向日葵是观察生殖核(雄配子)的好材料

向日葵(Helianthus annuns)的小孢子是具三细胞(核)的小孢子体(花粉),2个生殖核(细胞)细而长,1个营养核呈椭圆形,三核同时存在的时间长,容易区分,是观察三核小孢子体的一种好材料。     

Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.     

红豆草根瘤侵染细胞核在细胞凋亡中的超微结构变化

用透射电镜观察红豆草根瘤侵染细胞核在细胞凋亡过程中的超微结构,以探讨红豆草根瘤侵染细胞核在发育过程中的超微结构变化及其与细胞凋亡的关系.结果表明,红豆草根瘤侵染细胞核的超微结构随细胞发育程度不同而不同.在幼龄侵染细胞中,细胞核体积较大,近似圆形.在即将成熟和成熟的侵染细胞中,细胞核膜有内陷现象,其核仁常具有核仁泡和核仁联合体.在早期凋亡的侵染细胞中,细胞核体积减小,形状变得不规则,核膜出现大量内陷,在其表面形成许多大的突起和深的沟槽,有时还有内质网、线粒体、小液泡和细菌等位于核膜的内陷处,而且核仁也开始裂解.在后期凋亡的侵染细胞中,除细菌解体外,还出现核仁消失,核膜破裂,核质外流,并在细胞质中形成一些电子密度很高,无一定形状的团块状物质.     

NUCLEAR SYNCHRONY IN VALONIA MACROPHYSA(CHLOROPHYTA): LIGHT MICROSCOPY AND FLOW CYTOMETRY1

The coenocytic alga Valonia macrophysa Kützing was selected for an investigation of nuclear synchrony in the order Siphonocladales. Light microscopy reveals that nuclear synchrony is evident as patches of nuclei dividing simultaneously. Flow cytometry was utilized for the first time with a macroalga for cell-cycle analysis. Results indicate that nuclei in the entire cell exhibit a high degree of synchrony throughout the cell cycle. Also it appears that cells within a clonal culture are synchronous with each other, in their progression through the cell cycle. The advantages of using flow cytometry for cell-cycle analysis of coenocytic algae include the rapid collection of quantitative data on relative DNA content for a large number of nuclei.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

Numbers of myonuclei and satellite cell nuclei in latissimus dorsi muscles of the chicken

Summary In the 3-, 33- and 66-day-old chicken, two muscles, the oxidative slow tonic anterior latissimus dorsi and the glycolytic fast twitch posterior latissimus dorsi were compared by the measurement of muscle fibre diameter and the fraction of total muscle tissue nuclei which were either myonuclei or satellite cell nuclei. Between 3 and 33 days there was a period of rapid growth (more marked in the posterior latissimus dorsi) which coincided with a sharp fall in numerical density of myonuclei and satellite cell nuclei (number per cubic millimetre muscle tissue). The fraction of all nuclei which were satellite cell nuclei declined steadily.The higher levels of myonuclei and satellite cell nuclei in the anterior latissimus dorsi were thought to be a reflection of its oxidative metabolism and the presence of multiple endplates.The volume of sarcoplasm occupied by single myonuclei in anterior and posterior latissimus dorsi muscles was shown to be considerably greater than that occupied by nuclei in other cell systems.     

Fluorescent vital staining of plant sexual cell nuclei with DNA—specific fluorochromes and its application in gametoplast fusion

DNA－binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stined,the simultaneous visualization of fluorochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated.prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4,6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Purification of nuclei from a flagellate protozoan, Trypanosoma brucei

A simple and rapid technique is described for the isolation of nuclei from the flagellate protozoan Trypanosoma brucei. Cells were disrupted by nitrogen cavitation in the presence of hexylene glycol which enhances nuclear stability. Isolated nuclei were separated from nuclei trapped with cytoskeletal fragments and flagellae by Percoll density gradient centrifugation. Centrifugation in a medium with increased sucrose concentration greatly improved the separation of free nuclei from those trapped in cell debris. The isolation procedure resulted in the recovery of 34% of the nuclei present in the whole cell suspension. Electron microscopic and chemical analysis indicated that the recovered nuclei were in good condition and were highly purified.     

Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.     

White Blood Cells Image Classification Using Deep Learning with Canonical Correlation Analysis

White Blood Cells play an important role in observing the health condition of an individual. The opinion related to blood disease involves the identification and characterization of a patient's blood sample. Recent approaches employ Convolutional Neural Network (CNN), Recurrent Neural Network (RNN) and merging of CNN and RNN models to enrich the understanding of image content. From beginning to end, training of big data in medical image analysis has encouraged us to discover prominent features from sample images. A single cell patch extraction from blood sample techniques for blood cell classification has resulted in the good performance rate. However, these approaches are unable to address the issues of multiple cells overlap. To address this problem, the Canonical Correlation Analysis (CCA) method is used in this paper. CCA method views the effects of overlapping nuclei where multiple nuclei patches are extracted, learned and trained at a time. Due to overlapping of blood cell images, the classification time is reduced, the dimension of input images gets compressed and the network converges faster with more accurate weight parameters. Experimental results evaluated using publicly available database show that the proposed CNN and RNN merging model with canonical correlation analysis determines higher accuracy compared to other state-of-the-art blood cell classification techniques.     

The nuclear ceramide/diacylglycerol balance depends on the physiological state of thyroid cells and changes during UV-C radiation-induced apoptosis

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Autoradiography of Developing Syncytia in Cotton Roots Infected with Meloidogyne incognita

Cotton (Gossypium hirsutum) seedlings, uniformly infected with Meloidogyne incognita, were exposed for periods of 1-15 days to a nutrient solution containing tritium-labelled thymidine. Syncytium formation began with the amalgamation of cells near the nematode head, and was followed by synchronized mitoses of the nuclei which had been incorporated into a single cell. Syncytial nuclei synthesized DNA in roots harvested 3, 6, 9, 12, and 15 days after inoculation. Seedlings transferred from unlabelled to labelled nutrient solution 9 days after inoculation, and grown for 6 more days, contained some syncytial nuclei which did not become labelled. Giant-cell nuclei increased in size and, in many cases, all nuclei in one giant cell of a set showed active DNA synthesis at about the time the nematode molted to the adult stage.     

Cellular organization and appearance of differentiated structures in developing stages of the parasitic platyhelminth Echinococcus granulosus

Echinococcus granulosus is the causative agent of hydatidosis, a major zoonoses that affects humans and herbivorous domestic animals. The disease is caused by the pressure exerted on viscera by hydatid cysts that are formed upon ingestion of E. granulosus eggs excreted by canine. Protoscoleces, larval forms infective to canine, develop asynchronously and clonally from the germinal layer (GL) of hydatid cysts. In this report, we describe the cellular organization and the appearance of differentiated structures both in nascent buds and developed protoscoleces attached to the GL. Early protoscolex morphogenesis is a highly complex and dynamic process starting from the constitution of a foramen in the early bud, around which nuclei are distributed mainly at the lateral and apical regions. Similarly, distribution of nuclei in mature protoscoleces is not homogenous but underlies three cellular territories: the suckers, the rostellar pad, and the body, that surrounds the foramen. Several nuclei are associated to calcareous corpuscles (Cc), differentiated structures that are absent in the earlier bud stages. The number of nuclei is similar from the grown, elongated bud stage to the mature protoscolex attached to the GL, strongly suggesting that there is no significant cellular proliferation during final protoscolex development. The amount of DNA per nucleus is in the same range to the one described for most other platyhelminthes. Our results point to a sequential series of events involving cell proliferation, spatial cell organization, and differentiation, starting in early buds at the GL of fertile hydatid cysts leading to mature protoscoleces infective to canine.     

Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones

Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.     

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells

Summary Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the, amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.Abbreviation NEB nuclear-envelope breakdown