma within viral papillomas in a mare: immunohistochemical study

Present report describes the immunohistochemical diagnosis of poorly differentiated squamous cell carcinoma and involvement of papilloma virus in a mare. A five years old mare was brought to the Teaching Veterinary Clinical Complex with history of multi-lobulated haemorrhagic ulcerative mass in the vulvar region since last two months. The swollen region was aseptically prepared and the mass was resected out under local anaesthesia. Cytology of tumour revealed large cells with abundant basophilic cytoplasm and pleomorphic nuclei. Microscopically, presence of irregular cords of undifferentiated epidermal cells proliferating in downward fashion and invading dermis and subcutis were seen. AgNOR staining of section revealed 4 to 5 irregular, large dots or bizarre clusters in nuclei indicating malignancy. The PCR amplification was done by using specific primers for PVL1 gene of CPV that amplified a 480 base pair fragment of the Papilloma Virus L1 gene. Pancytokeratin immunolabeling was helpful in the diagnosing poorly differentiated squamous cell carcinoma. PCR confirmation suggested role of papilloma virus in induction and progression of squamous cell carcinoma.

AgNOR Staining in Prostatic Carcinoma - Comparative study with Bcl-2 and Gleason Score

AgNOR Staining in Prostatic Carcinoma - Comparative study with Bcl-2 and Gleason Score

The rRNA Gene Containing Marker Chromosome Associated with a Intellectual Disability: A Clinical Case Report

Marker chromosomes are structurally abnormal chromosomes that may be supernumerary in karyotype or replace one of the chromosomes. Marker chromosomes very frequently can be a cause of different pathologies, including a intellectual disability. This study considers the molecular cytogenetic analysis of an acrocentric marker chromosome in a patient with autism and a intellectual disability. With the help of the painting probe localization of human chromosomes, its origin from the pericentromeric regions of the p and q arms of chromosome 15 is shown. For more detailed detection of the region of origin of this chromosome, its microdissection following localization on both healthy control and patient metaphase plates was carried out. Its origin from the nucleolus-containing chromosome 15 is confirmed. Because of the rDNA-containing plasmid localization, two of the ribosome RNA gene clusters were detected on the patient’s chromosomes. Ag-NOR staining showed that both clusters are active. A possible contribution of the revealed chromosome pathology in clinical picture formation is discussed.

AgNOR Staining in Prostatic Carcinoma - Comparative Study with p53 and Gleason Score

AgNOR Staining in Prostatic Carcinoma - Comparative Study with p53 and Gleason Score

Estrogenic Activity of Ethanolic Extract of Papaya Peels (Carica Papaya L.) on Uterine Weight and Mammae Gland Proliferation on Ovariectomy Rats

Papaya bark is one of Indonesia's natural wealth that contains flavonoid compounds such as myricetin and kaempferol that included in the phytoestrogen compounds. The aim of this study is to examine the estrogenic effects of ethanolic extract of papaya peels (EEPP), on the development of mammae gland and the increasing of uterine weight. The in vivo test was performed with ovariectomy in Sprague Dawley female rats that caused the rats to be in an estrogen deficiency state. After 30 days of treatment, animals are sacrificed to take the uterus and mammae glands. Measurement of uterine weight and mammae gland are observed by hematoxylin-eosin staining method to know the lobulus development and AgNOR staining to determine the proliferation level of mammae gland epithelial cells. The test results showed that EEPP concentration of 500 and 1000 mg/kgBW were able to increase uterine weight and proliferation of mammae gland. From the results of this study, papaya bark has the potential to be one of the phytoestrogens compound to maintain female reproductive health and woman beauty.Keyword: ethanolic extract of papaya peels (EEPP), phytoestrogen, ovariectomized rats, uterine weight, mammae proliferation

AgNOR histochemical expression in benign prostatic hyperplasia and prostatic adenocarcinoma

Benign prostatic hyperplasia and prostatic adenocarcinoma were common diseases and usually occurred after the 5th decade of life. The problem in diagnosing using Hematoxylin and Eosin staining was how to differentiate whether it is benign or malignant zone. Therefore, proliferating markers, such as AgNOR, could be helping to over this difficulty. A descriptive study using consecutive sampling as the method of sample recruiting was conducted to describe AgNOR histochemical expression in benign prostatic hyperplasia and prostatic adenocarcinoma. AgNOR staining was done in 13 benign prostatic hyperplasia samples and 7 prostatic adenocarcinoma samples, which have been confirmed using p63 immunohistochemical staining before. Benign prostatic hyperplasia usually showed lower AgNOR proliferating activity while all of theprostatic adenocarcinoma (100%) had high AgNOR proliferating activity.