Mathematical analysis of the oncornavirus maturation process (virion RNA conversion and morphological condensation)

The rate of the maturation process of avian myeloblastosis virus experimentally estimated on the basis of genomic viral RNA conversion and morphological transition of virions was mathematically analysed. Three mathematical models were suggested and fitted to experimental data. It was found that: (a) model of simple kinetics (Model 1) does not agree with experimental data. Therefore, two hypotheses were considered in further mathematical modelling: (b) virions are identical in time of budding: maturation is dependent on the presence of a virion component which is degraded with time (Model 2). This model agrees with experimental data in all stages of the maturation process. (c) Virions are released from cells at different stages of assembly (Model 3). This model differs from experimental data especially in early stages of maturation. The hypothesis used for the construction of Model 2 seems to be the most plausible to explain the maturation process and is in agreement with data of murine leukemia virus maturation which was found to be accomplished by cleavage of p70 precursor protein.     

Alteration of vimentin intermediate filament expression during differentiation of human hemopoietic cells

We describe the alterations of vimentin intermediate filament (IF) expression in human hemopoietic committed precursors as they differentiate into mature cells of the erythroid, granulomonocytic, megacaryocytic and lymphoid lineages. A double labelling fluorescence procedure was used to identify hemopoietic cells expressing lineage-specific antigens and to decorate the vimentin IF network. Whereas very early progenitors from each lineage expressed vimentin, the density and organization of the network differed strikingly as the cells matured on a given pathway. T lymphocytes, monocytes and granulocytes retained vimentin expression at all stages of maturation. In contrast, megakaryoblasts lose vimentin expression at a very early stage of differentiation, erythroblasts at variable steps between the committed erythroid cell and the red cell. Finally, B lymphocytes tend to lose vimentin expression later when they mature into plasma cells.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stages as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7-day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/ maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Kinetics and cellularity of erythroblasts in the growing rat

Abstracts. Parameters of recognizable erythroid cell proliferation were measured in four groups of normal rats weighing 50, 100, 150 and 300 g in order to provide a comprehensive set of data suitable for a re-investigation of erythropoietic models. Total erythroblast cellularity was measured by the ⁵⁹Fe technique, DNA synthesis time by quantitative ¹⁴C-autoradiography, and the erythrocyte production rate was derived from the increase with time of the erythrocyte labelling index after repeated injections of ³H-leucine. Furthermore, the relative erythroblast density was determined in the various morphological compartments. From the total erythroid cell mass in DNA synthesis and the absolute erythrocyte production rate, figures were derived for the mean DNA synthesis time of erythroid cells and compared with the directly measured ones. The discrepancies in all weight groups between direct and indirect determination of DNA synthesis time were considerable. In a previous study re-evaluation of comparable data in literature had revealed comparable inconsistencies. Since a critical discussion of possible errors in the experimental techniques does not indicate data acquisition to be the principal source of disagreement it is concluded that the type of model applied to describe how cells pass the boundaries of morphological cell compartments is of high significance. Models based on a sequential flux of cells through the individual compartments are inadequate for evaluation of the presented set of data.     

Estimating the proportion of proliferating cells in a population.

In a population of cells that proportion which is actively engaged in the proliferative cycle is often estimated from the ratio of the observed labelling index to an expected labelling index, calculated, on the basis of all cells being in cycle, from the cell cycle phase durations and the age distribution. Ignoring the variability in cell cycle times may lead to large overestimates or underestimates in the expected labelling index. A method is given of obtaining a more accurate estimate of this variable, and hence of the proliferative proportion.     

Down-regulation of Myc is essential for terminal erythroid maturation

Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.     

CELL CLASSIFICATION AND KINETIC ASPECTS OF NORMOBLASTIC AND MEGALOBLASTIC ERYTHROPOIESIS

Mitotic indices and ³H-thymidine flash labelling indices have been determined in a total of 6000 erythroid cells from patients with megaloblastic anaemia (vitamin B₁₂ deficiency) and 4000 erythroid cells from patients with increased, normoblastic erythropoiesis. In the anaemic states there is a lack of mitoses in the more mature erythroid compartments relative to the number of mitoses in the early erythroid precursors; this must reflect skipped division and/or cell death in the later precursors. In order to further locate the deficit of mitoses, erythropoietic cells were subdivided in a way which aimed at stratifying them according to cell generations. It appears that there are four consecutive generations of recognizable proliferating red cell precursors. Balance considerations of mitotic figures suggest that in stressed normopoiesis all cells which enter generation III divide, whereas only about one-half of cells leaving generation III divide again in generation IV. In megaloblastic erythropoiesis it appears that only about one of three cells which leave generation III divide in generation IV. The data suggest that in megaloblastic anaemia, skipped division and/or cell death to a large extent take place in generation IV or at the transition from III to IV. In normoblastic erythropoiesis, the ratio labelled cells/mitotic cells is rather independent of cell maturation. In contrast, this ratio varies considerably in megaloblastic erythropoiesis, from 25:1 in early forms to 4-5:1 in late forms. As an explanation of the lack of mitoses, relative to cells in DNA-synthesis, in the early stages and the relative surplus of mitoses in the late stages it is proposed that cell cycle and cytological boundaries do not coincide in all cells. The present observations can be accounted for if a significant fraction of cells change their morphology (from basophilia to polychromasia) between their DNA-synthesis phase and the subsequent mitosis. It cannot be decided whether in addition there is a death function between DNA-synthesis and mitosis in the large basophilic megaloblasts, megaloblastic system could absorb a direct entry from the large basophilic cells amounting to perhaps about one-half of the flux through the S-pool of the large basophilic cells without being more dominated by very large cells than is actually the case; still, in large measure this will depend on the time from entry into the polychromatic pool until the subsequent mitosis or possible cell death. The alternative is a significant death function between S-phase and mitosis at the level of the large basophilic E1-E2 cells (generation I + II).     

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays

Abstract. Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bro-modeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G₂ for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.     

牛卵母细胞体外成熟的研究

牛卵母细胞的成熟过程中,包括细胞膜、细胞质、细胞核的成熟。其中细胞质的成熟最为复杂。线粒体、皮质颗粒数量的变化和位移,脂滴类型的变化和形态改变,空泡形态学的变化等是鉴别卵母细胞幼稚、成熟和老化的重要特征。透明带随着培养而外侧疏松,内侧致密,母卵细胞膜上伸出的微绒毛为膨大泡状和细长毛状两种。在培养１４小时后颗粒细胞与透明带脱离联系。根据综合指标判定,１８小时这前为成熟生长期,１８￣２６小时为成熟期,     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis

Abstract. Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

AN EVALUATION OF ERYTHROPOIESIS IN THE YOUNG RAT

Estimates of the cell population kinetic parameters have been obtained for the erythroid cells of the young growing rat using the technique of labelled mitoses and these results have been analysed by a computer programme. The phases of the cell cycle for the proliferating cells have been shown to be of shorter duration than generally reported. Together with the differential cell count and initial labelling index these data have enabled estimates of the growth fraction, birthrate, flow rate, number of divisions and transit time to be determined for each compartment.     

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis.

Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

Application of a DNA double labelling method for the flow cytometric analysis of recruitment of non-cycling cells in a mixed population of P and Q cells

Abstract. In this paper we describe the application of a non-radioactive DNA double labelling and staining method to an analysis of cell proliferation kinetics by flow cytometry, aimed at the direct measurement of recruitment rates in cell cultures. The method is based on the application of two halogenated deoxyuridines: iododeoxyuri-dine (IdUrd) and chlorodeoxyuridine (CldUrd) which are incorporated into DNA synthesizing cells. By applying two commercially available monoclonal antibodies both deoxyuridines can be detected separately. To measure recruitment all proliferating cells in a plateau phase culture were labelled first with IdUrd applied during a time interval approximately equal to the cell cycle time. Subsequently, recruitment induced by a medium change was analysed by flow cytometric assessment of incorporation of CldUrd in cells which had not taken up IdUrd.
Experiments designed to determine the toxicity of continuous labelling with IdUrd in different concentrations and of pulse labelling with CldUrd showed that there was no effect on the progression of cells through the cell cycle. The aim of this study is to test the sensitivity of the procedure to detect changes in proliferation kinetics, in particular the entrance of resting cells into the S phase. Although the cell culture model used is very simple, the results demonstrate clearly that a low rate of recruitment can be detected. It is suggested that the procedure described here is specific and sensitive enough to quantify changes in cell proliferation in tumours induced by various treatments and has advantages over other methods, which measure recruitment indirectly, or directly by using two radioactive thymidines.     

Evidences that hemoglobin switch in the chick embryo depends on erythroid cell line substitution

Chemical identifications of various hemoglobin types were performed on unfractionated erythroid cells derived from chicken embryos at 5 and 7 days of development and on purified primitive and definitive cells. Proteins were pulse-labelled in primitive erythroid cells at various times of culture to identify those actually synthesized. The data show that primitive cells contain and synthesize only embryonic hemoglobins at all stages of maturation and definitive cells contain adult and minor embryonic hemoglobins, but no major embryonic hemoglobins, not even in trace amounts. These results support a model for hemoglobin switch in the chicken embryo based on cell line substitution.     

Unique Pattern of Gene Expression in the Erythroid Precursor Cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU-E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt-genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt-genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU-E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt-genes may be induced after the stimulation of erythropoietin.     

Kinetics of spermatogenesis in the wild squirrel Funambulus palmarum (Linnaeus).

The paper describes in detail the morphology and kinetics of germ cell associations, pattern of mitotic divisions, frequency distribution of different cellular associations (stages) and percent degeneration of various germ cells in the squirrel in which spermatogenesis in adults occurs all year round. Eighteen steps of spermiogenesis were identified based on the development of the acrosomal system using PAS-haematoxylin. These were appropriately divided into Golgi, acrosome, cap and maturation phases. Thirteen types of cellular associations or stages (I-XIII) were characterized along the length of the seminiferous tubule which repeated itself in space and time constituting the seminiferous epithelial cycle (CSE). Of the 18 steps of spermiogenesis, the first 13 were associated with stages I-XIII, respectively, and the rest with the first 9 stages. Spermiation occurred in stage IX. Seven types of spermatogonia [A0, A1, A2, A3, A4, intermediate (In) and B type] were identified based on their shape, size and nuclear morphology. A0 spermatogonia are pale in appearance with homogeneously distributed chromatin surrounded by a thin nuclear membrane. These are present in all stages. A1 are oval in shape and possess a thicker nuclear membrane. They are found in stages VI-X. The chromatin material undergoes progressive condensation from A1 to A4 making the last generation of spermatogonia appear darker. The In spermatogonia which are derived from A4 are morphologically similar to them but smaller in size. The B-type spermatogonia derived from the In types possess a typically round nucleus with uniformly condensed chromatin material underneath the nuclear membrane. The spermatogonia divide mitotically at fixed stages of the CSE giving rise to their next generations. Thus, A-type spermatogonia divide at stages X, XIII/I, IV and V, while In divide at stage VI. During each CSE of the squirrel, 5 peaks of mitosis occur. There is a single generation of B-type spermatogonia. These differentiate into primary spermatocytes and undergo meiosis or maturation divisions which enter to form spermatids. The A4 which divide differentially in stage VI give rise to In- and A1-type spermatogonia. Therefore, A4 spermatogonia form renewing stem cells. Based on the above pattern of spermatogonial mitosis a model for stem cell renewal in the squirrel is proposed. The percentage degeneration of germ cells varied with the cell type. During a single CSE of the squirrel, a total of 42.09% germ cells were found to degenerate. An attempt is made to compare and contrast the kinetics of spermatogenesis in the wild squirrel with that of the other rodents studied so far.     

氯化高铁血红素诱导K562细胞红系分化对其细胞周期的影响

细胞周期的测量是细胞增殖动力学的研究基础。通过添加30μmol·L-1氯化高铁血红素(Hemin)诱导人慢性髓系白血病K562细胞红系分化,利用5-溴脱氧尿嘧啶核苷(BrdU)与7-AAD双染的方法检测Hemin诱导的K562红系分化细胞对细胞周期各期比例的影响,未诱导的K562细胞周期各期比例作为对照,检测发现Hemin诱导的K562红系分化细胞对其细胞周期相对值无明显影响。应用BrdU间隔染色结合流式细胞术的方法,通过分析BrdU间隔染色后BrdU阳性细胞群的动态变化规律,从而推算出K562红系分化细胞的倍增时间及细胞周期各期时长。根据测量结果发现,未诱导的K562细胞总倍增时间约为20 h,与通过生长曲线公式法计算倍增时间的结果相符,Hemin诱导的K562细胞的细胞周期倍增时长约为23 h。Hemin诱导的K562红系分化细胞较未诱导的K562细胞倍增时间与各期时长无明显差异。因此,Hemin诱导K562细胞红系分化对其细胞周期绝对值及相对值均无明显影响。