Karyometric observations of WISH cell cultures irradiated with 3 GHz microwaves.

WISH cell cultures 24 hours after passage were irradiated with 3 GHz microwaves (10 cm) at far field conditions in free space (anechoic chamber) for 30 minutes, at field power density 5 or 20 mW/cm2. Within 1,24 and 48 hours of the exposure to microwave fields the volumes of nuclei and nucleoli were measured with the use of a micrometer, and logvolumes and nucleo-nucleolar ratios were calculated. Under the applied irradiation conditions the culture medium temperature did not exceed 37 degrees C. In cultures irradiated at field power density 20 mW/cm2 increased number of cells with small nuclei and enlarged nucleoli was noted within 1 hour of the exposure. Within 24 and 48 hours after irradiation the nucleolar volume showed a slight decrease, whereas the nuclear volume increased. In cultures irradiated at field power density 5 mW/cm2 increased numbers of cells with enlarged nuclei and nucleoli were found. Analysis of the distribution curves of nuclear and nucleolar volumes suggests that non-thermal power densities of microwaves stimulate the metabolism of cell cultures. However, at higher power densities (20 mW/cm2) the stimulation phase is preceded by a period of reduced viability of cell cultures.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Quantitative studies of increased cell-to-substratum adhesion at low temperature

The cell-to-substratum adhesion of an established epithelial cell line cultured for 24 h on glass coverslips was determined at 4 degrees C, 8 degrees C and 37 degrees C using a miniaturised parallel-plate shearing apparatus. The measurements of the minimum shear necessary to dislodge the cells (minimum distraction force, MDF) demonstrated a three- to fourfold increase in the adhesion of the cells at 4 degrees C (6.17 Pa) compared to that at 37 degrees C (1.36 Pa). At 8 degrees C the MDF was 2.31 pascals. Part of the adhesion was resistant to mild trypsinisation. Trypsin-resistant adhesion (TRA) was stabilised by low temperature, and by treatment with concanavalin A (50 micrograms ml-1) or colchicine (200-400 microM). The effects of con A (140 micrograms ml-1) and low temperature (4 degrees C) were additive, giving a combined MDF of greater than 9.27 pascals. On the basis of their different temperature and protease susceptibility it is suggested that trypsin-sensitive adhesion (TSA) and TRA represent separate functional classes of cell-to-substratum attachment corresponding to 'frictional' and 'tractional' adhesion, respectively.     

Apoptosis induced by ultraviolet radiation is enhanced by amplitude modulated radiofrequency radiation in mutant yeast cells

The aim of this study was to investigate whether radiofrequency (RF) electromagnetic field (EMF) exposure affects cell death processes of yeast cells. Saccharomyces cerevisiae yeast cells of the strains KFy417 (wild-type) and KFy437 (cdc48-mutant) were exposed to 900 or 872 MHz RF fields, with or without exposure to ultraviolet (UV) radiation, and incubated simultaneously with elevated temperature (+37 degrees C) to induce apoptosis in the cdc48-mutated strain. The RF exposure was carried out in a special waveguide exposure chamber where the temperature of the cell cultures can be precisely controlled. Apoptosis was analyzed using the annexin V-FITC method utilizing flow cytometry. Amplitude modulated (217 pulses per second) RF exposure significantly enhanced UV induced apoptosis in cdc48-mutated cells, but no effect was observed in cells exposed to unmodulated fields at identical time-average specfic absorption rates (SAR, 0.4 or 3.0 W/kg). The findings suggest that amplitude modulated RF fields, together with known damaging agents, can affect the cell death process in mutated yeast cells. Bioelectromagnetics 25:127-133, 2004.     

A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport

A key factor in gene or drug therapy is the development of carriers that can efficiently reach targeted cells from a distal administration. In many gene/drug delivery studies, results obtained in 2D cultures fail to translate to similar results in vivo. In this work, we developed a perfusable 3D chamber for studying nanoparticle penetration and transport in cell-gel soft tissue cultures. The compartmented chamber is made of a polydimethylsiloxane (PDMS) top layer with the chamber features, created using micromachined lithography, bonded to a bottom glass coverslip. A solution of cells embedded in a hydrogel is loaded in the chamber between PDMS posts that serve as anchors to the cell-matrix at the gel-media interface. The chamber offers the following unique features: (i) rapid fabrication and simplicity in assembly, (ii) direct in situ cell imaging in a plane normal to the direction of flow or action, (iii) an easily configurable and controllable environment conducive cell culture under static or interstitial flow conditions, and (iv) facile recovery of live cells from chambers for post-experimental analysis. To assess the chamber, we delivered fluorescently labeled nanoparticles of three distinct sizes to cells-embedded Matrigels in the 3D chamber under flow and static conditions. Penetration of nanoparticles were enhanced under interstitial flow while live cell imaging and flow cytometry of recovered cells revealed particle size restrictions to efficient delivery. Although designed for delivery studies, the chamber is versatile and can be easily modified. Thus it may have broad applications for biological, tissue engineering, and therapeutic studies.     

Factor Xa generation at the surface of cultured rat vascular smooth muscle cells in an in vitro flow system.

The purpose of the present investigation was to explore the effects of well-defined flow conditions on the activity of tissue factor (TF) expressed on the surface of cultured rat vascular smooth muscle cells. Cells were cultured to confluence on Permanox brand slides and stimulated to express TF by a 90 min incubation with fresh growth medium containing 10 percent calf serum. The stimulated cells were then placed in a parallel plate flow chamber and perfused with Hank's Balanced Salt Solution containing factor VIIa, factor X (FX), and calcium. The chamber effluent was collected and assayed for factor Xa (FXa) and the steady-state flux of FXa was calculated. The flux values were 68.73, 94.81, 139.75, 138.19, 316.82, and 592.92 fmole/min/cm2 at wall shear rates of 10, 20, 40, 80, 320, and 1280 s-1, respectively. The FXa flux depended on the wall shear rate to a greater degree than predicted by classical mass transport theory. The flux at each shear rate was three to five times less than that calculated according to the Leveque solution. These features of the experimental data imply nonclassical behavior, which may partially result from a direct effect of flow on the cell layer.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm² shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional (3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.     

A new principle of cell cultivation: No air-liquid interface but gas exchange across a synthetic membrane

Summary A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O₂ and CO₂; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5-h period post-stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3-6 J kg-1. Extracellular hydrogen peroxide was detected using a luminol-based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV-vis spectrophotometry and HPLC, stress-related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 muM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.     

Conditions for isolation and culture of porcine myogenic satellite cells.

Myogenic satellite cells were isolated from semimembranosus muscles of 4-8 week-old pigs. Muscles were ground and incubated in 0.8 mg/ml Pronase solution for 40 min at 37 degrees C. Following enzymatic digestion, cells were separated from muscle debris by differential centrifugation and sequential filtering through 500 and 53 microns nylon mesh. Primary cultures grown in 16 mm diameter cell culture wells were used to evaluate five sera, media, and substrata for their ability to promote satellite cell proliferation and differentiation. Porcine satellite cell proliferation and myotube formation were optimized in cultures grown on gelatin-coated substratum in the presence of Minimum Essential Medium-alpha supplemented with 10% fetal bovine serum (FBS) (P less than 0.01). Maximum fusion was induced by 48 hr exposure to 2% FBS, horse serum, or lamb serum. These data 1) document the first evidence that myogenic satellite cells can be isolated from porcine skeletal muscle, and 2) identify culture conditions which optimize proliferation and myotube formation of porcine satellite cells.     

Increased survival of mesothelial cells from the peritoneum in peritoneal dialysis fluid

Peritonitis remains the most important factor in patient morbidity and technical failure associated with continuous ambulatory peritoneal dialysis (CAPD). In vitro examination of bacterial infection of cultured human peritoneal mesothelial cells (HPMC) is an attractive approach to the study of peritonitis in CAPD, yet there are few reports on this subject. Previous studies have shown two limitations: (i) cell cultures of HPMC lasted for days only when incubated in culture medium and (ii) short-term studies of <30 min were done in HPMC when incubated with peritoneal dialysis fluid (PDF). Human peritoneal mesothelial cells, maintained in a conventional single chamber culture system with PDF alone, were unable to survive more than 40 min. The present study was designed to prolong the viability of HPMC cultured in PDF, with the object of using cells under different conditions, such as that of simulating CAPD. HPMC were cultured using plastic microtiter plates, where they were grown to confluence and growth was arrested. PDF containing different concentrations of NaHCO3and human serum albumin was added. Cell viability after exposure for up to 24 h was measured by trypan blue, Cell Death Detection ELISA and Annex-V flow cytometry. The data confirmed the 'toxic' effect of PDF, with cell viability being <40% after 2 h incubation in 4.25% glucose in PDF. However, the survival time of HPMC increased significantly in 4.25% glucose PDF at a physiological pH and even further after the addition of human albumin. These experimental conditions simulating CAPD may allow future in vitro studies of mesothelial physiology and peritonitis related to CAPD treatment.     

PDGF stimulates transient phosphorylation of 180,000 dalton protein

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.     

Serum modulates the intracellular calcium response of primary cultured bone cells to shear flow

We investigated the effect of newborn bovine serum on the intracellular calcium [Ca²⁺]_i response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca²⁺]_i responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10–15 min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5 Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca²⁺]_i response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca²⁺]_i flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca²⁺]_i response in bone cells subjected to fluid flow-induced shear stress.     

Synthesis and photodynamic activity of zinc(II) phthalocyanine derivatives bearing methoxy and trifluoromethylbenzyloxy substituents in homogeneous and biological media

Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic studies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum yields (phi(F)) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell line. The production of singlet molecular oxygen, O(2)((1)Delta(g)), was determined using 9,10-dimethylanthracene yielding values of approximately 0.56 for both sensitizers. Under these conditions, the addition of beta-carotene (Car) suppresses the O(2)((1)Delta(g))-mediated photooxidation. In biological medium, no dark cytotoxicity was found for cells incubated with 0.1 microM of phthalocyanines 3 and 4 for 24 h. However, under similar conditions 0.5 microM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1muM of sensitizer, reaching values of approximately 0.05 nmol/10(6) cells after 3h of incubation at 37 degrees C. The cell survival after irradiation of the cultures with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15 min of irradiation). Also, these studies were performed treating the cells with 0.5 microM of ZnPc 3. In this case, an increase in the uptake (approximately 0.28 nmol/10(6) cells) was observed, which is accompanied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed.     

Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.     

In vitro stress measurements in the vicinity of six mechanical aortic valves using hot-film anemometry in steady flow

Based on hot-film anemometry, point velocity measurements in the total cross sectional area 1 and 2 diameters downstream of: Bj?rk-Shiley Standard, Convex-Concave and Monostrut, Hall-Kaster (Medtronic-Hall), St. Jude Medical and Starr-Edwards Silastic Ball aortic valves were made. The spatial distribution of Reynolds Normal Stresses (RNS) was visualized three-dimensionally in order to point out where and to what extent the highest RNSs were found. The measurements were made in steady flowing glycerol mixture at flow rates 10, 20 and 30 l. min-1 corresponding to mean velocities of 27, 54 and 81 cm s-1. The highest maximum RNS values were around 250 Nm-2 and were found downstream of the Bj?rk-Shiley Monostrut and Starr-Edwards Ball valves. The lowest maximum RNSs were found downstream of the St. Jude Medical and Hall-Kaster (Medtronic-Hall) valves (125-140 Nm-2). The Starr-Edwards valve had the highest mean RNS (117 Nm-2) followed by the Bj?rk-Shiley Monostrut (87 Nm-2). These simplified measurements of artificial heart valve performances concerning RNS, enhance the interpretation of results in more complicated flow models not to say in vivo.     

Heat-induced disturbances of intracellular movement and the consistency of the aqueous cytoplasm in HeLa S-3 cells: a laser-Doppler and proton NMR study.

Intracellular particle movements, of both saltatory and streaming types, in HeLa S-3 cells were simultaneously interrupted after 1 h exposure of cells to 43 degrees C, within 10 min at 44 degrees C and within 5 min at 45 degrees C. Intracellular movement inhibited after 15 min at 44 degrees C and 10 min at 45 degrees C was not reversible in cells rescued at 37 degrees C. Brownian motion was not observed in heat-treated cells while they were maintained at elevated temperatures, but became pronounced in blebbing which occurred shortly after they were returned to 37 degrees C. Returning these cells to 45 degrees C intensified the Brownian activity inside blebs, and rapidly induced cell lysis. The same heat-treated cells were simultaneously studied by laser-Doppler microscopy, which confirmed: a) that flow (cytoplasmic streaming) is completely arrested at 44 degrees C within 10 min, b) flow recovered in 10-15 min in cells rescued after 10-15 min at 44 degrees C, c) submicroscopic particles down to the size of water molecules had faster self-diffusion coefficients at 44 degrees C than at 37 degrees C. Proton nmr studies on cells exposed from 4 to 45 degrees C gave corrected relaxation times T1 and T2 which rose with temperature in a predictable manner. Inhibition of cellular movement at elevated temperatures was not specifically attributable to the depletion of intracellular ATP levels.     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.