Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Chronic Telogen Effluvium And Female Pattern Hair Loss Are Separate And Distinct Forms Of Alopecia: A Histomorphometric And Immunohistochemical Analysis.

Chronic telogen effluvium (CTE), a poorly understood condition, can be confused with or may be a prodrome to female pattern hair loss (FPHL). The pathogenesis of both is related to follicle cycle shortening and possibly to blood supply changes. To analyze a number of histomorphometric and immunohistochemical findings through vascular endothelial growth factor (VEGF), Ki-67, and CD31 immunostaining in scalp biopsies of 20 patients with CTE, 17 patients with mild FPHL and 9 controls. Ki-67 index and VEGF optical density were analyzed at the follicular outer sheath using ImageJ software. CD31 microvessel density was assessed by a Chalkley grid. Significant follicle miniaturization and higher density of nonanagen follicles were found in FPHL, compared with patients with CTE and controls. Ki-67+ index correlated positively with FPHL histological features. The FPHL group showed the highest VEGF optical density, followed by the CTE and control groups. No differences were found in CD31 microvessel density between the three groups. Histomorphometric results establish CTE as a distinct disorder, separate from FPHL from its outset. Its pathogenic mechanisms are also distinct. These findings support the proposed mechanism of 'immediate telogen release' for CTE, leading to cycle synchronization. For FPHL, accelerated anagen follicular mitotic rates and, thus, higher Ki-67 and VEGF values, would leave less time for differentiation, resulting in hair miniaturization.

Interplay Between Cell Growth And Cell Cycle In Plants.

The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.

Biased Random-key Genetic Algorithms For The Winner Determination Problem In Combinatorial Auctions.

Abstract In this paper, we address the problem of picking a subset of bids in a general combinatorial auction so as to maximize the overall profit using the first-price model. This winner determination problem assumes that a single bidding round is held to determine both the winners and prices to be paid. We introduce six variants of biased random-key genetic algorithms for this problem. Three of them use a novel initialization technique that makes use of solutions of intermediate linear programming relaxations of an exact mixed integer-linear programming model as initial chromosomes of the population. An experimental evaluation compares the effectiveness of the proposed algorithms with the standard mixed linear integer programming formulation, a specialized exact algorithm, and the best-performing heuristics proposed for this problem. The proposed algorithms are competitive and offer strong results, mainly for large-scale auctions.

Pituitary Macroadenoma Presenting As A Nasal Tumor: Case Report.

Pituitary macroadenomas are rare intracranial tumors. In a few cases, they may present aggressive behavior and invade the sphenoid sinus and nasal cavity, causing unusual symptoms. In this paper, we report an atypical case of pituitary adenoma presenting as a nasal mass. The patient was a 44-year-old woman who had had amenorrhea and galactorrhea for ten months, with associated nasal obstruction, macroglossia and acromegaly. Both growth hormone and prolactin levels were increased. Magnetic resonance imaging showed a large mass originating from the lower surface of the pituitary gland, associated with sella turcica erosion and tumor extension through the sphenoid sinus and nasal cavity. Histopathological analysis demonstrated a chromophobe pituitary adenoma with densely packed rounded epithelial cells, with some atypias and rare mitotic figures. There was no evidence of metastases. Macroadenoma invading the nasal cavity is a rare condition and few similar cases have been reported in the literature. This study contributes towards showing that tumor extension to the sphenoid sinus and nasopharynx needs to be considered and investigated in order to make an early diagnosis when atypical symptoms like nasal obstruction are present.

Microsatellite In Aeschynomene Falcata (leguminosae): Diversity, Cross-amplification, And Chromosome Localization.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.

Motivating Medical Students To Learn Basic Science Concepts Using Chronic Myeloid Leukemia As An Integration Theme.

To report on the use of chronic myeloid leukemia as a theme of basic clinical integration for first year medical students to motivate and enable in-depth understanding of the basic sciences of the future physician. During the past thirteen years we have reviewed and updated the curriculum of the medical school of the Universidade Estadual de Campinas. The main objective of the new curriculum is to teach the students how to learn to learn. Since then, a case of chronic myeloid leukemia has been introduced to first year medical students and discussed in horizontal integration with all themes taught during a molecular and cell biology course. Cell structure and components, protein, chromosomes, gene organization, proliferation, cell cycle, apoptosis, signaling and so on are all themes approached during this course. At the end of every topic approached, the students prepare in advance the corresponding topic of clinical cases chosen randomly during the class, which are then presented by them. During the final class, a paper regarding mutations in the abl gene that cause resistance to tyrosine kinase inhibitors is discussed. After each class, three tests are solved in an interactive evaluation. The course has been successful since its beginning, 13 years ago. Great motivation of those who participated in the course was observed. There were less than 20% absences in the classes. At least three (and as many as nine) students every year were interested in starting research training in the field of hematology. At the end of each class, an interactive evaluation was performed and more than 70% of the answers were correct in each evaluation. Moreover, for the final evaluation, the students summarized, in a written report, the molecular and therapeutic basis of chronic myeloid leukemia, with scores ranging from 0 to 10. Considering all 13 years, a median of 78% of the class scored above 5 (min 74%-max 85%), and a median of 67% scored above 7. Chronic myeloid leukemia is an excellent example of a disease that can be used for clinical basic integration as this disorder involves well known protein, cytogenetic and cell function abnormalities, has well-defined diagnostic strategies and a target oriented therapy.

Investigation Of Genetic Factors Underlying Typical Orofacial Clefts: Mutational Screening And Copy Number Variation.

Typical orofacial clefts (OFCs) comprise cleft lip, cleft palate and cleft lip and palate. The complex etiology has been postulated to involve chromosome rearrangements, gene mutations and environmental factors. A group of genes including IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, MSX1 and FGF has been implicated in the etiology of OFCs. Recently, the role of the copy number variations (CNVs) has been studied in genetic defects and diseases. CNVs act by modifying gene expression, disrupting gene sequence or altering gene dosage. The aims of this study were to screen the above-mentioned genes and to investigate CNVs in patients with OFCs. The sample was composed of 23 unrelated individuals who were grouped according to phenotype (associated with other anomalies or isolated) and familial recurrence. New sequence variants in GLI2, MSX1 and FGF8 were detected in patients, but not in their parents, as well as in 200 control chromosomes, indicating that these were rare variants. CNV screening identified new genes that can influence OFC pathogenesis, particularly highlighting TCEB3 and KIF7, that could be further analyzed. The findings of the present study suggest that the mechanism underlying CNV associated with sequence variants may play a role in the etiology of OFC.

Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system

Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB) is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed.

Número cromossômico em espécies brasileiras de Adesmia DC. (Leguminosae-Faboideae)

Chromosome numbers of 11 South-Brazilian species of Adesmia were determined. The cytological preparations were obtained by squashing cells of root tips, using the acetic-orcein method. The chromosome number for all the species studied was 2n=20, excepting A. incana var. incana with 2n=ca.40. The counts are new for nine species, and the other two agree with the literature. It is suggested x=10 as the basic number for the genus. Up to the present only four species were cited as polyploid.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Familial glioblastoma

The authors describe a family with three members affected by glioblastoma. The proband patient, a 7 year-old girl, developed a rare complication, a pulmonary metastasis. Chromosomal analysis of her peripheral blood lymphocytes showed a normal karyotype (46, XX), without structural abnormalities. Cytogenetic study of the tumor cells disclosed several abnormalities: 46, XX, 7q - / 46, XX, -2, 4p-, 7p-, +15/ 46, XX. Some aspects about genetics of glial neoplasms are discussed.

Karyological study of Amphisbaena ridleyi (Squamata, Amphisbaenidae), an endemic species of the Archipelago of Fernando de Noronha, Pernambuco, Brazil

The karyotype of Amphisbaena ridleyi, an endemic species of the archipelago of Fernando de Noronha, in State of Pernambuco, Brazil, is described after conventional staining, Ag-NOR impregnation and fluorescence in situ hybridization (FISH) with a telomeric probe. The diploid number is 46, with nine pairs of macrochromosomes (three metacentrics, four subtelocentrics and two acrocentrics) and 14 pairs of microchromosomes. The Ag-NOR is located in the telomeric region of the long arm of metacentric chromosome 2 and FISH revealed signals only in the telomeric region of all chromosomes. Further cytogenetic data on other amphisbaenians as well as a robust phylogenetic hypothesis of this clade is needed in order to understand the evolutionary changes on amphisbaenian karyotypes.

Astyanax hastatus Myers, 1928 (Teleostei, Characidae): a new species complex within the genus Astyanax?

Four populations of Astyanax hastatus Myers 1928 from the Guapimirim River basin (Rio de Janeiro State) were analyzed and three distinct cytotypes identified. These cytotypes presented 2n = 50 chromosomes, with 4M+8SM+10ST+28A (Cytotype A), 8M+10SM+14ST+18A (Cytotype B), 6M+8SM+4ST+32A (Cytotype C) and scanty heterochromatin, mainly located throughout pericentromeric regions of several chromosomal pairs. No homologies with the As-51 satellite DNA were observed in the three cytotypes, although all of them presented multiple 18S rDNA sites, as detected by both silver nitrate staining and FISH (fluorescent in situ hybridization). The application of the term "species complex" in Astyanax is discussed from a cytotaxonomic viewpoint.

Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.