The relationship between cell volume,ploidy, and functional activity in differentiating hepatocytes

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radio-immunoassay. The capacity of this cell type to store³⁵S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up^99mTC-colloid sulfur and to incorporate⁵⁹Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.     

The relationship between megakaryocyte ploidy and platelet volume

The origins and biologic significance of platelet heterogeneity in general, and platelet volume heterogeneity in particular, have been controversial scientific issues during the past decade. Although it has generally been held that specific megakaryocyte properties, especially ploidy level, are important determinants of platelet volume, the precise relationship between megakaryocyte properties and platelet properties is not well defined. The physiologic processes that specifically determine the relationship between megakaryocyte ploidy and platelet volume are unclear, and understanding of these processes has been further complicated due to the multiplicity of experimental and clinical models used to study the problem. Although it is generally true that increases in megakaryocyte ploidy are associated with increases in megakaryocyte volume, it is not well established that platelet volume is also increased during normal or abnormal thrombopoiesis as a direct result of a change in the ploidy level. Reexamination of earlier studies and some recent investigations suggest that changes in platelet volume and megakaryocyte ploidy are in fact dissociated in response to experimental thrombocytopenia. Critical review of the literature concerning the relationship between megakaryocyte ploidy and platelet volume reveals a limited number of conclusions that are well substantiated and emphasizes the relative lack of understanding about the events governing the complex process of platelet production and platelet heterogeneity.     

The relation between cell size, ploidy level and nitrate concentration in lettuce

In two experiments the relation between cell size, ploidy level and nitrate concentration of genotypes of lettuce ( Lactuca saliva L.) was investigated. Artificial increase of cell size by application of gibberellic acid to young plants decreased the percentage dry weight and the concentration of organic acids in the plant sap and increased the nitrate concentration of the shoot. Application of chlormequat chloride caused the opposite effects. Tetraploid genotypes of lettuce had larger cells, a lower nitrate concentration and a higher concentration of organic solutes than diploid genotypes. Genotypical differences in nitrate concentration could not be explained by differences in cell size, but were consistently related to differences in concentration of organic acids in plant sap. The percentage dry weight of genotypes was positively correlated with the concentration of organic acids in plant sap. However, differences in nitrate concentration were also found among genotypes with comparable dry matter content. These differences could be caused by differences in partitioning of photosynthates over structural components and organic acids in the vacuoles. Mono-saccharides seem to be less important than organic acids for explaining genotypical differences in nitrate level, because the concentration of monosaccharides was much lower than that of organic acids and because a negative relation between the concentration of monosaccharides and of nitrate was not found in every case. Genotypical differences in nitrate accumulation were not related to differences in osmolarity.     

The relationship between cell carbon and cell volume in freshwater algal species used in zooplanktonic studies

New records of cell carbon and cell volume are given for fivespecies of freshwater algae cultured optimally for use in physiologicalstudies on Daphnia spp. These are listed with other values forfreshwater algae from the literature, giving a total of 41 determinationsfor 25 species, together with details of culture media employed,formulae used for calculation of volume and techniques for carbondetermination. The following significant regression of cellcarbon on cell volume was fitted to 37 points: C=0.1204V^1.051(pg C,µm³) and the regression coefficient was found notto differ significantly from 1.0. Significant simple linearand polynomial regressions could also be fitted to the untransformeddata. There was a significant reduction of residual mean squarein the polynomial regressions when tested against the linearregressions and this demonstrated the curvilinearity of therelationship. The polynomial regressions predicted a more orless constant carbon-volume ratio of     

Quantitative analysis of the relationship between nucleotide sequence and functional activity.

Matrices can be used to evaluate sequences for functional activity. Multiple regression can solve for the matrix that gives the best fit between sequence evaluations and quantitative activities. This analysis shows that the best model for context effects on suppression by su2 involves primarily the two nucleotides 3' to the amber codon, and that their contributions are independent and additive. Context effects on 2AP mutagenesis also involve the two nucleotides 3' to the 2AP insertion, but their effects are not independent. In a construct for producing beta-galactosidase, the effects on translational yields of the tri-nucleotide 5' to the initiation codon are dependent on the entire triplet. Models based on these quantitative results are presented for each of the examples.     

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.     

The relationship between cell injury and osmotic volume reduction: II. Red cell lysis correlates with cell volume rather than intracellular salt concentration

It is possible to simulate freezing by suspending cells in progressively hyperosmotic solutions. It is not generally possible, however, to discriminate between cell volume reduction and solute concentration as the cause of injury since, in a normal cell behaving as an osmometer, volume is an obligate function of solution osmolality. The paper describes experiments in which osmolality and volume were disassociated by loading red cells with additional KCl by making them slowly permeable to potassium through treatment with valinomycin. It is shown that cell hemolysis is associated with the reduction of cell volume beyond some minimum volume regardless of the concentration of intracellular or extracellular electrolyte. Similarly, it is shown that hemolysis from thermal shock is related to a decrease in cell volume rather than to an increase in solute concentration.     

Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

The relationship between RNA content, cell volume and growth potential in ageing human embryonic mesenchymal cells

Modal cell volume and cellular RNA content were meaume was found to correlate with both culture replication rate and cellular RNA content. This observation suggests that cell volume changes may be related to the rate of cell division during ageing in vitro, and that both cell volume and RNA content may be closely controlled. There was no change in the relative proportions of the major rRNA species during the cultures' lifespan.     

Effects of anisosmotic medium on cell volume,transmembrane potential and intracellular K+ activity in mouse hepatocytes

Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V _m ) and intracellular K⁺ activity (a _K ⁱ ) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V _m increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV _m /controlV _m )] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea _K ⁱ did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K⁺ or 1mm quinine HCl or at 27°C resulted in no change inV _m compared with a 20-mV increase inV _m without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda _K ⁱ in response to anisosmotic medium; however,V _m behaves as an osmometer under these conditions. Also, increases inV _m by hypoosmotic stress were abolished by conditions or agents that inhibit K⁺ conductance.     

Nuclear DNA endoreduplication during petal development in cabbage: relationship between ploidy levels and cell size

The development of cabbage petals comprises two distinct phases: a cell division phase and a consecutive phase of cell expansion until the onset of opening. In this study, cytological changes characterizing the two phases of petal development were analysed. First, the mitotic activity and the surface area of epidermal cells during petal development were investigated. The DNA content of isolated nuclei from the different stages of petal tissues was determined by flow cytometric analysis. The results show that cell differentiation, leading to expanded cells, is characterized by endoreduplication. In the proximal part of the petal, after cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. By contrast, normal diploid nuclei remained in the distal part of the lamina in the mature petal. It is suggested that the developmental programmes of the cabbage petal may be a trigger for the initiation of endoreduplication. Correlation between ploidy levels and cell size is also discussed.     

Correlation between the number of centrioles and ploidy of mouse liver hepatocytes

Using the electron microscope it was shown that in interphase hepatocytes with ploidies equal to 2n, 2n.2, 4n, 4n.2 and 8n, the number of centrioles per cell exactly corresponded to the ploidy of the cell. Both in mononuclear and binuclear cells all the centrioles are accumulated in one complex in which each pair of centrioles forms a diplosome. In binuclear cells, the complex of diplosomes is situated at equal distances from each nucleus, thus making the cell centre. The involvement of the supernumerous centrioles in polyploid metaphase cells was detected for the regenerating liver of old mice. It was found that each mitotic pole had at least four centrioles. In the pole, a pair of centrioles forms diplosomes tightly connected to each other. It is suggested that the initially tetraploid cell might divide in this manner. In addition, a question is discussed on how the existence of centrioles can be associated with the mechanism of polyploidization.     

The relationship between settled volume and displacement volume in samples of freshwater zooplankton

The results of some volume estimates of zooplankton biomassare presented. The results suggest that, with freshwater zooplankton,the relationship between settled volume and displacement volumeremains relatively constant despite seasonal variations in speciescomposition.     

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression,grade and DNA ploidy in breast cancer

Quantification of c-erb B-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erb B-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erb B-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erb B-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erb B-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P=0.06). Immunohistochemistry revealed a significant relationship between c-erb B-2 expression and aneuploidy (P=0.04). Cytokeratinpositive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erb B-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erb B-2 expression at a level of more than 3200 molecules (P0.03). At the level of 3600 molecules significance was increased (P=0.004). These levels equated with between 15% and 19% of the samples being classified as c-erb B-2 positive. Application of these cut-off points showed no correlation between c-erb B-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P=0.0015), increased grade correlating with aneuploidy.     

Cell volume affects glycogen phosphorylase activity in fish hepatocytes

The activity of glycogen phosphorylase (GPase) in the active a-form (GPase a) is dependent on the hydration state of hepatocytes. We establish that GPase a catalysis in catfish (Ameiurus nebulosus) hepatocytes is a function of medium osmolarity and that a linear relationship exists between GPase a activity and osmolarity between 254 mosmol l^–1 and 478 mosmol l^–1. Exposure of isolated hepatocytes to hyperosmotic media increases enzyme activity up to 7-fold, indicative of covalent phosphorylation. GPase activation associated with cell shrinkage peaks within 10 min of exposure. The average degree of activation (2.7-fold-increase of GPase a) is only slightly less than in hepatocytes exposed to glucagon (3.1-fold-increase) under isosmotic conditions; with glucagon, the maximum is reached within 2 min. Phosphorylation status remains elevated during the entire 40 min experimental period; cells do not undergo regulatory volume increase (RVI) during this period and do not regain pre-exposure volume. We interpret the increased GPase a activity as an inherent response to hyperosmotic stress, likely brought about by molecular crowding. Activation of the enzyme results in increased glucose production from endogenous glycogen. Glucose is not retained in the liver cells, but may act as an oxidative substrate in extrahepatic tissues for the increased metabolic demand of ion regulation. Protein kinase A or intracellular Ca²⁺ make apparently small contributions to the activation of GPase, leaving us to speculate on alternate routes of enzyme activation. Conversely, hepatocyte swelling in hyposmotic medium leads to significant decreases in GPase a activity and curtailed glucose output. A minimum is attained in 10 min, and pre-insult rates are re-established within 40 min, somewhat lagging behind readjustment in cell volume by regulatory volume decrease (RVD). We conclude that cell swelling and subsequent RVD do not signify stress to the cells and metabolic demand may be decreased under cell swelling conditions. Alteration of GPase phosphorylation with extracellular osmolarity appears to be a general phenomenon, since we also find it in hepatocytes of another freshwater catfish (Clarias batrachus) and a marine scorpaenid (Sebastes caurinus).Abbreviations BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid - BSA bovine serum albumin - cAMP adenosine 3',5-cyclic monophosphate - GPase glycogen phosphorylase - MDH malate dehydrogenase - MHM modified Hanks medium - PKA c-AMP dependent protein kinase A - 8-Br-Rp-cAMPS 8-Bromo-Rp-3',5'-cyclic adenosine monophosphorothioate - RT room temperature - RVD regulatory volume decrease - RVI regulatory volume increaseCommunicated by L.C.-H. Wang     

The relationship between stem cell seeding efficiency and position in cell cycle.

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.