Relative Cell Size Research Articles

Rabbit esophageal cells show regulatory volume decrease: Ionic basis and effect of pH

Background: Regulatory volume decrease (RVD) after osmotic cellular swelling has been shown in several gastrointestinal epithelia but not in esophageal cells. In acid reflux disease, esophageal injury may be related in part to loss of RVD. Methods: Isolated basal esophageal cells were exposed to an external hyposmolar solution, and changes in relative cell size were assessed using a Coulter counter (Hialeah, FL) in the presence of K+ and Cl− transport inhibitors and at varying extracellular pH (pH0). Results: At pH0 7.4, a 30% hyposmotic dilution of the external solution caused an initial peak cell swelling (1.15 ± 0.05-fold) followed by a return to starting cell size by 5 minutes (RVD). RVD was inhibited by Ba2+ (4 mmol/L), quinine (1 mmol/L), or increasing the [K+]0 ≥ 10 mmol/L. RVD was also inhibited by depleting [Cl−]1 or in the presence of 0.5 mmol/L 4,4′-diisothiocyanastostilbene-2,2′-disulfonic acid disodium salt (DIDS) or 50 μmol/L diphenylamine-2-carboxylate, a Cl− conductance inhibitor. To test the effect of pH on RVD, cells in solutions at pH0 7.4, 7.0, or 6.8 were subjected to hyposmotic stress; RVD was significantly inhibited at pH0 6.8. This pH-dependent inhibition of RVD was reversed in the presence of valinomycin, a K+ ionophore. Conclusions: These studies show that isolated esophageal cells possess RVD mechanisms that are mediated by Cl−- and pH-dependent K+ effluxes. RVD appears to be inhibited by a decrease in pH0, suggesting the possibility that acid-induced esophageal injury results from inhibition of normal volume regulatory mechanisms.

Epidermal calcium-binding protein: a marker of early differentiation of basal layer keratinocytes of rats.

Epidermal calcium-binding protein (ECaBP) is present in the cells of the basal layer of the epidermis and other stratified epithelia. Since the basal layer compartment contains at least two types of cells: slow-cycling, poorly-differentiated, and actively proliferating, more differentiated cells, it was of interest to determine whether they both contained ECaBP. Basal and nearly suprabasal layer keratinocytes from newborn rat epidermis were fractionated into three fractions on the basis of cell size, using low-gravity sedimentation. The cell differentiation in each subgroup was estimated by cell size, morphology, cell cycle stage, RNA/DNA content, and the presence of specific keratins. The presence of ECaBP in these fractions was detected by immunocytochemistry and immunoblotting. Double staining with ECaBP antibodies and propidium iodide followed by flow cytometry was used to correlate ECaBP production and the stage of cell cycle. The relative cell size, measured by the light scattering was used to study the relationship between cell size and ECaBP production. The results show that small keratinocytes with low DNA and RNA content (G0 cells) do not express ECaBP. ECaBP was found only in intermediate size basal keratinocytes with higher DNA and RNA contents, corresponding to actively proliferating S phase cells. Large keratinocytes, which express suprabasal keratin and have low DNA and high RNA content, cease to express ECaBP. ECaBP may, therefore, be a useful marker for assessing the movement of cells from poorly differentiated reserve compartment towards proliferation and further differentiation in both physiological and pathological situations.

Effect of dilution rate on growth, productivity, cell cycle and size, and shear sensitivity of a hybridoma cell in a continuous culture

To study the effects of the growth rate of the hybridoma cell Mn12 on productivity, cell cycle, cell size, and shear sensitivity, six continuous cultures were run at dilution rate of 0.011, 0.021, 0.023, 0.030, 0.042, and 0.058 h(-1). This particular hybridoma cell appeared to be unstable in continuous culture with respect to specific productivity, as a sudden drop occurred after about 30 generations in continuous culture, accompanied by the appearance of two populations with respect to the cytoplasmic lgG content. The specific productivity increased with increasing growth rate. The shear sensitivity of the cell, as measured in a small air-lift loop reactor, increased with increasing growth rate. The mean relative cell size, as determined with a flow cytometer, increased with increasing growth rates. Furthermore, the fraction of cells in the S phase increased, and the fraction of cells in the G1/G0 phase decreased with increasing growth rates.

Cell Count and Size in Relation to Fruit Size Among Strawberry Cultivars

Fruit size, number of receptacle cells, and mean cell size were determined throughout development of secondary fruit of three day-neutral strawberry (Fragaria ×ananassa Duch.) cultivars grown in a greenhouse. Cells were counted after enzymatic separation of receptacle tissue, and mean cell volume was estimated from cell count and receptacle tissue volume. Size of mature fruit was small (3.8 g) in `Tillikum', medium (11.5 g) in `Tristar', and large (15.6 g) in `Selva'. Fruit size was correlated with the number of achenes per berry. Mature fruit of `Tillikum' had a lower fruit fresh weight per achene and lower achene population density (achenes per square centimeter) than the larger-fruited cultivars. The average number of cells per mature fruit was 0.72 × 106, 1.96 × 106, and 2.94 × 106 for `Tillikum', `Tristar', and `Selva', respectively. The relative difference among cultivars in the number of receptacle cells was established by the time of anthesis. In all cultivars, cell division was exponential for 10 days following anthesis and ceased by the 15th day. Mean cell volume increased slowly during active cell division, but rose rapidly and linearly for 10 days after cell division halted. Mean cell volume of all cultivars increased > 12-fold after anthesis and was ≈ 6 × 106 μm3 in mature fruit. The genotypic variation in the size of mature fruit was not the result of large differences in either duration of cell division after anthesis or mean cell volume, but rather was primarily due to differences in the number of receptacle cells established by anthesis.

Fetal dexamethasone exposure alters macromolecular characteristics of rat brain development: a critical period for regionally selective alterations?

Fetal glucocorticoid exposure retards postnatal growth and evokes abnormalities of nervous system structure and function. To examine the underlying mechanisms, we administered 0.2 or 0.8 mg/kg of dexamethasone to pregnant rats on gestational days 17, 18, and 19 and assessed brain region cell development with indices of DNA content (total cell numbers), DNA concentration (cell packing density), and protein/DNA ratio (relative cell size). Dexamethasone evoked deficits of pup body and brain region weights, but the brain regions displayed growth-sparing associated initially with preservation of cell numbers (normal or elevated DNA content and concentration), at the expense of relative cell size (decreased protein/DNA). Subsequently, brain cell acquisition lagged behind that of controls, with deficits in DNA and elevations of protein/DNA. In midbrain + brainstem and in cerebellum, cell markers returned to normal by weaning. However, the forebrain showed persistent elevations of DNA and reduced protein/DNA, indicative of replacement of neurons with glia. Because the treatment period coincided with the timing of neuronal cell replication in the forebrain, but not in the other regions, these results suggest that the critical period for lasting deficits of dexamethasone coincides with the peak of neuronal mitosis.

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures.

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension batch culture. The relative cell size, cytoplasmic and membrane IgG content and the viability were monitored. Besides, the specificity of the cytoplasmic and membrane IgG was ascertained by means of a synthetic peptide containing the antigenic epitope recognized by the antibody. Cell size was found to increase during the exponential growth phase. The viability as determined by FC follows a similar pattern with the viability data obtained by the conventional trypan blue exclusion test. The relative cytoplasmic and membrane IgG contents were high during the exponential growth and low during stationary phase. Measurement of cell cycle distribution and the antibody content in the culture fluid, indicated that the major part of the cytoplasmic IgG is secreted by cells in the G1-phase. It is concluded that flow cytometry is a useful tool to characterize hybridoma cell lines in a suspension batch culture.

Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts.

Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.

Fetal dexamethasone exposure impairs cellular development in neonatal rat heart and kidney: Effects on DNA and protein in whole tissues

Fetal glucocorticoid exposure causes postnatal growth retardation. To examine the mechanisms underlying effects on specific organ systems, we administered 0.2 or 0.8 mg/kg of dexamethasone to pregnant rats on gestational days 17, 18, and 19 and assessed three biochemical markers of cell development in heart and kidney of the offspring: DNA content per organ as an index of total cell numbers, DNA per g tissue as an index of cell packing density, and protein/DNA ratio as an index of relative cell size. In both tissues, DNA content became markedly subnormal during the first postnatal week, the ontogenetic period of rapid cell division. Partial recovery occurred by the end of the first postnatal month. In the heart, cell packing density was subnormal initially and the cells were significantly enlarged. In contrast, packing density was slightly elevated in the kidney; protein/DNA was increased by the low dose of dexamethasone, but markedly decreased by the high dose. These results suggest that tissue growth impairment caused by prenatal dexamethasone treatment reflects primary deficits in cell proliferation that extend to a variety of different cell types; however, consequent effects on cell packing density and cell size are dose-specific, possibly reflecting actions of glucocorticoids selective for certain cell types or phases of cell development.

Partitioning of cells in dextran—poly(ethylene glycol) aqueous phase systems : A study of settling time, vessel geometry and sedimentation effects on the efficiency of separation

The effect of prolonged settling times (up to 2 h), in high- and low-phase columns, on the cell partition ratios measured and on the separability of cell populations was examined. With closely related cell populations, modelled by rat erythrocytes in which subpopulations of red blood cells of distinct age were labeled isotopically, it was found that partitioning proceeds over the entire time period examined as evidenced by the continuous change in relative specific activity of cells in the top phase as the partition ratio falls. In control cell sedimentation experiments in top phase there was almost no change in the quantity of cells present when vertical settling ( i.e., high-phase columns) was used and no separation of specific subpopulations was found. In the horizontal settling mode the initially higher cell partition ratio, as compared to vertical settling, decreased to a greater extent with longer time intervals; a given purity of cells only being obtained at a lower partition ratio than in the vertical settling mode. Cell sedimentation in top phase was appreciable with time in the horizontal settling mode but did not result in a separation of cell subpopulations. The effect of relative cell partition ratios and sizes in high- and low-phase columns on the efficiency of separation was examined by use of rat or sheep 51Cr-labeled red cells mixed with an excess of human unlabeled erythrocytes. Rat and sheep red cells are appreciably smaller than human erythrocytes. Rat red cells have higher, and sheep red cells lower partition ratios than human erythrocytes. With vertical settling, over a 2-h period, there is no appreciable contribution to the change in relative specific activities by cell sedimentation. However, the more rapid sedimentation of the larger human red cells has, with time, a measurable effect on the relative specific activities obtained during cell partitioning when run in the horizontal mode: enhancing the rat—human and diminishing the sheep—human cell separations. Partitioning cells in high-phase columns is of advantage with respect to increasing separation efficiency and virtually eliminating the influence of other physical parameters ( e.g., cell size). Since the cell partitioning process continues for long periods of time, yielding ever-lower partition ratios with increasing proportions of cells with higher P values, a time may be selected which balances desired relative cell purity and yield.

A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production

A flow cytometric kinetic study of hybridoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody-producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourage consideration of structured kinetic studies which recognize the quality of the biophase.

Phenotypic analysis of mouse thymus development.

In this review, we have presented some of our data on the developing mouse thymus studied using mAbs to cell surface determinants and FMF. In the past, numerous parameters have been used in an attempt to describe the differentiation of T cells in the thymus. These parameters have included relative cell size, anatomical location, enzyme activity (TdT), expression of surface molecules such as Thy-1, Lyt-1, H-2, PNA, T200, TL, and CD4/CD8, and the ability to express IL-2R [45, 46]. None of these parameters has been adequate to describe how the thymus generates functional ‘mature’ T cells, which now include CD4/CD8 single-positive and double-negative cells, from its small numbers of elusive precursors [17, 45, 47]. However, given the irrefutable evidence that the thymus does influence the repertoire of TcR expressed by cells generated therein [48], analysis of gene regulation and surface expression of TcR molecules by thymocytes is certainly the most promising approach for the elucidation of the mysterious nature of intrathymic T cell differentiation.

Quantitation of structural features characterizing weight- and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits.

The structural organization of articular cartilage from the medial femoral condyle of young adult rabbits has been examined after processing according to an improved fixation procedure. By using recently developed stereological methods, a quantitative analysis of chondrocyte number, surface area, volume, and matrix volume per cell was carried out in the light microscope; at the electron microscopic level, quantities of cytoplasmic components within chondrocytes (including organelles) were estimated. These measurements were made for each of the four zones from the (articular cartilage) surface down to the tidemark, and the results (for each zone) were compared between weight- and less-weight-bearing regions. In general, articular cartilage revealed considerable heterogeneity in structure throughout its depth. The number of cells per unit volume is maximal beneath the surface and decreases toward the tidemark. The size of chondrocytes, and the mean matrix volume surrounding each, increases from the surface toward the deeper zones. Comparison between weight- and less-weight-bearing regions reveals striking differences. The numerical volume density of cells in the superficial zone of regions bearing high physiological load is less than half of that in less-weight-bearing regions, chondrocyte death being principally responsible for this reduction. A comparison between the midzones of weight- and less-weight-bearing areas reveals that the former is characterized by a decrease in cell density and an approximately threefold increase in cell size in relation to the latter. The increase in cell volume is attributable principally to an accumulation of intermediate filaments and glycogen particles, and represents an adaptation to increased functional requirements. Near the tidemark, numerical volume densities of chondrocytes in both weight- and less-weight-bearing locations are similar, but the larger cell size in the former still persists.

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Elastic Behavior of Open‐Cell Alumina

The elastic constants of open‐cell aluminas were measured as a function of their relative density and cell size. The density exponent was found to be in excellent agreement with previous theoretical work that considered cell strut bending as a major deformation mode. The measured constants in the elastic behavior relationships were found to be less than previously suggested, and it was found that this factor is sensitive to the microstructure of the cell struts, especially porosity within these struts. The materials studied were found to contain soem closed cells, but these were not found to have a major effect on the elastic behavior. The values of Young's modulus and the shear modulus depended on the relative density in the same way, and this is consistent with previous theoretical work which showed that Poisson's ratio should be independent of density.

B cells as antigen-presenting cells: Antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of IA b. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-γ did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.

Relation between cell size and detonation parameters in overdriven gaseous detonations

An experimental study of the cellular structure of overdriven detonation waves which are caused by the transition process from deflagration to detonation has been conducted. The results, relating cell size and detonation parameters, are presented in this paper. For mixtures of 2H 2 + O 2, 2H 2 + 5air, and 2H 2 + O 2 + 3Ar it has been found that the relation between the number of transverse cells in a square tube, N, and the detonation pressure P is approximately linear, and the slopes of the ( N, P P CJA ) lines are almost the same for different mixtures and initial pressures. A simple formula, therefore, was obtained to estimate overdriven detonation pressure from cell size. The dependence of cell size on detonation velocity was also observed experimentally and analysis of the results has shown that L ≅ constant. Therefore, the conclusion for CJ gaseous detonation is that this result can be used to predict overdriven cases.

PRODUCTION AND PROPERTIES OF MONOCLONAL ANTIBODIES AGAINST HUMAN ECTO-5′-NUCLEOTIDASE: 77

After immunization of mice with partially purified human placental 5′-nucleotidase hybrid cells were produced following the standard protocol. Culture supernatants were screened for the ability to inhibit enzyme activity. From 18 different hybrids showing significant inhibition 5 were established as clones. Properties of the MABs were studied in ascites fluid and in purified Ig-preparations. Inhibition of the MABs is directed against the enzyme in membrane bound and solubilized form and there is no tissue specificity. Similar inhibitory titers were observed with the MABs coated on plastic. The MABs belong to the IgG 1 and IgG 2b subclasses. Some of them show a marked temperature dependence in their inhibitory potency. They are not directed against the sugar moiety of the 5′-N molecule. In fluorescence studies with PBL a smaller subpopulation, compared to polyclonal antisera, shows a positive reaction with the MABs. The titers for optimal fluorescence are much lower than for inhibition. Results of flow-cytometric studies relating cell size to positive reaction with the MABs will be reported.

Cinétiques de croissance de Escherichia coli et de Saccharomyces cerevisiae en culture sur membranes filtrantes

The growth kinetics of Escherichia coli and Saccharomyces cerevisiae cultivated on Millipore membrane filters (MF) was studied. Exponential growth kinetics was observed for both microorganisms, but in the case of E. coli cultivated on 0.8 micron and 1.2 micron MF, a decrease in the growth rate compared to that observed in liquid medium was noted. On the other hand, S. cerevisiae was unable to grow on a MF belonging to some batches, whereas E. coli showed normal behavior towards these MF. These data underline the importance of the effect upon growth kinetics of the relative cell size proportionally to the size of the MF pores.

Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves

As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monococcum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts.