Blood volume measurement in baboons: simultaneous estimation of red cell volume and plasma volume

A method is described for frequent sequential blood volume estimation in baboons using 32P for red cell volume measurements and 125I-albumin for simultaneous plasma volume measurements. Values for red cell, plasma, and total blood volumes are reported. Close correlations of the volumes to bodyweight were demonstrated. Circulatory half-lives of the isotopes, determined from disappearance curves, confirmed their suitability for serial measurements in these baboons.     

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.     

EGFR signaling promotes basal autophagy for lipid homeostasis and somatic stem cell maintenance in the Drosophila testis

ABSTRACT

In contrast to stress-induced macroautophagy/autophagy that happens during nutrient deprivation and other environmental challenges, basal autophagy is thought to be an important mechanism that cells utilize for homeostatic purposes. For instance, basal autophagy is used to recycle damaged and malfunctioning organelles and proteins to provide the building blocks for the generation of new ones throughout life. In addition, specialized autophagic processes, such as lipophagy, the autophagy-induced breakdown of lipid droplets (LDs), and glycophagy (breakdown of glycogen), are employed to maintain proper energy levels in the cell. The importance of autophagy in the regulation of stem cell behavior has been the focus of recent studies. However, the upstream signals that control autophagic activity in stem cells and the precise role of autophagy in stem cells are only starting to be elucidated. In a recent publication, we described how the Egfr (epidermal growth factor receptor) pathway stimulates basal autophagy to support the maintenance of somatic cyst stem cells (CySCs) and to control lipid levels in the Drosophila testis.     

Changes in cell volume of bacteria and heterotrophic nanoflagellates in a hypereutrophic pond

Changes in cell volume of planktonic bacteria and heterotrophic nanoflagellates (HNF) were examined in a hypereutrophic pond from April to October, 1997. There were marked changes in the abundance of bacteria, HNF and ciliates and in protistan bacterivory during this period. The cell volume of free-living bacteria (0.121 ± 0.031 m³, mean ± SD) was large relative to that reported in the literature. The cell volumes of HNF was 71.1 ± 24.8 m³. Both cell volumes did not follow a seasonal trend. The dominant size class of bacteria was seasonally variable, whereas density of filamentous bacteria was relatively high between August and September. Biomass of filamentous bacteria accounted for up to 33.6% of total bacterial biomass. A correlation analysis for cell volume of bacteria and HNF, density of filamentous bacteria and some microbial variates was performed. The positive correlations detected (p<0.05) were between density of bacteria and cell volume of HNF, and between density of filamentous bacteria and cell volume of HNF.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Lipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.     

Correlation among corpus allatum volume,cell size,and juvenile hormone biosynthesis in ovariectomized adult Blattella germanica

The corpora allata (CA) of both intact and ovariectomized Blattella germanica adult females exhibited a high degree of bilateral symmetry in the rate of juvenile hormone (JH) biosynthesis, the mean size of CA cells, and gland volume (81.3%, 98.3%, and 100% respectively with less than a twofold difference between the two glands in CA pairs). This permitted us to split each CA pair randomly, measure JH biosynthesis in one gland, and dissociate the other gland into a cell suspension in which the size of CA cells was measured. In ovariectomized females, changes in CA volume and the spontaneous and farnesoic acid (FA)-stimulated rates of JH biosynthesis, measured from the same glands, were well correlated (r = 0.78, for both correlations). Similarly, the mean volume of CA cells in one gland increased in relation to increases in both the spontaneous and FA-stimulated rates of JH biosynthesis by the contralateral member of the pair (r = 0.83 and r = 0.91, respectively). Concurrent changes in CA cell size and activity suggest that in the CA of B. germanica cellular growth and degradation are involved in the regulation of JH biosynthesis.     

Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.     

Saturated free fatty acids,palmitic acid and stearic acid,induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cell

In men, obesity has generally been associated with reduced plasma testosterone levels and with elevation of the plasma free fatty acids (FFAs). In this study, we investigated the effects of saturated FFAs including palmitic acid (PA) and stearic acid (SA), and polyunsaturated FFA arachidonic acid (AA) on the survival of rat testicular Leydig cell cultured in vitro. PA and SA markedly suppressed Leydig cell survival in a time- and dose-dependent manner. In contrast, AA stimulated the cell proliferation at 5-10 times of physiological concentration. The suppressive effect of PA and SA on cell survival was caused by apoptosis evidenced by DNA ladder formation and Annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effect of PA was possibly mediated by ceramide generation because it could be completely blocked by ceramide synthase inhibitor fumonisin B1 and exogenous ceramide itself could directly induce apoptosis in vitro. Surprisingly, the apoptosis induced by PA could be partly prevented by AA. These results indicate that PA and SA induce apoptosis in testicular Leydig cells by ceramide production and these apoptotic effects may be a possible mechanism for reproductive abnormalities in obese men, and AA can partly prevent the apoptotic effect induced by saturated FFA.     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Interrelations among Na and K content,cell volume,and buoyant density in human red blood cell populations

Summary This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na+K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966.Scand. J. Lab. Invest. 18:167–180) cell water and cell volume was found to vary directly with the summed content of Na+K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na+K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.     

Cadmium-induced changes in parietal cell structure and functions of rats

The aim of this study was to determine the cadmium (Cd)-induced functional and structural changes in gastric parietal cells of male rats exposed to high Cd for 30 d. In the present study, control animals were fed with normal food and tap water; the remaining animals received Cd (15 ppm CdCl₂) in drinking water for the same period. Receiving Cd for 30 d increased the mean blood Cd level, the mean tissue Cd content, and the mean blood pressure (p<0.01, p<0.001, p<0.01, respectively). The basal acid output fell; however, the increases in stimulated acid output were not statistically significant. Light and electron microscopic examination revealed respectively that (1) Cd decreases the mean parietal cell number per unit from the control value of 23.46±3.84 to 19.46±2.12 (p<0.05) and it affected preferentially the cells located at the distal half of the zymogenic unit and (2) in parietal cells, the Cd-induced alterations were characterized with swollen canalicular profiles, broken-down tubulovesicles, or degenerated mitochondria. We concluded that Cd augments the elimination rate of parietal cells by increasing the alteration rate and reduced basal acid output can be explained easily with the loss of parietal cell population.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

Long chain‐polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of nystatin on intracellular contents and membrane transport of alkali cations,and cell volume in HeLa cells

Summary Nystatin (50 g/ml) had strong influence on the intracellular contents and membrane transports of monovalent ions and water in HeLa cells. The nystatin-induced changes in the intracellular ion content and cell volume were inhibited by sucrose, and Donnan and osmotic equilibria were attained. Using cells under conditions for these equilibria, the concentrations of intracellular impermeant solutes, their mean valence, the differences of their intra- and extracellular osmotic concentrations, and the circumferential tension of the cell membrane were determined. Stimulation by nystatin of the influx of one cation species, e.g. Rb, was inhibited by another cation species, e.g. Na. The stimulatory effect of nystatin on cation fluxes was reversible within 1 hr after ionophore addition, and after 1-hr treatment the intracellular contents of Na and K became proportional to their extracellular concentrations, provided that the sum of these concentrations was constant (300mm). Similar proportionality was also observed in the presence of choline, provided that the choline concentration was less than those of the alkali cations. The implications of these results in relation to the osmotic properties of cultured cells, and the experimental regulation of alkali cations in the cells, are discussed.     

Multiple effects of mercury on cell volume regulation, plasma membrane permeability, and thiol content in the human intestinal cell line Caco-2

In a previous study, we characterized Cd–Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation value of 0.3 μmol/L ²⁰³Hg was measured in the presence of 100 μmol/L unlabeled Hg in the serum-free exposure medium without modification in the initial uptake rate. This phenomenon was eliminated at 4^∘C. Mercury induced an increase in tritiated water and [³H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 μmol/L Hg and 2 mmol/L N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects. Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport. During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and consequently cell capacity. Deceased 25 March 2004     

Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

This study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA?+?OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA?+?OA enhanced cell proliferation in a dose-dependent manner (10 to 100?µM), whereas it lowered at 250?µM. In addition, a cell-cycle analysis showed that 100?µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA?+?OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.     

Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation in Cunninghamella echinulata

Aims: To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Methods and Results: Cunninghamella echinulata grown on a TWH medium rapidly took up glucose and produced large amounts of lipids. However, when some quantities of the organic nitrogen were removed from TWH (by acid followed by alkaline precipitation of proteins) the uptake of glucose was dramatically reduced and large quantities of fungal biomass having low lipid content were produced. Nevertheless, when glycerol was used as carbon source instead of glucose, the uptake rate as well as the biomass production and the lipid accumulation processes were unaffected by the TWH organic nitrogen removal. Finally, when the fungus was grown on a glucose supplemented TWH medium that contained no assimilable organic nitrogen (after further precipitation of proteins with methanol), the produced biomass contained non-negligible quantities of lipids, although glucose uptake remained low. Lipid analysis showed that the produced lipids comprised mainly of neutral lipids, which were preferentially consumed during lipid turnover. Lipid production on the original TWH medium having glucose as carbon source was 0·48 g of lipid per gram of dry biomass, corresponding to 8·7 g of lipid per litre of growth medium. The produced lipids contained 11·7%γ-linolenic acid (GLA), hence the GLA yield was more than 1 g l⁻¹. Conclusions: Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Significance and Impact of the Study: Agro-industrial wastes containing organic nitrogen, such as tomato waste, are produced in vast amounts causing severe environmental problems. These wastes could be used as fermentation feedstock to produce microbial lipids.     

Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b-) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (-/-)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (-/-) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (-/-) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (-/-) and WT mice after luminal hypotonic challenge, but VDAC-1 (-/-) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (-/-) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.