Caracterização do papel de Nop53 na montagem da subunidade ribossomal maior em Saccharomyces cerevisiae / Characterization of the role of Nop53 in the assembly of the large ribosomal subunit in Saccharomyces cerevisiae

Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2

Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle

Synaptic configuration of quadrivalents and their association with the XY bivalent in spermatocytes of Robertsonian heterozygotes of Mus domesticus

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic pro- phase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17. RESULTS: Electron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent. CONCLUSIONS: The spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.

Parkinson's disease and nucleolar stress / 中华医学遗传学杂志

Parkinson's disease (PD) is a common age-related neurodegenerative disorder characterized mainly by motor dysfunction resulting in bradykinesia, rigidity, tremor, gait impairment, and postural instability. The classic pathogenic feature of PD is preferential loss of dopaminergic neurons in the substantia nigra. Downregulation of rRNA transcription is one of major mechanisms to maintain cellular homeostasis under stress conditions. Nucleolar stress has emerged as a component of the degenerative process caused by impaired rRNA transcription and altered nucleolar integrity. Recent study has indicated that the response to stress conditions and quality control mechanisms are impaired in PD, and that metabolic stress may be a trigger mechanism for PD. This review aims to present evidence for a role of nucleolar stress in PD and has summarized mechanisms by which nucleolar stress may play a role in the progression of PD.

Avaliação da técnica de coloração AgNOR em testículos de ovinos / Evaluation of the AgNOR staining method in ovine testicles

A coloração pela prata das regiões organizadoras de nucléolos (NORs) é caracterizada por marcar proteínas ligadas ao ácido ribonucleico ribossômico, avaliando a proliferação em células normais ou neoplásicas. Objetivou-se estudar, em testículos de ovinos obtidos em matadouro, a validade do uso da técnica de coloração pela prata (AgNOR) na identificação das regiões organizadoras de nucléolo (NORs) em células saudáveis da linhagem espermatogênica. Utilizaram-se 43 pares de testículos de ovinos mestiços entre seis e 10 meses de idade. Testes de Wilcoxon e Spearman foram empregados, com nível de 5%. As médias das NORs nas células das gônadas direita e esquerda foram, respectivamente: espermatogônia (8,77±1,14 e 9,04±0,96), espermatócitos (4,99±2,00 e 6,20±2,07; P<0,05), Leydig (8,05±2,82 e 7,89±2,29) e Sertoli (8,07±1,88 e 7,61±2,16; P<0,05). Houve correlação (P<0,05) entre os lados para o número de NORs: espermatócitos x Leydig (0,49); espermatócitos x Sertoli (0,49) e Leydig x Sertoli (0,96). Conclui-se ser válido o emprego da técnica AgNOR para avaliar o potencial proliferativo das células saudáveis em testículos de ovinos com prática execução e baixo custo.(AU)

The silver staining technique for AgNOR nucleolar organizer regions (NORs) is characterized by marking proteins linked to the ribosomal ribonucleic acid, evaluating cell proliferation. The aim was to study the validity of the AgNOR staining technique in the testicular cell proliferation in crossbred ovine. Forty-three pairs of ovine testicles between 6 and 10 months old were collected. Wilcoxon and Spearman tests were used with a significance level of 5%. The mean NORs count in cells of the right and left gonads were respectively: spermatogonia (8.77±1.14 and 9.04±0.96), spermatocytes (4.99±2.00 and 6.20±2.07, P<0.05), Leydig (8.05±2.82 and 7.89±2.29) and Sertoli cells (8.07±1.88 and 7.61±2.16; P<0.05). There was a correlation between the mean values for the right and left sides for the number of NORs (P<0.05) between Leydig x spermatocytes (0.49); spermatocytes x Sertoli (0.49) and Sertoli x Leydig (0.96). The study demonstrates that the AgNOR staining technique is indicated to evaluate the cell proliferative potential in ovine testis with practical implementation and low cost.(AU)

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1 / 中华病理学杂志

<p><b>OBJECTIVE</b>To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein.</p><p><b>METHODS</b>Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope.</p><p><b>RESULTS</b>GFP was fused with the native TMSG-1(aa1-380) or different fragments including T1 (aa1-70), T2 (aa1-128), T3 (aa129-380), T4 (aa71-128), T5 (aa71-179) and T6 (aa71-380). Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fused with fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aa1-380) or fragment T1 (aa1-70), T2 (aa1-128), T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm. Fragment T4(Δ119-128) was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4(Δ119-128) remained in the nucleus, but no longer in the nucleolus.</p><p><b>CONCLUSIONS</b>There is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.</p>

Morfometría del nucleolo en la diferenciación celular del epitelio mamario / Nucleolus morphometry in the cellular differentiation of mammary epithelium

Antecedentes: El proceso biológico de diferenciación celular es la traducción de múltiples procesos nucleares y citoplasmáticos que determinan cambios complejos y fundamentales en la ultraestructura, bioquímica y fsiología celular, los cuales pueden ser cuantifcados mediante técnicas morfométricas. Objetivo: Evidenciar en términos cuantitativos y morfológicos las variaciones experimentadas por los nucleolos pertenecientes a células mamarias de la línea HC11 tanto normales como en mecanismo de diferenciación. Método: Se estudió a nivel de la microscopía electrónica de transmisión los tipos celulares en etapa de proliferación (HC11 GM) en comparación con células en estadio de diferenciación (HC11 IM), cuantifcando las variaciones de los nucleolos y su relación con estructuras involucradas en síntesis proteica. Resultados: Se evidencian diferencias estadísticamente signifcativas referentes al área, volumen y longitud entre los nucleolos pertenecientes al tipo celular normal-proliferante y el que se encuentra en proceso de diferenciación. Conclusión: Las células mamarias en proceso de diferenciación presentan una notable disminución de sus nucleolos, y sus ribonucleoproteínas constitutivas generarán básicamente ribosomas adheridos al retículo endoplasmático rugoso, sintetizando proteínas de exportación.

Background: The biological process of cell differentiation is the traslation of multiple nuclear and cytoplas-mic processes that determine complex and fundamental changes in ultrastructure, biochemistry and cell physiology, which can be quantifed using morphometric techniques. Objective: To show in quantitative and morphological terms changes experienced by the nucleolus belonging to HC11 line mammary cells both, in proliferating and differentiation process. Methods: A study at the level of transmission electron microscopy of cell types in stage of cell proliferation in comparison with stage of differentiation was designed to quantify variations of nucleolus and their relation to structures involved in protein synthesis. Results: Marked differences in the area, volume and length of the nucleolus were found between normal-proliferating cell types and those in mechanism of differentiation. Conclusion: The mammary cells in differentiation process show a dramatic decline in its nucleoli and their ribonucleoproteins generate basically ribosomes attached at endo-plasmic reticulum, synthesizing export proteins.

Cytogenetical and ultrastructural effects of copper on root meristem cells of Allium sativum L

Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.

p53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Association of nucleolar organizing regions and Ki-67 expression with recurrence rate of hair follicle tumor in dogs / Associação das regiões organizadoras de nucléolos e da expressão do Ki-67 com a taxa de recorrência dos tumores de folículo piloso em cães

Mitotic index, nuclear diameter, number of nucleolar organizing regions, and Ki-67 expression, in hair follicle tumors of 82 dogs were evaluated. Tissue specimens were used to prepare sections for histological staining for number of nucleolar organizing region and immunohistochemical staining for Ki-67. Tumors were classified as trichoblastoma (n=32), benign trichoepithelioma (n=30), pilomatricoma (n=7), malignant trichoepithelioma (n=6), infundibular keratinizing acanthoma (n=5), and tricholemmoma (n=2). Head, dorsum, and limbs were the most affected sites. Malignant trichoepithelioma presented significantly higher mitotic index, number of nucleolar organizing regions and Ki-67 expression. Regarding benign neoplasms, trichoblastoma presented significantly higher mitotic index and number of nucleolar organizing regions. Ki-67 expression did not differ among hair follicle benign neoplasms. Recurrence was only observed in two cases, with one benign trichoepithelioma and one malignant trichoepithelioma. In the two cases, nodules have not been removed with clean surgical margin. It was concluded that in benign neoplasms of hair follicles, the number of nucleolar organizing regions and Ki-67 expression were significantly smaller than in malignant neoplasm. Clean surgical margins are suggested to be more responsible to tumor recurrences than the number of nucleolar organizing regions, expression of Ki-67, and the mitotic index.

O objetivo deste trabalho foi avaliar o índice mitótico, o diâmetro nuclear, o número de regiões organizadoras de nucléolos e a expressão do Ki-67 em 82 tumores de folículo piloso de cães, entre 2000 e 2006. Os tumores foram classificados como tricoblastoma (n=32), tricoepitelioma benigno (n=30), tricoepitelioma maligno (n=6), pilomatricoma (n=7), acantoma infundibular ceratinizante (n=5) e tricolemoma (n=2). A cabeça, o dorso e os membros foram os locais mais frequentemente acometidos. O tricoepitelioma maligno apresentou índice mitótico, número de regiões organizadoras de nucléolos e expressão do Ki-67 significativamente maiores quando comparado aos outros tipos de tumores. Dentre as neoplasias benignas, o tricoblastoma apresentou índice mitótico e número de regiões organizadoras de nucléolos significativamente maiores. A expressão do Ki-67 não diferiu entre os tumores benignos de folículo piloso. A recorrência foi observada apenas em dois casos, incluindo um tricoepitelioma benigno e um tricoepitelioma maligno. Em dois casos, os nódulos não foram removidos com margem cirúrgica completa. Desta forma, conclui-se que nas neoplasias benignas de folículo piloso, o número das regiões organizadoras de nucléolo e da expressão de Ki-67 foram significativamente menores que em neoplasias malignas. Além disto, sugere-se que a margem cirúrgica completa está mais associada à recorrência dos tumores que as regiões organizadoras de nucléolo, a expressão de Ki-67 e o índice mitótico.

Chromosome number and cytogenetics of Euphorbia heterophylla L

Euphorbia heterophylla L. (Euphorbiaceae) is a herbaceous species of great economic importance due to its invasive potential and consequent damage to agriculture and pasture land. For the first time, we provide information on its chromosome number, morphology, and behavior of mitotic chromosomes. Seeds were germinated and submitted to four treatments to obtain metaphases: 0.5% colchicine for 2 to 5 h, at ambient temperature; 0.5% colchicine for 16 to 24 h; 0.0029 M 8-hydroxyquinoline (8-HQ) for 2 to 5 h at ambient temperature, and 0.0029 M 8-HQ for 16 to 24 h at 4°C. The material was then fixed in methanol:acetic acid (3:1) and kept at -20°C for 24 h. Roots were macerated in the enzyme solution of Flaxzyme™ (NOVO FERMENT™)-distilled water (1:40) at 34°C for 2 h and later fixed again. Chromosome preparations were obtained by the dissociation of the apical meristems. The best chromosome preparations were obtained with the use of 8-HQ for 21 h 30 min at 4°C. E. heterophylla showed 2n = 28 chromosomes. The short arm of the largest pair of chromosomes of the complement (pair number 1) displayed a secondary constriction while the nucleolus was observed in the interphasic cell. Structural rearrangements were also observed in the E. heterophylla L. genome. The genomic instability associated with polyploidy may be the result of selection shaped by environmental adaptations and/or human-induced manipulation through agricultural practices.

[Evaluating correlation between NRBC count and fetal distress]

Nucleated red blood cells [NRBC] are occasionally observed in blood of newborns and some recent studies have reported of a relationship between NRBC count and fetal distress and hypoxia. To investigate the correlation between NRBC count and fetal distress. This was a case-control study conducted at Kosar medical centre in Qazvin, Iran. During a 6-month period in 2005, fifty women of unifetal pregnancy at third trimester of their gestation were chosen and the NRBC counts of their newborns who suffered fetal distress [case group] were evaluated. The control group composed of 100 women at their third trimester of pregnancy whose fetuses showed no sign of any distress. Data were analyzed using X2 and t test. The mean NRBC count in fetal distress group was 2406.6 +/- 2470.7 and in control group 673.43 +/- 709.9. Statistically, increased NRBC count in fetal distress group was found to be significant [p=0.000]. NRBC count in fetal distress group was significantly increased hence it could be used as a marker to evaluate the fetal distress and hypoxia in infants

[Predictive value of nucleated red blood cells count in cord blood of newborn]

The screening of infants who need to be admitted immediately following birth but without application of invasive procedures is of prime importance. The aim of this study was to evaluate the value of nucleated red blood cells [nRBCs] count of cord blood in predicting the need for admission to NICU or neonatal ward. This was a case-control study performed on 100 live, newly born full-term infants [70 healthy infants and 30 infants admitted to NICU or neonatal ward] at Vali-e-Asr Hospital of Zanjan [Iran] in 2005. Umbilical cord blood was collected at delivery time to measure the nRBCs count. Data were collected through questionnaires and further analyzed by SPSS using chi square and Mann-Whitney Tests. The mean nRBCs counts in admitted neonates [case group] and healthy infants [control group] failed to show a statistically significant difference however, by omitting the cases for whom negative nRBCs counts were reported, a significant difference between two groups was observed. The number of abnormal nRBCs, the mean number of abnormal nRBCs, and the number of absolute abnormal nRBCs [nRBCs>1000] in cord blood of the case group were significantly higher than those in control group. The sensitivity and specificity of nRBCs count were 33.3% and 100%, respectively. Although the nRBCs count alone could not be considered as an ideal screening tool for those group of neonates with clinical complications however, it seems that the nRBCs count could be a helpful diagnostic parameter in predicting a need for admission

El nucléolo como un regulador del envejecimiento celular / The nucleolus as a regulator of cellular senescence

El nucléolo, considerado únicamente como el sitio de síntesis de los ribosomas, actualmente representa una estructura nuclear dinámica que participa en la regulación de importantes procesos celulares. Numerosas evidencias han demostrado que el envejecimiento celular es una de las diversas funciones que son controladas por el nucléolo. Las mutaciones en las proteínas de localización nucleolar promueven el envejecimiento prematuro en levaduras y humanos. La carencia de represión en la transcripción de genes que codifican para el ARNr que se encuentran dañados, y las mutaciones en las helicasas del ADN encargadas de minimizar la formación de círculos extra-cromosómicos del ADN que codifica para el ARNr, provocan modificaciones en la estructura del nucléolo e inducen envejecimiento prematuro en levaduras. De igual manera, en los humanos la carencia de las helicasas del ADN localizadas en el nucléolo y que participan en el mantenimiento de la integridad genómica, favorecen el desarrollo de aquellas enfermedades asociadas con el envejecimiento acelerado. Además, la presencia de algunos componentes de la telomerasa en el nucléolo, indica que parte de la biosíntesis de esta enzima se realiza en esta estructura nuclear, sugiriendo una conexión entre el nucléolo y la síntesis de los telómeros en la regulación del envejecimiento celular. Por otra parte, el nucléolo secuestra proteínas para regular su actividad biológica durante el inicio o término de la vida replicativa celular.

The nucleolus has been considered originally only as the site for the ribosome synthesis, but now it is well known that it represents a dynamic nuclear structure involved in important cellular processes. Several evidences have demonstrated that the nucleolus regulates the cellular senescence. Specific mutations on the DNAs codifying for nucleolar proteins induced premature senescence from yeast to human. The failure to repress the genes transcription codifying for damaged rRNA, and the mutations in DNA helicases, which minimizes the formation of DNA extra-chromosomal circles codifying for rRNA, modify the nucleolar structure and induce premature senescence in yeast. Similarly, in humans, the reduction of these DNA helicases levels, which are localized in the nucleoli and participate in maintenance of genomic integrity, helps to the development of those diseases associated with premature senescence. Furthermore, the presence in the nucleolus of some telomerase components, indicates that part of the biosynthesis of this enzyme occurred in this nuclear structure; suggesting a communication between the nucleolus and the synthesis of the telomeres in the regulation of cell senescence. On the other hand, the nucleolus sequesters proteins to regulate its own biological activity, from the start to the end of cellular replication. In addition this nuclear structure is involved in the biosynthesis of most cellular ribonucleoprotein particles, as well as in cell cycle regulation, making it central to gene expression. In conclusion, the nucleolus became a multifunctional subnuclear structure involved from cell proliferation to cell senescence.

Analysis of the nucleolar cycle in the seminiferous epithelium of rodents

The nucleolus is a subcompartment of the nucleus and the site of ribosome biogenesis. During the mitotic and meiotic cell cycles, a disorganization and later reorganization of the nucleolar material occur, an event called nucleologenesis. In the spermatogenesis of mammals and other vertebrates, there is evidence of the disorganization of the nucleolus at the end of meiosis I, which supplies material for the cytoplasmic formation of an organelle called the “chromatoid body” (CB). The CB is a structure characteristic of spermatogenic cells and seems to be responsible for RNA metabolism in these cells and for some events of spermiogenesis, such as the formation of the acrosome, cellular communication between spermatids, and the formation of the spermatozoon middle piece and tail. The aim of this paper was to obtain information about the cytochemical and ultrastructural nature of the nucleolar cycle and the distribution of cytoplasmic RNAs in the seminiferous tubule cells of Rattus novergiucus, Mus musculus and Meriones unguiculatus. The testis was fixed in Bouin and Karnovsky solutions for conventional histological analysis and for cytochemical study that included: periodic acid-Schiff, hematoxylin-eosin, Feulgen reaction, silver-ion impregnation, Gomori’s reticulin stain, toluidine blue, modified method of critical electrolyte concentration, and basic and acid fast green. The blocks of testis fixed in glutaraldehyde were used for ultrastructural analysis by transmission electron microscopy. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All the techniques used provided information on the origin and function of the CB in the spermatogenic cells. Therefore, considering the persistence of the RNA and nucleolar ribonucleoproteins during spermatogenesis of Rattus novergicus, Mus musculus and Meriones unguiculatus, our findings corroborate the statement that these molecular complexes are very important in the spermiogenesis phases...

Relation of human zygote morphology and implantation of the transferred embryo / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the relationship between human embryo implantation rates and the zygote morphology and establish zygote morphologic indices that indicate the embryo implantation potential after in vitro fertilization and embryo transfer (IVF-ET).</p><p><b>METHODS</b>Sixty-two patients with IVF-ET were enrolled in this study, who were below 35 years of age with endometrium thickness no greater than 8 mm on the day of HCG injection. Embryo transfer was performed on day 3 after oocyte retrieval, and 30 patients with successful implantation of all the embryos transferred (implantation rate of 100%) were allocated into the implantation group, and the other 32 patients with implantation failure (implantation rate of 0) served as the control group. The zygote morphologic characteristics were analyzed for the pronuclei, nucleolar precursor bodies (NPB), polar body, cytoplasmic halo, color and granulation of the cytoplasm, and the results were compared between the two groups.</p><p><b>RESULTS</b>The implantation rate was significantly higher for embryos with the two pronuclei in close vicinity, central position of the pronuclei in the cytoplasm and comparable size of the two pronuclei. Embryos developed from zygotes with linear arrangement of 3 to 7 NPB in moderate sizes tented to have a higher implantation rate (P<0.05). The implantation rate could be obviously lowered when the cytoplasm contained excessive cytoplasmic granularity (P<0.05). The other morphologic characteristics of the embryos such as the polar bodies, color of the cytoplasm, cytoplasm halo, or vacuoles in the cytoplasm did not significantly impact on the implantation rate.</p><p><b>CONCLUSION</b>The morphology of the two pronuclei reflects the quality of the zygote and may help predict the developmental potential of the embryo chosen for transfer on day 3 in IVF.</p>

Nucleophosmin mutations in hematological malignancies - review / 中国实验血液学杂志

Nucleophosmin (NPM) is a protein that shuttles between the nucleus, nucleoplasm and cytoplasm. NPM gene mutations and aberrant cytoplasmic NPM localization have been recently described in acute myelogenous leukemia (AML) with normal karyotype and in a few myelodysplastic syndromes. Expression of NPM mutant reduces the ability of Arf to initiate a p53 response and to induce cell cycle arrest. Clinical research has revealed that NPM mutations are relative to prognosis and can be used to monitor and quantify minimal residual disease (MRD) in AML patients with normal karyotype, therefore, these findings indicate that nucleophosmin mutations might contribute to illustration of myeloid leukemogenesis. In this paper, the research progress of nucleophosmin mutations in haematological malignancies was reviewed.

New evidence for nucleolar dominance in hybrids of Drosophila arizonae and Drosophila mulleri

Drosophila mulleri (MU) and D. arizonae (AR) are cryptic species of the mulleri complex, mulleri subgroup, repleta group. Earlier cytogenetic studies revealed that these species have different regulatory mechanisms of nucleolar organizing activity. In these species, nucleolar organizing regions are found in both the X chromosome and the microchromosome. In the salivary glands of hybrids between MU females and AR males, there is an interspecific dominance of the regulatory system of the D. arizonae nucleolar organizer involving, in males, amplification and activation of the nucleolar organizer from the microchromosome. The authors who reported these findings obtained hybrids only in that cross-direction. More recently, hybrids in the opposite direction, i.e., between MU males and AR females, have been obtained. The purpose of the present study was to evaluate, in these hybrids, the association of the nucleoli with the chromosomes inherited from parental species in order to cytogenetically confirm the dominance patterns previously described. Our results support the proposed dominance of the AR nucleolar organizer activity over that of MU, regardless of cross-direction.

Oxidative stress-induced accumulation of heat shock protein 70 within nucleolus / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of oxidative stress on the accumulation of heat shock protein 70 (HSP70) within C2C12 myogenic cells.@*METHODS@#Heat shock response (42 degrees C for 1 h and recovery for 12 h at 37 degrees C) was used to induce the expression of heat shock protein 70. We constructed a recombinant plasmid of HSP70 with enhanced green fluorescent protein (EGFP). After being transfected transiently into C2C12 cells, immunoblotting was used to detect the expression of HSP70 induced by heat shock response and transfection. Immunocytochemistry, fluorescent microscopy and immunoblotting were used to detect the translocation of HSP70.@*RESULTS@#Immunoblotting showed that the overexpression of HSP70 was induced by heat shock response and transient transfenction. HSP70 localized within the cytoplasm of the normal cells, but HSP70 translocated from the cytoplasm to the nucleus and the nucleolus at 1 h after the treatment of oxidative stress (0.5 mmol/L H2O2) by using immunocytochemistry, fluorescent microscopy and immunoblotting for cellular partial proteins.@*CONCLUSION@#Oxidative stress may induce the accumulation of heat shock protein 70 within the nucleolus.

Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry.

The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver