The 3D structure of human chromosomes in cell nuclei

The spatial arrangement of some genetic elements relative to chromosome territories and in parallel with the cell nucleus was investigated in human lymphocytes. The structure of the chromosome territories was studied in chromosomes containing regions (clusters) of highly expressed genes (HSA 9, 17) and those without such clusters (HSA 8, 13). In chromosomes containing highly expressed regions, the elements pertaining to these regions were found close to the centre of the nucleus on the inner sides of chromosome territories; those pertaining to regions with low expression were localized close to the nuclear membrane on the opposite sides of the territories. In chromosomes with generally low expression (HSA 8, 13), the elements investigated were found symmetrically distributed over the territories. Based on the investigations of the chromosome structure, the following conclusions are suggested: (1) Chromosome territories have a non-random internal 3D structure with defined average mutual positions between elements. For example, RARα, TP53 and Iso-q of HSA 17 are nearer to each other than they are to the HSA 17 centromere. (2) The structure of a chromosome territory reflects the number and chromosome location of clusters of highly expressed genes. (3) Chromosome territories behave to some extent as solid bodies: if the territory is found closer to the nuclear centre, the individual genetic elements of this chromosome are also found, on average, closer the centre of the nucleus. (4) The positions of centromeres are, on average, nearer to the fluorescence weight centre of the territory (FWCT) than to genes. (5) Active genes are not found near the centromeres of their own territory. A simple model of the structure of chromosome territory is proposed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Specificity of arrangement of human chromosomes in the nucleus of a moving cell

Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 10, pp. 479–481. October, 1989.     

The eleven stages of the cell cycle,with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis

Since we had subdivided the cell cycle into 11 stages—four for mitosis and seven for the interphase—and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041–3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: ‘free,’ ‘attached’ to chromatin accumulations, and ‘compacted’ in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin ‘aggregates’ appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin ‘bulges’ also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into ‘chromomeres,’ a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular ‘bands’ at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The “nucleoli” at stages I–III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the ‘prenucleolar bodies,’ which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase “pause,” during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the “chromosome condensation” period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II–IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. The process continues until, soon after S ends, that is in G2, every segment of nucleofilament has become part of aggregates and bulges which thus include wholly new nucleofilaments. Then, the bonds tying aggregates and bulges to the matrix are broken, leaving them free at prophase (stage V) to approach one another, adhere, and thus build chromomeres. At prometaphase (substage VIa), a last adhesion process unites the chromomeres of a row which thus build metaphase chromosomes. Meanwhile, nucleoli lose the histone free-DNA patches at stage IV and disappear at the end of stage VIa. 3) Stages VIb and VII constitute the “climax” of the cell cycle, a 20-min period during which metaphase chromosomes at the equatorial plate split into daughter chromosomes. 4) From stage VIII to XI, the “chromosome decondensation” period extends over about 2 hr during which progressive dissociation of the stage VIII chromosomal mass eventually yields free and attached nucleofilaments as well as the three heterochromatins. Meanwhile lucent vesicles fuse into prenucleolar bodies which finally coalesce into nucleoli. Anat. Rec. 252:426–443, 1998. © 1998 Wiley-Liss, Inc.     

D-type cyclins in adult human testis and testicular cancer: relation to cell type,proliferation, differentiation,and malignancy

D-type cyclins are proto-oncogenic components of the ‘RB pathway’, a G1/S regulatory mechanism centred around the retinoblastoma tumour suppressor (pRB) implicated in key cellular decisions that control cell proliferation, cell-cycle arrest, quiescence, and differentiation. This study focused on immunohistochemical and immunochemical analysis of human adult testis and 32 testicular tumours to examine the differential expression and abundance of cyclins D1, D2, and D3 in relation to cell type, proliferation, differentiation, and malignancy. In normal testis, the cell type-restricted expression patterns were dominated by high levels of cyclin D3 in quiescent Leydig cells and the lack of any D-type cyclin in the germ cells, the latter possibly representing the only example of normal mammalian cells proliferating in the absence of these cyclins. Most carcinoma-in-situ lesions appeared to gain expression of cyclin D2 but not D1 or D3, while the invasive testicular tumours showed variable positivity for cyclins D2 and D3, but rarely D1. An unexpected correlation with differentiation rather than proliferation was found particularly for cyclin D3 in teratomas, a conceptually significant observation confirmed by massive up-regulation of cyclin D3 in the human teratocarcinoma cell line NTera2/D1 induced to differentiate along the neuronal lineage. These results suggest a possible involvement of cyclin D2 in the early stages of testicular oncogenesis and the striking examples of proliferation-independent expression point to potential dual or multiple roles of the D-type cyclins, particularly of cyclin D3. These findings extend current concepts of the biology of the cyclin D subfamily, as well as of the biology and oncopathology of the human adult testis. Apart from practical implications for the assessment of proliferation and oncogenic aberrations in human tissues and tumours, this study may inspire further research into the emerging role of the cyclin D proteins in the establishment and/or maintenance of the differentiated phenotypes. Copyright © 1999 John Wiley & Sons, Ltd.     

Regulation of cathepsin E expression during human B cell differentiation in vitro

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up-regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.     

三七皂甙R1诱导HL—60细胞系分化的形态和功能变化

用三七皂甙R_1(80μg/ml)处理HL-60细胞,发现68%的HL-60细胞向中性粒细胞分化,其中晚幼粒细胞32%,杆状核粒细胞30%,分叶核粒细胞6%;进一步实验显示分化后细胞硝基蓝四氮唑(NBT)还原能力、吞噬功能、细胞膜补体受体以及酸性磷酸酶(ACP)和β葡萄糖醛酸酶(β-Gr)活性都明显增高。结果表明,三七皂甙R_1能诱导HL-60细胞向中性粒细胞分化。     

Regulation of human B-cell activation,proliferation, and differentiation by soluble factors

This review describes a series of studies performed in our laboratory which have focused on the activation and subsequent proliferation and differentiation of human B lymphocytes. Utilizing polyclonal signals which activate B cells by interacting with their surface membrane Ig, we have examined the events in the transition of a resting B lymphocyte to an Ig-secreting cell. A major theme in these studies is the role of soluble factors in B cell proliferation and B cell differentiation. Specific B cell growth and differentiation factors are described. Data on their sources, biochemical characterization, and methods of assay are included. Additionally, the potential role of interleukin 2 in human B cell function is discussed. The recognition that B cells exist in a variety of activation states and that transition between different states is dependent upon different signals was the impetus for a series of studies which more precisely delineated these states and the signals involved. These findings and the observations from other investigators led to a proposed model of the sequential steps in a factor-dependent B cell differentiation pathway. This model is discussed and related to the mechanisms of T cell activation and growth. Finally, the effects of two pharmacologic agents, glucocorticoids and cyclosporin A, on human B cell function are described and discussed in the context of this model.     

Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components—a finding resulting from the biological follow-up of unbiased human genetic studies.     

Cell cycle analysis and detection of proliferative cell nuclear antigen of the endometrium after hormone replacement therapy

Background: To understand the effect of sequential combined hormone replacement therapy (HRT) on the postmenopausal endometrium. Methods: Sonographic endometrial thickness, endometrial histopathology, flow cytometric cell cycle analysis and the level of proliferative cell nuclear antigen (PCNA) were studied. Results: One hundred and thirty-eight postmenopausal women were enrolled in this study. Among which, 97 women had their endometrium being adequately obtained; the most frequent type of histopathology was normal endometrium (91.8%). Endometrial hyperplasia was found in seven patients (7.2%), including typical simple hyperplasia (n=1, 1%), focal simple hyperplasia (n=5, 5.2%) and complex hyperplasia without atypia (n=1, 1%). The proliferative fractions (PF; S plus G₂–M phase) of cells from normal and hyperplastic endometrium of menopausal women after HRT were 8.18 and 8.95%, respectively, which were lower than those from 29 premenopausal women without HRT. The level of PCNA of normal and hyperplastic endometrium in postmenopausal women after HRT was about 80 and 84%, respectively, of that from premenopausal endometrium. Conclusions: Our study showed the PF of the cell cycle and the level of PCNA were not increased in the menopausal endometrium under HRT as compared to the premenopausal controls.     

Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells

We have monitored the organization of DNA topoisomerase II (Topo II) in relation to chromatin disaggregation during mitogen stimulation of lymphocytes and to the mitotic chromosome condensation cycle by immunofluorescence microscopy with isozyme-specific antibodies. Labelling for both Topo II and Topo II was diffusely nucleoplasmic and non-nucleolar in resting lymphocytes and the pattern changed little during stimulation. Topo II labelling intensity increased in parallel with the extent of cell stimulation, but a fraction of fully stimulated cells was labelled very brightly. Topo II labelling intensity was also greater in stimulated cells, but all partially and fully stimulated cells were labelled at the same, higher, intensity. In addition, anti-Topo II detected a few small spots within nucleoli of stimulated cells that coincided with regions containing fibrillarin. In lymphocytes and HeLa, chromosome association of Topo II began in prophase and lasted throughout mitosis. In contrast, Topo II stayed nucleoplasmic in prophase, was diffusely cytoplasmic during mitosis, and was first detected post-mitotically in nuclel with decondensing chromosomes and a reformed nuclear envelope. The results are consistent with a role for Topo II, but not for Topo II, in mitotic chromosome condensation, and indicate that the isotypes may play independent roles in the reorganization of chromatin structure during lymphocyte mitogenic activation.accepted for publication by T. D. Allen     

Phenotypic changes and cell cycle activation in early tubulointerstitial injury of rat adriamycin nephrosis

Tubular response, including phenotypic changes against a variety of injuries, is an initial event that promotes tubulointerstitial injuries. Using the progressive kidney disease model of rat adriamycin (ADR) nephrosis, the present study focused on the cell cycle activation and phenotypic changes that occur in the tubuli in early tubulointerstitial injury in ADR nephrosis. At 12 weeks, experimental animals developed overt nephrosis with tubulointerstitial injury, which correlated well with the degree of proteinuria and incidence of glomerulosclerosis. Initial pathology of the tubuli showed a slight dilatation of tubuli, which tended to occur in individual nephrons. Immunohistochemistry demonstrated that vimentin-positive tubuli and osteopontin (OPN)-positive tubuli were associated mostly with proliferating cell nuclear antigen expression. Protein levels of OPN in the renal cortex were correlated with the level of proteinuria by western blotting. Vimentin- and OPN-expressing tubuli were tightly associated with a peritubular influx of alpha-smooth muscle actin (SMA)-positive cells or ED-1-positive cells. In addition, we found thrombomodulin+/ TUNEL+ (dUTP-biotin nick-end labeling) peritubular endothelial cells and ED-1+/alpha-SMA+ cells at an early stage among interstitial inflammatory cells. These results suggest that cell cycle activation in tubular cells forms the background for the phenotypic tubular changes that are involved in chronic tubulointerstitial injury in ADR nephrosis.     

Metastasizing APUD cell tumours of the human gastrointestinal tract. Light microscopic and karyometric studies

A total of 11 metastasizing gastrointestinal APUD cell tumours from biopsy and autopsy files were reclassified according to Soga and Tazawa as well as to WHO Histologic Classification of Tumours. The much higher proportion of APUD cell tumours in autopsies (11 cases from 1000 autopsies in comparison with 22 cases from 22 000 biopsies) demonstrate that the majority of them will not be discovered during the patient's life. EC cell carcinoids (type A) predominate in both non-metastasizing and metastasizing gastrointestinal APUD cell tumours. Metastases from EC cell carcinoids occurred only in regional lymph nodes and in the liver. The APUD cell tumours originating in the pancreas represent the most frequently metastasizing gastrointestinal carcinoids. Besides in the liver and in regional lymph nodes metastases from pancreatic APUD cell tumours were seen in the skin, the brain and the skeleton. One case with two (pancreatic, bronchial) competitive primary APUD cell tumours and a skin metastasis was studied by means of automated cell image analysis. Cell populations of these three tumour sites were characterized by morphometric and densitometric nuclear parameters. It could be demonstrated that the skin metastasis consisted of a cell population, which occurred as a subpopulation in the primary tumour of the bronchus. The results of karyometric investigations supported the hypothesis that single components of tumours can metastasis selectively.     

体外构建人羊膜间充质干细胞膜片及成骨分化潜能

文题释义：细胞膜片技术：该技术避免了蛋白酶的消化和外源性支架材料的应用,通过细胞外基质分泌形成膜片组织,然后将膜片用于修复组织缺损和改善器官功能。该技术保留了大量自体细胞分泌的细胞外基质,为细胞的增殖和分化提供与体内极度相似的微环境,目前该技术已经用于临床眼角膜和食管损伤的修复。人羊膜间充质干细胞：取自于废弃的胎盘,贴壁生长,具有低免疫原性和生长周期短等特点,不仅具有成体间充质干细胞的特性还具有部分胚胎间充质干细胞的特性。  摘要背景：人羊膜间充质干细胞属于成体干细胞,其来自于废弃的胎盘,来源广泛,可以无创获取,具有免疫原性低、生长周期短等特点,是组织工程种子细胞的重要来源,目前人羊膜间充质干细胞已经用于临床糖尿病的治疗。目的：探索一种简便的方法构建人羊膜间充质干细胞膜片,并研究其成骨分化潜能。方法：将第3代人羊膜间充质干细胞高密度接种于普通培养皿中,加入成膜片诱导培养基以构建人羊膜间充质干细胞膜片,通过组织学染色以及扫描电镜观察细胞膜片的特性。取第3代人羊膜间充质干细胞高密度接种于培养皿中,加入成膜片诱导培养基培养7 d,再换用成骨诱导培养基培养14 d以构建成骨诱导的人羊膜间充质干细胞膜片。通过茜素红染色、免疫组化染色、碱性磷酸酶活性、RT-PCR以及Western blot检测人羊膜间充质干细胞膜片的成骨分化潜能。结果与结论：①苏木精-伊红染色可见人羊膜间充质干细胞膜片由多层细胞累积而成,细胞分布均匀;②扫描电镜观察可见人羊膜间充质干细胞膜片呈复层结构,胞外有大量的胞外基质产生,细胞包埋于胞外基质中;③人羊膜间充质干细胞膜片成骨诱导14 d,茜素红染色后可见橘红色沉淀,免疫组化染色后细胞周围有大量Ⅰ型胶原产生;④与未诱导的人羊膜间充质干细胞膜片相比,成骨诱导14 d后人羊膜间充质干细胞膜片碱性磷酸酶活性显著升高(P < 0.01),Ⅰ型胶原、骨钙蛋白、Runt相关转录子2的mRNA和蛋白表达量显著升高(P < 0.05);⑤该实验应用一种简便、经济的方法在普通培养皿上成功构建了人羊膜间充质干细胞膜片,体外研究证实人羊膜间充质干细胞膜片具有良好的成骨分化潜能。ORCID: 0000-0003-2163-3897(邹刚)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Induction of the ganglion cell differentiation program in human retinal progenitors before cell cycle exit

Background: Despite the disease relevance, understanding of human retinal development lags behind that of other species. We compared the kinetics of gene silencing or induction during ganglion cell development in human and murine retina. Results: Induction of POU4F2 (BRN3B) marks ganglion cell commitment, and we detected this factor in S‐phase progenitors that had already silenced Cyclin D1 and VSX2 (CHX10). This feature was conserved in human and mouse retina, and the fraction of Pou4f2⁺ murine progenitors labeled with a 30 min pulse of BrdU matched the fraction of ganglion cells predicted to be born in a half‐hour period. Additional analysis of 18 markers revealed many with conserved kinetics, such as the POU4F2 pattern above, as well as the surprising maintenance of “cell cycle” proteins KI67, PCNA, and MCM6 well after terminal mitosis. However, four proteins (TUBB3, MTAP1B, UCHL1, and RBFOX3) showed considerably delayed induction in human relative to mouse retina, and two proteins (ISL1, CALB2) showed opposite kinetics, appearing on either side of terminal mitosis depending on the species. Conclusion: With some notable exceptions, human and murine ganglion cell differentiation show similar kinetics, and the data add weight to prior studies supporting the existence of biased ganglion cell progenitors. Developmental Dynamics 243:712–729, 2014. © 2013 Wiley Periodicals, Inc.     

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.