AgNOR staining and quantification

Nucleolar organiser regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localised throughout the nucleolar area, and are called "AgNORs". The NORs' argyrophilia is due to a group of nucleolar proteins, which have a high affinity for silver (AgNOR proteins). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR protein amount than corresponding non-malignant cells. Moreover, in cancer tissues AgNOR protein expression was found to be strictly related to the cell duplication rate. Over the past 12 years, the "AgNOR method" has been applied in tumour pathology for both diagnostic and prognostic purposes. However, the lack of a standardised silver-staining protocol has led to much misinterpretation of actual structures evaluated in individual studies. Indeed, the absolute AgNOR scores reported by different authors for the same types of tumour are scarcely comparable and the results produced by these investigations sometimes seem to be conflicting. In order to achieve definitive standardisation of the AgNOR method and produce comparable data in all laboratories, the "International Committee on AgNOR Quantitation" was founded, and during the first Workshop "AgNORs in Oncology" held in Berlin in 1993 guidelines for AgNOR protein evaluation were first defined. The present paper discusses the main technical aspects of NOR silver-staining, and critically evaluates the methods commonly employed for AgNOR protein quantification in routine cyto-histopathology.     

Dynamic changes of nucleolar DNA configuration and distribution during the cell cycle in Allium sativum cells

To investigate the correlation between subnucleolar structure and function, the precise distribution and configuration of nucleolar DNA during the cell cycle of Allium sativum were determined using the NAMA-Ur DNA-specific staining technique. We showed that nucleolar DNA is present in two forms: compacted chromatin clumps and a decondensed DNA cloud. The form of the DNA within the nucleolus varied greatly as the cell cycle progressed. During telophase, chromosomes extended into the prenucleolar body. In early G1 phase, DNA was only located in the fibrillar centers in the form of the condensed chromatin clump, while in mid-G1, S and G2 phases, the two forms of DNA were distributed in the fibrillar centers (FC) and dense fibrillar component (DFC). In prophase of mitosis, nucleolar DNA, along with FC and DFC, was linked into a network structure and condensed into a large chromatin clump. The area of the DNA cloud in the dense fibrillar component changed during different phases of the cell cycle. Our results demonstrated that the configuration of nucleolar DNA undergoes a series of decondensations and condensations during the cell cycle to fulfill the function of the nucleoli during the different phases.     

Ventral and dorsal streams processing visual motion perception (FDG-PET study)

Background

Cullin ubiquitin ligases are activated via the covalent modification of Cullins by the small ubiquitin-like protein nedd8 in a process called neddylation. Genetic mutations of cullin-4b (cul4b) cause a prevalent type of X-linked intellectual disability (XLID) in males, but the physiological function of Cul4B in neuronal cells remains unclear.

Results

There are three major isoforms of Cul4B (1, 2, and 3) in human and rodent tissues. By examining the endogenous Cul4B isoforms in the brain, this study demonstrates that Cul4B-1 and Cul4B-2 isoforms are unneddylated and more abundant in the brain whereas the lesser species Cul4B-3 that misses the N-terminus present in the other two isoforms is neddylated. The data suggest that the N-terminus of Cul4B inhibits neddylation in the larger isoforms. Immunostaining of human NT-2 cells also shows that most Cul4B is unneddylated, especially when it is localized in the process in G0-synchronized cells. This study demonstrates that Cul4B accumulates during mitosis and downregulation of Cul4B arrests NPCs and NT-2 cells in the G2/M phase of the cell cycle. In both human and rodent brain tissues, Cul4B-positive cells accumulate ??-catenin in the dentate subgranular zone and the subventricular zone. These Cul4B-positive cells also co-express the MPM-2 mitotic epitope, suggesting that Cul4B is also necessary for mitosis progression in vivo.

Conclusions

This study provides first evidence that unneddylated Cul4B isoforms exist in the brain and are necessary for mitosis progression in NPCs. The data suggest that unneddylated Cul4B isoforms specifically inhibits ??-catenin degradation during mitosis. Furthermore, unneddylated Cul4B may play a role in addition to cell cycle since it is exclusively localized to the processes in starved NT-2 cells. Further analyses of the different isoforms of Cul4B will help understand the cognitive deficits in Cul4B-linked XLID and give insights into drug and biomarker discoveries.     

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.     

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera)

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species (Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as "persistence of the nucleolar material", and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene-diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.     

P53及其相关蛋白对X射线照射肝癌细胞周期的调节

X射线照射人肝癌细胞HepG2， 照射后细胞存活随照射剂量增大明显下降。 流式细胞术分析， 不同剂量组照射后24 h均发生G2期阻滞。 照射后不同时间组的细胞周期分布也有不同， 照射后12 h， 有显著的S期延迟。 Western Blot 显示照射后24 h P53， MDM2， P21蛋白表达上升， 并有时间效应: P53在照射后24 h之内始终维持较高表达, MDM2和P21分别在照射后6和12 h的表达最高。 X射线照射通过影响P53及其相关蛋白的表达影响细胞周期。 HepG2 cells were irradiated with X ray at the doses of 0, 1.0, 2.0, 4.0 or 8.0 Gy and separately maintained in DMEM at 37 ℃ for 0, 6, 12 or 24 h. Colony forming assay showed that cell survival decreased with the irradiation dose increasing. Cell cycle was detected by FACS, the arrest of S phase was found after 12 h irradiation and arrest of G2 phase took place at 24 h after all irradiation doses, which suggested that cell cycle distribution was different in groups gathered after different maintaining time. The results of Western blotting showed that the expression of P53, MDM2 and P21 increased more after irradiation than the control. The expression of P53 remained high at 24 h after irradiation, while the levels of MDM2 or P21 arrived at the highest at 6 h or 12 h after irradiation respectively. The expressions of P21 after irradiation were in corresponding with the cell cycle distribution in the groups of different maintaining time. In conclusion, irradiation change the distribution of cell cycle by effecting the expression of P53 and its related proteins.     

Higher NORs-expression in lymphocyte of trisomy 21 babies/children: in vivo evaluation

Extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo regulation of these genes is not known. The objective of this work is to compare the NORs expression in interphase nuclei in non-stimulated lymphocytes of DS patients and healthy controls. Because the AgNOR staining is the indicator of the active rRNA genes, comparison of the image analysis values of AgNORs area between DS's and healthy controls' interphase lymphocytes is considered to be sufficient to evaluate the level of rDNA activities in the two groups. The Nucleolus Organizer Regions area/Total Nuclear area (NORa/TNa) was calculated using a computer program designed by us. 100 consecutive NORa/TNa per individual were evaluated. We report that 24 DS children's peripheral lymphocytes show significantly higher NORa/TNa mean value (6.32 +/- 1.77%) than that of the 20 healthy controls' cells (5.31 +/- 1.34%) (2-tailed Mann-Whitney U test, z = 19.4, P = 0.000). The same is true for the nucleolus (AgNOR spot) number per nucleus. The mean value of nucleoli number per nucleus in DS lymphocytes was significantly higher than that of the controls: z = 14.6, P = 0.000. In conclusion, extra rRNA genes on the chromosome 21 are not down-regulated in DS patients' lymphocytes. Rather, extra NORs expressions in 'in vivo' condition contribute to the increase of AgNORs area and AgNOR spots number per nucleus. This is the first work on the comparison of NORs activities in resting (non-stimulated) interphase lymphocytes between DS and healthy controls.     

NOR expression increases in interphase lymphocytes of Down syndrome babies/children as AgNORs surface, according to the mitogen concentration in the culture medium

The extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo/in vitro regulation of the activity of these genes is not fully understood. The objective of this work was to compare the NORs expression pattern in interphase lymphocytes of DS patients with regular trisomy 21 and control individuals according to phytohemagglutinin (PHA) concentration (0.37, 0.75, 1.48 and 2.21 ml) per 100 ml of medium. Because the AgNOR staining is an indicator of the active rRNA genes, comparison of the image analysis values of the AgNOR area in 72 h cultivated lymphocytes for each concentration of PHA between DS patients (N=30) and controls (N=24) provided a plausible conclusion on the regulation of the extra rRNA genes in DS lymphocytes. The nucleolus organizer regions area/total nuclear area (NORa/TNa) was calculated using an in-house computer program. Fifty consecutive interphases per PHA concentration were analysed for each individual, for determination of the NORa/TNa. In contrast to healthy controls, NORa/TNa of lymphocytes from DS patient babies/children (0-8 years old) increased gradually in parallel with the PHA concentration in the culture medium: 10.44+/-1.72% for 0.37 ml of PHA, 11.74+/-1.93% for 0.75 ml of PHA, 13.25+/-2.03% for 1.48 ml of PHA and 13.43+/-2.08% for 2.21 ml of PHA per 100 ml of medium. Contrary to control cells (in which the NORa/TNa ratio according to PHA concentration in the culture medium remains constant), DS interphase lymphocytes in culture do not down-regulate their NOR expression. These results obtained from interphase NORs are consistent with the previous results obtained by evaluating the mean of AgNOR+ chromosome number in metaphase cells, also in relation to the mitogen concentration in the culture medium.     

碳离子辐照对人肺癌细胞A549细胞周期进程的影响

选取对数生长期人肺癌细胞A549接受0—6.0 Gy 碳离子照射， 用克隆形成法检测细胞的存活率； 并于照射后12和24 h收集细胞， 用流式细胞术检测细胞周期各时相的细胞百分比， 观察不同剂量碳离子辐照对A549细胞周期进程的影响。 结果显示: 0—6.0 Gy 碳离子照射后细胞存活率显著下降； 照射后12 h细胞发生G0/G1期阻滞， 而照射后24 h， 1.0 Gy 照射组细胞在G0/G1期阻滞， 2.0—6.0 Gy 照射组细胞在G2/M期阻滞。 上述结果表明， 在A549细胞接受碳离子照射后的12 和24 h内， 1.0 Gy 照射可持续激活细胞G1期检查点， 而2.0—6.0 Gy 碳离子照射后其细胞周期进程是随时间变化的。 To investigate the effects of cell cycle progression of A549 cell induced by 12C6+ ion irradiation at different doses, the survival fractions of the A549 cells were determined by colony forming assay; cell cycles were analyzed by FACS at 12 h or 24 h after irradiation. The results showed that the percentage of survival in the A549 cells decreased with irradiation doses. Compared with control group， the percentage of the cells in G0/G1 phase significantly increased at 12 h after irradiation with different doses of 12C6+ ions. However， at 24 h after irradiation the percentage of the cells in G0/G1 phase significantly increased with 1.0 Gy 12C6+ ions， while the cells showed increasing percentage in G2/M phase with 2.0, 4.0 or 6.0 Gy 12C6+ ions. The results suggested that G1 cell cycle checkpoint was activated in 12—24 h after irradiation with 1.0 Gy 12C6+ ions， but after irradiation with 2.0—6.0 Gy 12C6+ ions， the cell cycle progression of the A549 cells changed with time.     

AgNORs in breast tumours

There is evidence that the quantitative distribution of AgNOR proteins is a proliferation-related parameter that can be used as a prognostic index in tumour pathology. In breast cancer, some authors found a significant prognostic correlation of AgNOR protein quantity, whereas other did not. However, in all the reports dealing with AgNOR area (as opposed to count) this parameter was always turned out to be an independent prognostic indicator. The present study tests the significance of AgNOR proteins in a large series of primary breast carcinomas, exploring the associations between the AgNOR protein amount, as evaluated by image cytometry, and the other well-established prognostic markers commonly considered for breast cancer, along with patients' survival. Our results demonstrated a highly significant association between AgNOR protein quantity and tumour prognosis. Moreover, when the AgNOR area values were entered in multivariate analysis together with the other predictive parameters commonly considered in breast carcinomas, they showed an independent prognostic value together with Ki67-labelling index (LI), N-status and tumour size. Considering node-negative and -positive cases separately, the AgNOR protein area and Ki67-LI both come out as a independent predictors only in the latter group: the short follow-up time of our series (36 months median) could be responsible for this discrepancy.     

Nucleolin level in plant root meristematic cells under chilling stress and recovery

Nucleolin and its homologues are multifunctional proteins which reside mainly in nucleoli of yeast, animal and plant cells. Hence, they are generally implicated in many stages of ribosome biosynthesis. In this study nucleolin was identified in root meristematic cell nucleoli of soybean plants subjected to chilling stress, recovered after chilling and under control conditions with the use of the immunogold electron microscopy technique. Soybean nucleoli exhibited various metabolic activities under these conditions (St?piński, 2004). Current studies showed that the level of nucleolin, expressed as a number of gold grains per μm², varied in particular subnucleolar regions in the soybean root meristematic cell nucleoli. Labeling density changed in these regions when plants were subjected to the given treatment. Most abundantly this protein was present in dense fibrillar component (DFC) around fibrillar centers (FCs) in the nucleoli of recovered plants, while in the nucleoli of stressed plants this region contained the lowest level of nucleolin. It can be supposed that nucleolin participates in ribosome biogenesis and its level is correlated with metabolic activity of soybean nucleoli – the more active nucleoli, the higher level of nucleolin and vice versa.     

Prognostic relevance of AgNORs in tumor pathology

The importance of the analysis of the silver-stained nucleolar organizer regions (AgNORs) for prognostic purposes in tumor pathology has been reviewed. Current available data from the literature demonstrate that the evaluation of the quantity of interphase AgNORs is an independent prognostic factor in several types of human tumors. Results of our investigations indicate that AgNORs are the most powerful variable predicting survival in patients with pharyngeal carcinoma, multiple myeloma, male breast and prostate carcinoma. The combination of AgNOR counts and histologic pattern allows the stratification of patients with multiple myeloma, pharyngeal and prostate carcinoma into low- and high-risk groups, which could benefit from different therapy. Moreover, AgNOR analysis predicts response to treatment in adult patients with acute myelogenous leukemia, and appears as an independent prognostic factor in a prospective study on renal cell carcinoma. Therefore, AgNOR analysis is a really important prognostic factor for several human neoplasias. The experimental and theoretical justifications for AgNORs as a prognostic factor are also reviewed, in particular the strict correlation between AgNOR quantity and tumor cell doubling time. Lastly, the lack of prognostic significance of AgNOR analysis in some circumstances is critically discussed.     

Cytoskeleton and Chromosome Damage Leading to Abnormal Mitosis Were Involved in Multinucleated Cells Induced by Silicon Nanoparticles

Recently, amorphous silicon nanoparticles (SNPs) are widely used in a variety of fields, especially in biological and medical science. Thus, the adverse effect of these nanoparticles should be carefully investigated. The multinucleation effect of SNPs was firstly reported in our previous studies, while the relative mechanism is still unclear. Therefore, the purpose of this study is to investigate the mechanisms with regard to the formation of multinucleated cells. Two sizes of amorphous SNPs (Nano‐Si64 and Nano‐Si46) are carefully characterized. Cytotoxicity and rate of multinucleated cells are firstly determined after human hepatic L‐02 cells are treated with two SNPs for 24 h. Then cell fusion and abnormal mitosis, two ways could form multinucleated cells, are investigated, respectively. Results indicated that SNPs produce a dose‐dependent and size‐related multinucleation effect in L‐02 cells. Abnormal mitosis instead of cell fusion is the main reason for the formation of multinucleated cells caused by SNPs. Both two SNPs could affect the quantity and distribution of cytoskeleton through extra ROS and Ca²⁺ leading to abnormal mitosis and cytokinesis. Additionally, chromosome damage resulting in corresponding G2/M cell cycle arrest should be another aspect, which finally leads to the formation of multinucleated cells in L‐02 cell line.     

Chromatoid body: remnants of nucleolar proteins during spermatogenesis in triatomine (Heteroptera, Triatominae)

In this study, we analyzed fibrillarin nucleolar protein expression in CBs of spermatogenic cells from testicular follicles of Triatoma sordida and Triatoma infestans. In the structural and ultrastructural analysis, it was used impregnation by silver ions, immunocytochemistry, immunofluorescence and transmission electron microscopy using antibodies against fibrillarin. Regarding the results, the fibrillarin nucleolar protein marked the nucleus and some cytoplasmic spots of germ cells during spermatogenesis in triatomines. These data suggest that fibrillarin could be a constituent of the CB that was most likely derived from nucleolar fragmentation. This is the first time that fibrillarin protein expression has been shown in the CB during spermatogenesis progression in triatomines. The knowledge regarding CB constituents may help to expand the understanding of the physiological role of this structure and the role that it plays in the reproductive biology of triatomines, which are vectors of Chagas Disease.     

Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

Background  

In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events.