Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro

Pulse labelling experiments with [³H] thymidine (dT) and double labelling experiments with [³H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 ± 2.7% (x?± SEM) and T_s= 7.2 ± 0.7h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (T_c) of 11.2–14.2 h. The longer T_c values (18–20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G₁/S and S/G₂ borders of in vivo vs. in vitro studies.     

Mitotic sympathetic neuroblasts initiate axonal pathfinding in vivo

Neuronal precursor proliferation and axodendritic outgrowth have been traditionally regarded as discrete and sequential developmental stages. However, we recently found that sympathetic neuroblasts in vitro often elaborate long neuritic processes before dividing. Furthermore, these “paramitotic” neurites were maintained during cell division and neuritic morphology was consistently preserved by daughter cells after mitosis. This inheritance of neuritic morphology in vitro raised the possibility that proliferating neuroblasts engage in axodendritic outgrowth. To determine whether mitotic superior cervical ganglion (SCG) neuroblasts are engaged in pathfinding in vivo, we have combined retrograde axonal tracing of efferent nerve trunks with bromodeoxyuridine (BrdU) labeling of cells in S‐phase. In fact, about 13% of BrdU(+) cells were retrogradely labeled, indicating that mitotic neuroblasts often have extraganglionic axonal projections. Moreover, the presence of axons during S‐phase was observed at two developmental ages (E15.5 and E16.5), implicating an ongoing function of paramitotic axons during neuronal ontogeny. Using a calculation to account for experimental limitations, we estimate that virtually all mitotic SCG neuroblasts have direct access to extraganglionic signals during development. We conclude that mitotic neuronal precursors in vivo engage in pathfinding, raising the possibility that interaction of proliferating populations with distant signals actively coordinates cell division and neural connectivity. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 366–374, 1999     

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4°C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling. Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4°C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriusly compromises the results of this technique.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

Abstract. In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G₂ phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6–12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine ([³H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [³H]TdR labelling was simulated. The presence of two distinct sub-populations in G₂ phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G₁ phase is suggested. The sizes of the slowly cycling fractions in G₁ and G₂ showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3–5%) was arrested in G₂ phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G₂ cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Kinetic heterogeneity of an experimental tumour revealed by BrdUrd incorporation and mathematical modelling

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA-BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell-cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA-BrdUrd fluorescence distribution. Sequential DNA-BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (T _pot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.     

Comparison of isotopic and immunohistochemical methods of studying epithelial cell proliferation in hamster tongue

Our purpose was to validate different approaches to the study of cell proliferation in stratified squamous epithelia, using oral mucosa as a model. Dorsal and ventral tongue from the hamster were examined following in vivo labelling with tritiated thymidine and bromodeoxyuridine (BrdUrd), and in vitro labelling with BrdUrd. These were compared with direct immunolabelling of fixed tissue sections with monoclonal antibody PC10. For the former methods S phase cells were quantified following autoradiography or immunohistochemistry. We conclude that the proliferative status of simple, flat, lining mucosae such as ventral tongue can be derived by all three prelabelling methods and, on average, 18–19 cells per surface millimetre length were in DNA synthesis. On the other hand dorsal tongue epithelium, which is thicker, has an undulating morphology and a complex cell renewal pattern, gives different results with the three labelling methods. In both sites the proliferating cell nuclear antigen (PCNA) index was fourfold that obtained by nucleotide labelling. This is consistent with PCNA marking proliferative cells in other phases of the cell cycle in addition to the S phase. Thus, there are potential differences between the information on proliferative status derived by PCNA immunohistochemistry and other established cell cycle markers, which need to be taken into account in the interpretation of epithelial cell kinetic data in health and disease.     

Chromatin-bound PCNA as S-phase marker in mononuclear blood cells of patients with acute lymphoblastic leukaemia or multiple myeloma

Objectives: Proliferating cell nuclear antigen (PCNA) has often been used as a marker to aid assessment of tumour growth fraction. This paper addresses the question of whether it can be used as an S‐phase marker, when the non‐chromatin‐bound form of the protein is removed by pepsin treatment. Materials and methods: Cytofluorometric measurements were carried out after immunofluorescence staining of PCNA and counterstaining of DNA. S‐phase fraction was determined with the help of windows on PCNA versus DNA scattergrams, or mathematically from DNA histograms. Results: S‐phase fractions obtained using the two methods correlated well, but did not always agree, exact discrepancies depending on the mathematical model used for histogram analysis. Conclusions: Determination of S‐phase fractions with the help of PCNA immunofluorescence staining is possible, and probably more reliable than calculation of S‐fractions from DNA histograms. It thus offers an alternative to assays involving BrdU labelling in vivo.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

Summary A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37° C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ

Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments, where also differences between cell types were noted. We asked whether similar differences between species and between cell types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to 7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in vivo. Accepted: 5 January 2000     

Cell proliferation kinetics of six xenografted human cervix carcinomas: comparison of autoradiography and bromodeoxyuridine labelling methods

Abstract. Cell kinetic and histologic parameters of six xenografted tumours with volume doubling times ranging from 6 to 43 d were investigated in order to obtain kinetic information on a panel of tumours to be used in radiobiological studies. The six tumours covered a range of histologies and their DNA indices varied from 2–7 to 1–4. The length of the cell cycle (T_c), potential doubling time (T_pot) and labelling index (LI) were determined by continuous labelling with [³H]TdR and autoradiography in three tumours. T_c varied from 30 to 40 h. Determinations of the length of the S phase (T_s) were found to be less reliable by this method. Data on T_s and LI were also determined in all six tumours using bromodeoxyuridine (BrdU) labelling and the single sample method; values of T_pot were slightly longer than those obtained via the autoradiographic method. In addition, multiple samples were taken after BrdU labelling. T_c was determined by fitting the data obtained from mid-S, mid-G₂ and mid-G₁ windows to curves described by a damped oscillator. Data obtained via the mid-S window were found to be most reliable. Generally, cell cycle times obtained by the BrdU method were longer than those observed with the autoradiographic method. Differences between the two methods could be explained by inaccuracies in the determination of T_s, LI and T_c and differences in the experimental approach. We consider the BrdU labelling method to be a suitable alternative for the time-consuming autoradiography, if data on T_s or T_pot are sufficient. Due to difficulties in the reproducibility of the immunofluorescence staining and asynchronization of cells approximately 10 h after labelling, the method of windows analysis was affected by similar problems to those observed in interpretation of percentage labelled mitosis (PLM) curves. However, the method may serve as an alternative to determine cell cycle times in vitro and, if improved technically, in vivo. Careful comparison of the data obtained from mid-S, mid-G₁ and mid-G₂ windows may increase the reliability of the determination of cell kinetic parameters.     

THE EFFECT OF VINCRISTINE ON MOUSE JEJUNAL CRYPT CELLS OF DIFFERING CELL AGE: DOUBLE LABELLING AUTORADIOGRAPHIC STUDIES USING 3H- AND 14C-TdR

The mechanism of action of the alkaloid vincristine (VCR) has been investigated in vitro on HeLa cells in culture and in vivo on jejunal crypt cells of the mouse. The in vitro experiments with HeLa cells show that VCR affects not only mitotic but also interphase cells. The VCR-affected cells first continue their passage through the cell cycle undisturbed but after reaching mitosis they are arrested in metaphase. This agrees well with the results obtained by Madoc-Jones & Mauro (1968) and Madoc-Jones (1973) on synchronized cell cultures. Until now there has been no investigation of the mechanism of action of VCR in vivo. This is due to the absence of a suitable technique for synchronization in vivo. The present study is based on a method which permits the assessment of the VCR sensitivity as a function of the cell age without synchronization in the usual sense. The jejunal crypt epithelium of the normal mouse was double labelled with ³H- and ¹⁴C-thymidine (TdR) in such a way as to produce a narrow subpopulation of crypt cells with a maximum age difference of 1 hr. On autoradiographs these cells can be distinguished by their characteristic labelling from other cells. As this ‘pseudo’-synchronized subpopulation passes through the cycle the effect of VCR can be studied, i.e. one can analyse the effect in well-defined time intervals of the cycle. The results show that the effect of VCR is the same in vivo as in vitro. The crypt cells which are affected by VCR in interphase continue their passage through the cycle, but upon entering mitosis they are arrested in metaphase. VCR has, at the concentration used in the present study, no effect on the duration of the S and G₂ phases. The necrotic cells seen after VCR application are formed from arrested metaphases.     

A COMPARISON OF AXONALLY TRANSPORTED PROTEINS IN THE RAT SCIATIC NERVE BY IN VITRO AND IN VIVO TECHNIQUES

An in vitro system for studying fast axonal transport in mammalian nerves has been developed. The viability of in vitro nerve preparations was established on the basis of three criteria: electron microscopy, electrical properties, and the activities of two marker enzymes, 5'-nucleotidase and total ATPase. The specific activity of transported proteins was greater using the in vitro procedure, and the level of locally incorporated radioactivity lower, when compared to in vivo transport experiments. Separation of solubilized transported proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate showed that a large number of polypeptides are transported. Using a double label procedure which employed L-[³H]methionine and L-[³⁵S]methionine, proteins transported in vitro and in vivo were compared. No differences in the electrophoretic distribution of transported proteins from the two systems was seen. The major component of transported proteins electrophoresed with an apparent molecular weight of 105,000 ± 24,000. Using the in vitro system, transported proteins were compared to those labelled locally in either Schwann cells or cells of the dorsal root ganglion. Large differences in the labelling patterns were observed in both comparisons. We conclude that in vitro procedures provide a valid means of studying rapid axoplasmic transport. The proteins carried by rapid axoplasmic transport differ from those synthesized in either the Schwann cells of the sciatic nerve or the cells of the dorsal root ganglion.     

Cell division and cell enlargement during potato tuber formation

Cell division and cell enlargement were studied to reveal the developmental mechanism of potato tuberization using both in vivo in vitro culture systems. Distribution of cells in S-phase was visualized by immunolabelling of incorporated bromodeoxyuridine (BrdU). Mitosis was detected in DAPI (4,6-di-amidino-2-phenylindole) or toluidine blue-stained sections. Timing and frequency of cell division were determined by daily cell counting, and cell enlargement was deduced from measurements of cell diameters.Under in vivo conditions, lateral underground buds developed into stolons due to transverse cell divisions and cell elongation in the apical region of the buds. At the onset of tuber formation, the elongation of stolons stopped and cells in pith and cortex enlarged and divided longitudinally, resulting in the swelling of the stolon tip. When tubers had a diameter of 0.8 cm, longitudinal divisions had stopped but randomly oriented division and cell enlargement occurred in the perimedullary region and continued until tubers reached their final diameter.In vitro tubers were formed by axillary buds on single node cuttings cultured under tuber-including conditions. They stopped growing at a diameter of 0.8 cm. Pith and cortex were involved in tuberization such as that found during the early stage of in vivo tuberization (<0.8 cm in diameter). The larger size of in vivo tubers is, however, due to further development of the perimedullary region, which is lacking in vitro conditions.Keywords: Cell division, cell enlargement, DNA synthesis, in vitro culture, potato, tuber formation.      

Variation of H1 Content throughout the Cell Cycle in Regenerating Rat Liver

Histone H1⁰ a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1⁰ content. These observations suggest that H1⁰ may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1⁰ decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1⁰ in the nucleus. We defined new protocols to analyze H1⁰ content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1⁰ and the position of cells in the cell cycle showed that no significant difference in H1⁰ content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1⁰ and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1⁰ and that the decrease of H1⁰ is not a prerequisite of cell division. We propose that the accumulation of H1⁰ is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Resonance Raman quantification of the redox state of cytochromes b and c in‐vivo and in‐vitro

We observe the redox state changes with respiration of cytochromes b and c in mitochondria in a living Saccharomyces cerevisiae cell as well as in isolated mitochondria with the very use of Raman microspectroscopy. The possibility of monitoring the respiration activity of mitochondria in vivo and in vitro by Raman microspectroscopic quantification of the cytochrome redox states is suggested. It will lead to a new means to assess mitochondrial respiration activity in vivo and in vitro without using any labelling or genetic manipulation. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Circatidal variation in epithelial cell proliferation in the mussel digestive gland and stomach

Epithelial cell renewal in mussel (Mytilus galloprovincialis, Lmk) digestive gland and stomach was investigated by bromodeoxyuridine (BrdU) immunohistochemistry. Mussels were exposed to 4 mg BrdU/l seawater continuously. Starting at 6 h after treatment, samples were collected every 2 h for 2 days and BrdU labelling was estimated by direct counting at the light microscope, with values being noted per thousand BrdU-positive cells. BrdU-positive reaction was observed in the nuclei of digestive, basophilic, duct and stomach cells, and in haemocytes. Cell renewal in digestive diverticula was synchronised following a circatidal pattern: BrdU labelling increased during low tide and decreased during high tide. Clearcut mitotic figures were identified in digestive cells, thereby confirming that mature cell types proliferate, in agreement with results from immunohistochemistry for proliferating cell nuclear antigen and BrdU. Epithelial cell renewal in the stomach also appeared to be synchronised.This investigation was funded by the Basque Government (GVPI95-36 and GVP99-1) and by a grant to Consolidated Research Groups (UPV/EHU)     

CELL SPECIFICITY OF CHALONE-TYPE INHIBITORS OF DNA SYNTHESIS RELEASED BY BLOOD LEUCOCYTES AND ERYTHROCYTES

Inhibitors of DNA synthesis released into balanced salt solution by rat erythrocytes and by rat leucocytes have been found to possess target-cell-specific properties which would be expected of chalones. When assayed in short-term in vitro cultures the erythrocyte product reduced DNA synthesis (as measured autoradiographically) in erythroblasts present in populations of bone-marrow cells but did not affect the DNA synthesis in myeloid or lymphoid cells. The leucocyte product, under the same culture conditions, reduced DNA synthesis in leucocyte precursor cells. The grain counts over nuclei of different cell types were recorded as well as the DNA labelling index. Results so far obtained cannot ascribe the erythrocyte-chalone-produced reduction in labelling index to a blockage of entry into S phase. This cell-specific inhibitor may reduce continuing DNA synthesis in S phase cells to undetectable levels, compared with synthesis in control media. The leucocyte product, however, most probably prevents entry of leucocyte precursor cells into S phase. Possible relevance of these inhibitors as components of physiological control mechanisms or as therapeutic agents is discussed.