First chromosomal analysis of Gymnorhamphichthys britskii: the remarkable lowest diploid value within the family Rhamphichthyidae (Gymnotiformes)

Gymnorhamphichthys britskii is a Neotropical electric fish of family Rhamphichthyidae described from the Paraná-Paraguay system. This study reports the first karyotypic description of G. britskii collected from the upper Paraná river basin, which presented 2n=38 chromosomes, karyotype composed of 14 metacentric, 8 submetacentric, 2 subtelocentric and 14 acrocentric chromosomes, and fundamental number as 62 for both sexes. Heteromorphic sex chromosomes were absent. A single pair of nucleolar organizing regions (NORs) was detected in the submetacentric chromosome pair number 9 by silver staining and confirmed by the 18S rDNA probe. The 5S rDNA was located in a single chromosome pair. Heterochromatic regions were clearly observed in the short arms of the NOR-bearing chromosome pair and in the telomeric positions of most acrocentric chromosomes. Besides the present data are valuable to help in understanding karyotypic evolution in Rhamphichthyidae, data from NORs confirmed the tendency of this family in presenting simple NORs sites, similar to the other Gymnotiformes clades. Yet, the presence of a large heterochromatic block in the NOR-bearing chromosome can be used as cytogenetic markers for G. britskii, and that centric fusions appear to be an important mechanism in the karyotype evolution and differentiation among Gymnotiformes species.(AU)

Gymnorhamphichthys britskii é um peixe neotropical da família Rhamphichthyidae descrita no sistema Paraná-Paraguai. Este estudo relata a primeira descrição cariotípica de G. britskii coletado na bacia do alto rio Paraná, que apresentou 2n = 38 cromossomos, cariótipo composto por 14 metacêntricos, 8 submetacêntricos, 2 subtelocêntricos e 14 acrocêntricos, e número fundamental 62 para ambos sexos. Cromossomos sexuais heteromórficos estavam ausentes. Um único par de regiões organizadoras de nucléolos (RONs) foi detectado no par de cromossomos submetacêntricos número 9 por coloração com prata e confirmado pela sonda DNAr 18S. O DNAr 5S foi localizado em um único par cromossômico. Regiões heterocromáticas foram claramente observadas nos braços curtos do par de cromossomos que carrega a RON e nas posições teloméricas da maioria dos cromossomos acrocêntricos. Além dos dados presentes serem valiosos para auxiliar na compreensão da evolução cariotípica em Rhamphichthyidae, dados de RONs confirmaram a tendência desta família em apresentar sítios simples de RONs, semelhantes aos demais clados de Gymnotiformes. No entanto, a presença de um grande bloco heterocromático no cromossomo portador da RON, pode ser usado como marcador citogenético para G. britskii e as fusões cêntricas parecem ser um mecanismo importante na evolução e diferenciação cariotípica entre as espécies de Gymnotiformes.(AU)

Interspecific chromosomal divergences in the genus Characidium (Teleostei: Characiformes: Crenuchidae)

Karyotypes of seven fish species of the genus Characidium, three of them studied for the first time, were characterized using conventional cytogenetic techniques (Giemsa staining, Ag-NOR, and C-banding). All species presented a diploid number of 2n=50, with only metacentric and submetacentric chromosomes, as observed in all Characidium species studied. In two species cells with one to three B chromosomes were observed. All species analyzed have a single NOR-bearing chromosome pair with morphological differences among them. Characidium cf. zebra shows heterochromatic blocks restricted to the pericentromeric regions of all chromosomes denoting the absence of a sex chromosome system. On the other hand, the species Characidium lanei, C. pterostictum, C. lauroi, C. oiticicai, C. schubarti, and Characidium sp., besides presenting pericentromeric heterochromatic blocks, exhibited large interstitial and/or terminal heterochromatic blocks, and a ZZ/ZW sex chromosome system. The constitutive heterochromatin seems to play a relevant role in the chromosome differentiation process of the studied species, mainly in relation to the sex chromosomes. The geographical isolation of the rivers in which the species were sampled, associated with their way of life restricted to headwaters environments, may have favored the process of fixation of different karyotypes found in each of the analyzed species.

Os cariótipos de sete espécies de peixes do gênero Characidium, três estudadas pela primeira vez, foram caracterizados com o uso das técnicas citogenéticas convencionais (Giemsa, Ag-RONs e bandamento-C). Todas as espécies apresentaram número diplóide de 2n=50 cromossomos, com predominância de cromossomos dos tipos meta e submetacêntricos. Nesse estudo foi também observada a presença de até três cromossomos B em células de duas espécies, C. oiticicai e C. pterostictum. O bandamento C e o tratamento com nitrato de prata revelaram significativas diferenças nos cariótipos das espécies analisadas. A espécie Characidium cf. zebra apresenta heterocromatina restrita às regiões pericentroméricas dos cromossomos e ausência de heteromorfismos cromossômicos relacionados à diferenciação sexual, enquanto as espécies Characidium lanei, C. pterostictum, lauroi, C. oiticicai, C. schubarti e Characidium sp., evidenciaram, além de blocos pericentroméricos também observados em Characidium cf. zebra, grandes blocos heterocromáticos intersticiais e/ou terminais e sistema cromossômico de diferenciação sexual do tipo ZZ-ZW. A heterocromatina constitutiva parece exercer papel relevante no processo de diferenciação cromossômica destas espécies, principalmente em relação à diferenciação de cromossomos sexuais. O isolamento geográfico dos rios em que essas espécies foram amostradas, bem como o seu modo de vida restrito às regiões de cabeceira, podem ter favorecido o processo de diferenciação cromossômica e a fixação dos cariótipos particulares encontrados em cada uma das espécies analisadas.

Cytogenetic analysis of three sea catfish species (Teleostei, Siluriformes, Ariidae) with the first report of Ag-NOR in this fish family

Despite their ecological and economical importance, fishes of the family Ariidae are still genetically and cytogenetically poorly studied. Among the 133 known species of ariids, only eight have been karyotyped. Cytogenetic analyses performed on Genidens barbus and Sciades herzbergii revealed that both species have 2n = 56 chromosomes and Cathorops aff. mapale has 2n = 52 chromosomes: Genidens barbus has 10 Metacentrics (M), 14 Submetacentrics (SM), 26 Subtelocentrics (ST), and 6 Acrocentrics (A), Sciades herzbergii has 14M, 20SM, 18ST and 4A, whereas Cathorops aff. mapale has 14M, 20SM, and 18ST. The nucleolus organizer regions (NORs) were found in a single chromosome pair on the short arm of a large-sized ST pair in Genidens barbus and on the short arm of a middle-size SM pair in Cathorops aff. mapale. Multiple NORs on the short arms of two large-sized ST pairs were found in Sciades herzbergii. The occurrence of diploid numbers ranging from 2n = 52 through 56 chromosomes and the presence of different karyotypic compositions, besides the number and position of NORs suggest that several numeric and structural chromosome rearrangements were fixed during the evolutionary history of this fish family.

Karyology of the Atlantic forest rodent Juliomys (Cricetidae): a new karyotype from southern Brazil

Juliomys is a small rodent from the family Cricetidae which inhabits the Atlantic forest and forests from Argentina to eastern Brazil. The three species recognized so far have different karyotypes. In this paper, we describe a new karyotype with 2n = 32, FN = 48 found in Juliomys specimens from a high-altitude area in the Atlantic forest of southern Brazil. The karyotype was analyzed after G- and C-banding and silver staining of the nucleolus organizer regions (Ag-NOR) and its G-banding patterns were compared with those of the newly described species Juliomys ossitenuis (2n = 20, FN = 36). The 2n = 32 karyomorph presented peculiar features and was very different from those of the other species of the genus: J. pictipes (2n = 36, FN = 34), J. rimofrons (2n = 20, FN = 34) and J. ossitenuis (2n = 20, FN = 36). Differences were mostly due to centric and tandem fusions, pericentric inversion and loss of heterochromatin. The karyotype represents a powerful tool to differentiate Juliomys species and our data suggest that the karyotype described herein belongs to a new species.

Patterns of ribosomal DNA distribution in hylid frogs from the Hypsiboas faber and H. semilineatus species groups

We performed a comparative analysis of the nucleolus organizer region (NOR) distribution in the karyotypes of hylid frogs from two different taxonomic groups, Hypsiboas faber and H. semilineatus. Silver nitrate staining of NORs (Ag-NORs) and fluorescence in situ hybridization (FISH) with a rDNA probe were used to investigate the chromosomal location of rDNA loci in two species. The karyotype of H. semilineatus and the Ag-NORs distribution of the four species are presented herein for the first time. After conventional staining, the four species presented very similar karyotypes with 2n = 24, but Ag-NORs analyses revealed species-specific characteristics. H. albomarginatus, H. faber and H. semilineatus had one pair of interstitial Ag-NORs in the short arm of pairs 2, 11, and in the long arm of pair 7, respectively. H. pardalis presented telomeric NORs in the long arm of pair 11. Ag-NORs were heteromorphic in three of the species (H.pardalis, H. semilineatus and H. albomarginatus) and FISH confirmed the differential activity of rDNA genes in H. semilineatus. In the present study, 2n = 24 karyotypes could be distinguished by their Ag-NORs distribution. Our results further the knowledge about the cytogenetics of hylids from Brazil.

Astylus variegatus (Coleoptera, Melyridae): Cytogenetic study of a population exposed to agrochemical products

In this work we describe the cytogenetic analyses performed in specimens of Astylus variegatus (Germar, 1824) collected in two localities: one area of natural vegetation and one of agricultural crops, where agrochemical products were used. Astylus variegatus had karyotypes with 2n(male) = 16+Xy p and 2n (female) = 16+XXp, with exclusively metacentric chromosomes. Pachytene spermatocytes showed synapsed autosomal bivalents and non-associated sex chromosomes. In diplotene, the autosomal bivalents exhibited one or two terminal chiasmata and the Xy p had a typical parachute configuration. In meiotic cells of some specimens, an extra chromosome, interpreted as a B chromosome, was observed. C-banding showed constitutive heterochromatin in the pericentromeric region of all chromosomes, with the exception of the y p. Silver nitrate staining revealed one nucleolus organizer region (NOR) on the terminal region of the short arm of the second autosome pair. Silver staining of meiotic cells confirmed the NOR pattern detected in mitotic cells and revealed an argentophilous material on the Xy p. A cytogenetic comparison between the two populations of A. variegatus showed a statistically significant divergence (chi2 = 117.10; df = 1) in the number of aneuploid cells and a higher frequency of B chromosome in the population exposed to agrochemicals.

Preponderance of GC-rich sites in silver-stained nucleolus organizing regions of Rita rita (Hamilton) and Mystus gulio (Hamilton) (Bagridae, Pisces), as revealed by chromomycin A3-staining technique and scanning electron microscopic studies

The karyotypes of two species of catfish, Rita rita (Hamilton) (2n = 54; 14m + 34sm + 6st; NF = 102) and Mystus gulio (Hamilton) (2n = 58; 30m + 12sm + 2st + 14t, NF = 100) were studied through Giemsa-, silver- and chromomycin A(3)-staining techniques. The silver-stained karyotypes in both sexes of R. rita and M. gulio revealed that the nucleolus organizing regions were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes, placed at positions Nos. 2 and 1, respectively, which was confirmed by scanning electron microscopy. Staining with a GC-specific fluorochrome, chromomycin A(3), produced bright fluorescence in the Ag-positive nucleolus organizer regions, suggesting thereby that nucleolus organizing regions actually included GC-rich sites of active r-RNA genes in metaphase chromosomes of these two bagrids. Further such studies are needed due to the extreme paucity of data on fish.

Studies on the Cytotoxicity and Antineoplastic Activity of Methyl Gallate / 대한해부학회지

In order to develop antitumor agent which indicates weak side effects and strong antitumor activity, cytotoxicity and antitumor effects of MG was evaluated by MTT assay and SRB assay of colorimetric assay methods on the cultured NIH 3T3 fibroblasts and human oral epithelioid carcinoma cells (KB cells). KB cells were cultured in EMEM and RPMI 1640 media containing 10% fetal bovine serum, antibiotics and fungizone. After incubation for 24 hrs, the cells were treated with MG by dose dependent manner for 48 hrs under the same condition. The MTT and SRB quantity were measured by ELISA reader (Spectra Max 250, USA). The microscopic study was carried out to observed morphological change, Ag-NORs (argyrophylic nucleolar organizer region) number and PAS positive reacton of cultured KB cells. The results were as follows; 1. The MTT50 were 16677 micrometer in NIH 3T3 cell and 65.55 micrometer in KB cells treated with MG. 2. The SRB50 were 8701.23 micrometer in NIH 3T3 cell and 168.81 micrometer in KB cells treated with MG. 3. The number of Ag-NORs was not significant in cultured NIH 3T3 cells, but the number of Ag-NORs was significantly decreased in cultured KB cells treated with MG. 4. PAS reaction of cultured NIH 3T3 cells was not changed, but PAS reaction of cultured KB cells was changed to negative reaction. 5. Morphological changes of cultured NIH 3T3 cells treated with MG was weak, but KB cell treated with MG was severe. These results indicate that MG have noncytoxicity on NIH 3T3 fibroblasts and high cytotoxicity on KB cells.

Histologic Grading of Astrocytic Neoplasms in Conjunction with Evaluation of Proliferative Activity Using Ag-NORs Count PCNA Expression, and Flow CYtometric DNA Analysis

Although the histologic grade of astrocytic neoplasms of the brain have been used as a prognostic factor, the lack of an objective criteria is possible to create the disagreement of classification. We evaluated 25 cases of astrocytic neoplasms of brain to document the usefulness of prolifera-tive potential of tumor as a prognostic indicator and the correlation with histologic grade by Nils Ringertz. The Ringertz's classification was relatively simple in an application among the variable systems and easy to define the differentiate from grade to grade. The examined cases were com-prised of 7 astrocytomas, 9 anaplastic astrocytomas and 9 glioblastoma multif6rmes. The prolife-rative potential of tumors were measured by Ag-NORs count, PCNA labeling index and flow cytometric analysis. The mean numbers of Ag-NORs per cell and PCNA labeling index were sig-nificantly differ among each histologic grade. In addition, abnormal DNA content and high prolif-erative index were frequently identified in anaplastic astrocytoma and glioblastoma multiforme. Therefore, the Ag-NORs counts, PCNA labeling index, DNA index and proliferative index were well correlated with the histologic grade.

Comparative Study of the Breast Carcinoma by the Measurement of Nucleolar Organizer Regions and the Flow Cytometric DNA Analysis

It is important to predict the prognosis of patient with cancer. Well known parameters predicting the prognosis are staged and degree of differentiation. Recently, the prognostic and diagnostic significance of silver-stained nucleolar organizer regions(Ag-NORs) and flow cytometric DNA analysis are introduced. This study aims to detect the diagnostic significance of Ag-NORs in breast lesions, and to correlate the number of Ag-NORs and cellular ploidies in breast carcinoma. In addition, the existence of heterogeneity according to the sample in the same patient is studied Sixty six paraffin blocks(two primary lesions and the two metastatic lymph nodes in 13 patients, and one primary lesion and one metastatic lymph node I 10 patients) of breast carcinoma, 30 paraffin blocks of fibroadenoma and 23 paraffin blocks of normal breast are studied. The numbe of Ag-NORs increase from normal breast(mean 1.45) through fibroadenoma(mean 1.60) to carcinoma(mean 2.43). It was significantly different between each lsions(P<0.0005). The number of Ag-NORs increased in the aneuploidy carcinoma than that in the euploidy one(P<0.05). But, there was difference between the number of Ag-NORs and DNA index according to the sample area in the same patient with carcinoma. In conclusion, the number of Ag-NORs is diagnostically significant in breast lesions and is correlated with cellular ploidies in breast carcinoma. In addition, Ag-NORs and cellular ploidies are heterogenous in the same patient.

Pathological Predictor for Prognosis in Gastrointestinal Mesenchymal Neoplasms

To evaluate the prognostic predictor and clinicopathologic characteristics of the gastrointestinal (GI) mesenchymal neoplasm, we examined 75 cases of GI mesenchymal tumors surgically resected during 8 years from 1983 to 1990. Various histological parameters referrable to the prognosis, including the Ag-NORs count, were analysed. Fifty cases were followed-up for 1 to 7 years. Sixteen out of these fifty cases died during this period. The location of tumor was the stomach in 33 cases, the small intestine in 31 cases and the large intestine in 11 cases, and the tumor size was variable from 2 to 35 cm in diameter. The GI mesenchymal neoplasm appeared as an extraluminal mass in 50 cases, an intramural mass in 17 cases, and an intraluminal mass in 8 cases. Each tumor was composed of spindle or epithelioid cells, the former cell type being more common than the latter (45 vs 30 cases). Mitotic count of the tumor showed the best correlationship with the survival of patients(p0.05). These results indicate that the mitotic count is the most valuable pathological predictor for the prognosis in GI mesenchymal neoplasms.

Nucleolar Organizer Regions in Normal Tissue and Hyperplastic and Neoplastic Lesions

For the identification of proliferating cells in tissue, the argyrophilic method for the demonstration of nucleolar organizer regions (Ag-NORs) have been described. To evaluate the applicability of Ag-NORs in surgical pathology, the authors have done Ag-NORs staining on 144 cases of routinely processed, formalin-fixed paraffin sections of various tissues; 15 normal tissues, 12 reactive and hyperplastic lesions, 30 benign neoplasms, 4 borderline lesions, and 83 malignant tumors. The results were summerized as follows; 1) In normal tissues, the mean numbers of Ag-NORs were highter in labile cells, especially in actively proliferating cells such as germ cells of testis, crypt epithelial cells in gastrointestinal mucosa, and lymphocytes from germinal center of tonsil and lymph node, than those of stable cells. 2) The mean numbers of Ag-NORs in reactive and hyperplastic lesions, benign neoplasms, and borderline lesions were similar to those of normal labile cells. 3) The mean numbers of Ag-NORs in carcinomas and sarcomas, (usually more than 2) significantly exceeded those of normal and non-malignant conditions. However, certain cases of carcinomas such as papillary carcinomas of thyroid, mucinous carcinoma of stomach, bronchioloalveolar carcinoma of lung, and adenoid cystic carcinoma of lung, and some of the leiomyosarcoma, malignant fibrous histiocytoma, and malignant schwannoma showed relatively lower numbers of Ag-NORs. 4) In non-Hodgkin's lymphomas, the high grade lymphomas showed more Ag-NORs than the low grade ones. From above results, it is suggested that the Ag-NORs technique is helpful in differentiation between malignant and non-malignant lesions. However, further evaluation on the significance of Ag-NORs upon the behavior of the cancer is to be made.