FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid–specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

流式细胞仪在生物学中的应用

简要论述了流式细胞仪（flow cytometry,FCM）的工作原理,并对其在生物学基础科学研究中的应用进行阐述,包括对细胞凋亡、细胞周期、免疫细胞、细胞受体的研究应用。     

Unknown G0/G1 Cell Subpopulation Revealed by Hydrochloric Acid/Acridine Orange Staining

An unknown cell subpopulation was observed in mouse and rat thymus, spleen and bone marrow cells, as well as in human peripheral blood mononuclear cells (resting and stimulated by PHA) using equilibrium HCl/acridine orange staining. This subpopulation includes cells with decreased green and unchanged red fluorescence. The staining does not affect cells in S- and G₂/M-phases. The mechanism and biological meaning of the effect await further investigation.     

DNA content analysis of insect cell lines by flow cytometry

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flow Cytometry studies of marine phytoplankton populations

Abstract

Flow cytometry has been applied to the study of phytoplankton populations and cell components. In this work, cells cycle studies on three marine diatoms cultures were carried out by a flow cytometer. Phaeodactylum tricomutum, Chaetoceros simplex and Thalassiosira allenii, showed different DNA patterns in G1, G2, S, phases. This situation may be related to the specific algal physiology. Cultures of P. tricomutum were maintained in 4 media with different silicates concentrations. The lack of silicates did not seem produced a significative cells arresting in the cell cycle phases. During the experiment, the cell fraction in S phase, decreased in all media tested. These preliminary results could be further developed to apply flow cytometry to environmental problems in order to identify general algal groups and study algal physiology in different growth conditions.     

On the trophic fate of Phaeocystis pouchetii (Hariot). II. Grazing rates of Calanus hyperboreus (Krøyer) on diatoms and different size categories of Phaeocystis pouchetii

Experiments were conducted with CIV and C V copepodites of Calanus hyperboreus (Krøyer) to determine if they would feed on the prymnesiophyte Phaeocystis pouchetii (Hariot). We used analysis of gut pigment to estimate ingestion and clearance rates. In applying this methodology we have demonstrated that pigments can be completely extracted from whole animals within 90 min, and that laborious procedures of tissue homogenization and centrifugation are not required. We conducted two experiments. In the first experiment Stage IV copepodites were exposed to ≈1 mg C·1⁻¹ of either P. pouchetii flagellates, small colonies (25–200 μm), large colonies (> 200 μm) or mixed diatoms > 25 μm (primarily Chaetoceros socialis Lauder and Nitzschia grunowii Hasle). Ingestion rates and daily rations were almost four times greater on both sizes of colonies than on either Phaeocystis pouchetii flagellates or mixed diatoms. Daily rations of copepodites feeding on colonies ranged from 8.1 to 12.4% · day⁻¹, well within the range previously reported for Calanus hyperboreus or sympatric copepods of similar size. From the second experiment we determined that Stage V copepodites obtained a daily ration of 6.2 to 10.8% · day⁻¹ when feeding on small colonies of Phaeocystis pouchetii. We conclude that a diet of P. pouchetii colonies should sustain the metabolic and growth requirements of Calanus hyperboreus copepodites.     

鼻咽癌相关基因NGX6对鼻咽癌细胞周期的影响

为了探讨鼻咽癌（NPC）候选抑瘤基因NGX6对NPC细胞的细胞周期进程及细胞周期素的影响,阐明它的作用机制,通过建立稳定表达NGX6的鼻咽癌HNE1细胞株,采用细胞免疫组织化学,流式细胞仪检测与分析细胞周期及细胞周期素的改变,用western blot验证它对细胞周期的影响。结果显示稳定表达NGX6的HNE1细胞较对照组细胞周期中G0/G1期比值明显增加,而S期比例减少。细胞凋亡率无明显变化。流式细胞仪检测发现cyclinD1、A和E的表达明显减少,以cyclinD1的改变最为明显。Western blot检测也发现cyclinD1的表达明显下调。以上结果说明NGX6主要通过下调cyclinD1的表达,延缓细胞周期的G1→S的进程,从而抑制NPC细胞的过度增殖。     

用流式细胞光度术研究PGE2对小鼠骨髓CFU—GM细胞周期的影响

在一定PGE_2浓度(4.8×10~(-9)mol/L)作用下,小鼠骨髓细胞CFU-GM经4.5d和7d培养后,其增殖状态下的细胞G_n/G_r期细胞数均比对照组增加,S期细胞数减少,G_2 M期细胞数也有下降,但不明显。在不同PGE_2浓度(2.8×10~(-9)～2.8×10~(-6)mol/L)作用下,经4d培养,随着PGE_2浓度增加,G_n/G_1期细胞数递增,而S期细胞数却随PGE_2浓度增加而减少,G_2 M细胞数也减少,但与PGE_2剂量关系不明显。以上实验结果提示,PGE_2主要抑制G_1期细胞向S期细胞的转化,阻断S期细胞生长。 此外,通过流式细胞光度术(FCM)对小鼠骨髓细胞CFU-GM集落细胞的前向角和90°散射光测定,与对照组比较,前者无变化,后者变化较明显。此结果表明,PGE_2对细胞内部颗粒的折光度有影响。     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: a population analysis

Single-cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G(1), S(1) and G(2) + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single-cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G(1) phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G(1) phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G(2) + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.     

STRATEGIES AND KINETICS OF PHOTOACCLIMATION IN THREE ANTARCTIC NANOPHYTOFLAGELLATES1

Three Antarctic nanophytoflagellates (two cryptophyte species and a Pyramimonas sp.) were compared for their capacity to phiotoacclimate and for their kinetic responses in changing photic environments. Division rate, cell size cellular fluorescence, and chlorophyll a content were measured steady and transient states of semi-continuous cultures maintain at 1.0° C. Of all parameters tested, cell size was most affected by irradiance. Acclimation kinetics were modeled using a first-order equation. Rates of change in cell size following shifts in irradiance were comparable with rates of change in chemical composition reported for temperate algae. Response rates of cellular in vivo red and orange fluorescence were lower. In many cases, however, responses could not be described by the first-order kinetic model. Division rates remained high for approximately 3 days following a shift down in irradiance, after which new division rates were established. The nanoflagellates studied here appear to respond to small irradiance perturbations at low rates. However, they may fail to adapt and abrupt changes in photon flux density (PFD). When shade-adapted (25 μmol, m^?2, m^?2, s^?1) cells were exposed to high PFD (400 μmol, m^?2, s^?1) for 1–3 days, cell were incapable of readapting division rate and pigment content to the initial irradiance condition (25 μmol, m^?2, s^?1) for about 1 month following the shift-down step. The ecological role of the kinetics of photoacclimation in nanophytoflagellate growth performance in Antarctic ecosystems is discussed.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Flow Cytometry of Mitotic Cells

We have developed a procedure using flow cytometric measurement of a mitosis-specific antigen that may be used to count mitotic cells and sort them from nonmitotic cells. The procedure may also be used in conjunction with measurement of cellular DNA content and of bromodeoxyuridine incorporation into cellular DNA to assign cells to the G1/G0, S, G2, or M phase of the cell cycle.     

利用流式细胞仪研究拟南芥叶发育过程中细胞周期的调控

叶的形态建成依赖于细胞不断地分裂增殖和不同类型细胞的特化。在叶发育早期,叶细胞主要通过旺盛的有丝分裂来增加原基中细胞的数目。随着叶片的生长,叶细胞自顶部向基部逐渐退出有丝分裂进入内复制来增加细胞的倍性,同时伴随细胞的扩展和分化。本文介绍利用流式细胞仪研究双子叶模式植物拟南芥叶发育过程中细胞周期调控的方法和具体研究实例。我们发现至少存在3种类型的细胞周期异常的拟南芥叶发育突变体。此外,我们还介绍利用流式细胞仪测定DNA复制效率的方法。