Characterization of murine and humanized anti-CD33, gelonin immunotoxins reactive against myeloid leukemias

M195 antibodies recognize CD33, an antigen present on acute myeloid leukemia blasts as well as some myeloid progenitor cells, but not on the ultimate hematopoietic progenitor stem cell. Immunotoxins (IT) reactive with human myeloid leukemias were constructed by conjugating gelonin, a single-chain ribosome-inactivating protein, to murine and genetically engineered, humanized M195 antibodies via anN-succinimidyl-3-(2-pyridyldithio)-propionate linkage. No losses of gelonin cytotoxic activity or M195 binding activity were observed after conjugation of up to two toxin molecules per antibody. Toxin conjugates displayed specific, potent toxicity for CD33⁺ cells. The murine and humanized IT were not toxic to CD33^– cells and were 600 and 4500 times more potent, respectively, than free gelonin in inhibiting CD33⁺ HL60 cells. Treatment of HL60 cells with 1 g/ml HuM195-gelonin resulted in more than 1000 times lower colony formation; normal bone marrow mononuclear cell colonyforming units treated with HuM195-IT were reduced by a factor of 10. HL60 leukemia cells could be effectively purged from an excess of normal bone marrow cells. Exposure of target cells to IT for as little as 30 min was as effective as continuous exposure of IT for up to 6 days. However, measures of the efficacy of the immunotoxin were directly related to the length of time of observation after IT exposure and were inversely related to cell concentration. M195-gelonin immunoconjugates are potential candidates for therapeutic use in in vivo or ex vivo bone marrow purging of myeloid leukemias.These studies were supported in part by the Lucille P. Markey Charitable Trust, ACS Grant No. IM551, NIH PO1CA33049, NIH RO1CA55349. Research conducted, in part, by the Clayton Foundation for Research. David A Scheinberg is a Lucille P. Markey Scholar     

Establishment and characterization of a cell line derived from bovine tracheal glands

Summary Bovine tracheal submucosal gland cells have been isolated by enzymatic digestion and serially propagated in tissue culture for more than 12 mo. (40 passages). The cells exhibit an epithelioid appearance at confluence and contain alcian blue (pH 2.5)/periodic acid-Schiff-positive material within cytoplasmic granules. By electron microscopy numerous osmiophilic secretory granules are seen. Maximal growth is observed when the cells are grown on human placental collagen-coated culture vessels in medium supplemented with 20% fetal bovine serum. Scintillation spectrometry revealed that radiolabeled precursor (³⁵SO₄) was incorporated into high molecular weight molecules and released from cells. Isoproterenol (10⁻⁶ to 10⁻³ M) stimulated the release of³⁵SO₄. The maximal response to isoproterenol was completely inhibited by the β-adrenergic antagonist propranolol. It is concluded that the cultured cells retain features of tracheal gland cells and may serve as a useful model of synthesis and secretion of macromolecules by tracheal gland cells. This study was supported in part by NIH Program Project grant HL-24136, by a National Cystic Fibrosis Foundation Research Development Grant, and by a grant from Cystic Fibrosis Research, Inc. Dr. Finkbeiner is a recipient of NIH Clinical Investigator Award HL-01387.     

Effects of hypoxia on lipolysis in isolated rat myocardial cells

Summary The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO₄, 1 mM DL-carnitine, 1.5 mM CaCl₂ and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N₂–5% CO,. Control (normoxic) incubations were carried out under air-5% CO₂ atmosphere. Basal glycerol release increased from 46.6 ± 3.0 nmol/106 cells · 30 min in normoxia to 64.5 ± 4.3 nmol/10⁶ cells · 30 min in hypoxia (p < 0.05). Addition of isoprenaline (10 M) resulted in a significant (p < 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 ± 2.5 nmol/10⁶ cells · 30 min) than in normoxia (12.2 ± 2.7 nmol/106 cells · 30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 M), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.     

Cyclic AMP stimulation of calcium efflux from kidney,liver, and heart mitochondria

Summary The effect of cyclic AMP on subcellular calcium turnover was studied in isolated kidney, liver and heart mitochondria. The calcium concentration of the incubating medium was determined by fluorometric methods after its separation by millipore filtration. Liver and kidney mitochondria take up calcium in exchange for H⁺ and lower the medium calcium to 1 to 40×10^–6 m in less than 2 min. Cyclic AMP produces an instantaneous release of calcium from mitochondria and a rise in the steady-state calcium concentration of the medium. A new medium calcium level of 0.7 to 3×10^–4 m is achieved in less than 3 sec and is proportional to cyclic AMP concentrations between 10^–7 and 3×10^–6 m. Cyclic AMP is inactive above 5×10^–6 m and below 10^–7 m. Cyclic IMP, 5 AMP, dibutyryl cAMP are inactive at any concentration. Cyclic GMP is active at 10^–5 m and competitively inhibits cyclic AMP action. The same staedy-state calcium level is reached from higher or, lower calcium concentrations, i.e. whether cyclic AMP is added before or after the addition of calcium to the mitochondrial suspension. At low calcium or phosphate concentrations, the calcium released by cyclic AMP is immediately reaccumulated by the mitochondria is less than 2 min with a further release of H⁺. This pulse can be repeated by sequential additions of cyclic AMP. The transient or sustained response to cyclic AMP depends on the medium calcium x phosphate product and presumably on the presence or absence of calcium phosphate precipitate inside the mitochondria. These results support the hypothesis that cyclic AMP regulates cytoplasmic calcium by controlling the mitochondrial calcium efflux rate. This mechanism may be involved in the regulation of calcium transport and in some hormonal effects mediated by cyclic AMP.     

Effects of atmospheric small negative ions on the oxygen consumption of mouse liver cells

The effects of small negative air ions on the oxygen uptake of isolated mouse liver cells were studied by exposing the liver cells to varying ion concentrations. For concentrations of the order of 1–2 × 10⁵ ions/cm³, the oxygen uptake was always higher than in the normal atmospheric conditions of 3–8 × 10²/ions/cm³. For intermediate concentrations varying effects of activation and inhibition were observed. A statistical analysis showed that the oxygen uptake increased by approximately 14% when liver cells were exposed to ion concentrations of values 1–9 times the normal, by approximately 9% when exposed to 10–99 times the normal, and by approximately 38% when exposed to 100–999 times the normal. The significance and possible implications of the results are discussed.     

The effect of different algal densities of Scenedesmus acuminatus on the population growth of Moina micrura Kurz (Crustacea: Anomopoda,Moinidae)

Six densities (0.5 × 10⁶, 1.0 × 10⁶, 1.5 × 10⁶, 2.0 × 10⁶, 3.0 × 10⁶, and 4.0 × 10⁶ cells ml^–1) of the micro-alga Scenedesmus acuminatus, were fed to the cladoceran, Moina micrura, in 40-litre glass aquaria. Moina population increased with increasing cell densities of Scenedesmus only up to treatment 3 (i.e. 1.5 × 10⁶ cells ml^–1) where a peak population of 11303 individuals per litre was obtained. Moinapopulation growth was inhibited at higher algal densities. The percentage of egg-bearing females and the number of eggs per egg-bearing females, followed a similar pattern. Comparatively, the peak production density of approximately 11000 Moina per litre, is interesting from a mass production point of view and indicates that S. acuminatus is a satisfactory micro-alga food for M. micrura.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

Summary A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5×10^–18 mol/m³/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0×10^–18 mol/m³/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Aquatic surface respiration,an adaptive response to hypoxia in the guppy,Poecilia reticulata (Pisces,Poeciliidae)

Synopsis Aquatic respiration at the air-water interface, herein termed aquatic surface respiration (ASR), is used by the guppy,Poecilia reticulata (Poeciliidae) to meet oxygen demand in hypoxid water. A specific position in which the head contacts the surface and the jaws open just beneath the surface is adopted. ASR is initiated at a P0₂ of about 50 torr and the percent time spent rises in a steep, linear fashion as PO₂ decreases. Below 4 torr more than 90% of the animal's time is spent in ASR. Males spend less time in ASR than do females. The percent time in ASR increases with increasing size of female guppies, but decreases with increasing size of male guppies. At low oxygen (18 torr) laboratory-born guppies derived from stocks likely to experience deoxygenation spend less time in ASR than do guppies derived from stocks less likely to experience deoxygenation. The percent time in ASR increases with temperature when PO₂ is held constant. Acclimation to low oxygen decreases the percent time in ASR.Guppies not permitted ASR die in 6–41 h at 14–17 torr and 10–15 min at 1–4 torr. Guppies performing ASR survive the duration of experiments at 13–35 torr (13 days), 14–17 torr (96 h), and 1–4 torr (9.5 h). Activity, courtship, and dive duration in response to a shadow stimulus are all reduced by low oxygen. ASR is an effective alternative to aerial respiration as an adaptation to hypoxic waters, but is probably energetically and temporally more costly.     

Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichpolusia ni cells in suspension culture

Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×10⁵ and 1.7×10⁵ units/ 10⁶ cells¹ respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×10⁶ cells/ml) than the serum- based media (1.5±5×10⁶ cells/ml in GTC100 and 2±1×10⁶ cells/ml in TNM-FH). However, using a cell density of 5×0⁵ cells/ml, the productivity per cell varied, from a low of 4.5×10⁴ units in EX-CELL-400 medium to a high of 7.6×10⁴ units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.     

Unit gravity sedimentation separation of cells comprising the caput epididymidis of the rat

Summary Cells from the rat caput epididymidis were separated by unit gravity sedimentation. Purest 12-ml fractions contained 88–95% basal cells or 64–76% principal cells. Ultrastructure of separated cells was similar to that of cells in intact tissue. Viability of separated cells was excellent as determined by dye exclusion tests and cellular ATP content. By combining fractions pools containing 4.0±0.9 × 10⁶ cells (86±8% basal cells) and 1.4±0.4 × 10⁶ cells (56±7% principal cells) were obtained. Thus, studies on the function of basal and principal cells from the rat caput epididymidis should be possible.This research was supported by NIH Grant HD-07244 and was authorized for publication as Paper No. 5231 in the Journal Series of the Pennsylvania Agricultural Experiment Station. We thank Mrs. J. Eastman for expert technical assistance     

Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Oxygen gradients in animal-cell bioreactors

An estimation is made of oxygen gradients in animal-cell bioreactors, using straightforward engineering calculations. Three types of bioreactor are considered: stirred vessel, bubble column and air lift, of sizes between 0.01 and 10 m³. First, the gradient is estimated in the stagnant layer surrounding a cell (15 m), a microcarrier (185 m) with 300 cells attached to it, a macroporous support (1.25 mm) containing 185,00 cells and one (6 mm) containing 4.25 million cells. It is assumed that oxygen consumption is 10^–16 mole O₂·cell^–1·s^–1, while mass transfer coefficients are obtained from Sherwood relations. Circulation and liquid-retention times of the bioreactors are compared with the oxygen-exhaust times of suspensions with 10¹², 10¹³ and 10¹⁴ cells/m³ to estimate if oxygen gradients are likely to exist in the bulk-liquid phase. Finally, the gradient in the liquid film surrounding air bubbles is estimated using k _l A-values obtained from empirical correlations. It is clear from all these estimations that in many situations severe gradients can be expected. The question remains, however, whether gradients should be avoided as much as possible, or may be tolerated to a certain extent or even created on purpose because of possible beneficial effects.     

The effect of oxygen concentration on the decomposition of organic materials in soil

Summary 1. Techniques are described for relating the oxygen concentrations in the soil water on the surfaces of micro-organisms to their metabolizing activities.2. Studies were made on the decomposition of organic materials in water-saturated crumbs (mean radius 1.55 × 10^–1 cm) of a loam soil.3. Respiration of water-saturated crumbs was not inhibited unless the oxygen concentration was less than about 10^–6 M. Evidence was obtained that above a similar low oxygen concentration there was no inhibition of respiration in soils of widely different type.4. Anaerobic decomposition of the soil organic matter was very slow. Anaerobic decomposition of casein digest was more rapid than that of any other material tested; the products were water soluble and included 83 µ-equivalents of volatile fatty acid per mg of -amino-N decomposed.5. Casein digest percolation of soil crumbs under air resulted in the formation of micro-organisms that respired at 70 per cent of their maximum rate when the oxygen concentration was about 2.7 × 10^–6 M.6. No products of anaerobic casein digest decomposition could be detected on percolating casein digest through soil crumbs when 80 per cent of the soil contained no oxygen and the maximum concentration in any part of the soil was about 3 × 10^–5 M.7. The kinetics of oxygen uptake consequent on the decomposition of casein digest and of other simple organic compounds in soil crumbs were similar and were only slightly affected by reduction of oxygen partial pressure in the atmosphere from 15 to 1.7 cm of mercury.8. It is concluded that change-over from aerobic to anaerobic metabolism of organic materials takes place in widely different soils at an oxygen concentration less than about 3 × 10^–6 M.     

Basal oxygen consumption,ventilatory frequency,and heart rate during the protracted spawning run of the Southern Hemisphere lamprey Geotria australis

Summary Basal oxygen consumption, ventilatory frequency, and heart rate were recorded at four different times during the unusually protracted 15–16-month spawning run of the Southern Hemisphere lamprey Geotria australis. At 15°C, the mean basal oxygen consumption of G. australis caught immediately after they had left the sea and embarked on the spawning run (45 l · g^-1 · h^-1) was less than in young adults about to commence their marine feeding phase (64 l · g^-1 · h^-1), but greater than in large ammocoetes (26.5 l · g^-1 · h^-1). Basal oxygen consumption fell progressively during the spawning-run of to 33 l · g^-1 · h^-1 after 5 months and 25 l · g^-1 · h^-1 after 10 months, before rising to 35 l · g^-1 · h^-1 after 15 months when the animals were approaching sexual maturity. The downwards trend in basal oxygen consumption contrasts with that recorded during the spawning run of Lampetra fluviatilis. Furthermore, these values for spawning-run of G. australis are far lower than those measured at any time during the upstream migration of L. fluviatilis or during the parasitic phase of landlocked Petromyzon marinus. A low and declining metabolic rate during much of the spawning run of G. australis would facilitate the conservation of energy reserves during this very long non-feeding period. Trends shown by ventilatory frequency and heart rate essentially parallel those of basal oxygen consumption. The Q₁₀s for basal oxygen consumption, ventilatory frequency and heart rate over the temperature range 5–25°C were 1.6, 1.6, and 1.7, respectively. The trends shown by basal oxygen consumption during metamorphosis and the upstream migration did not parallel those exhibited by circulating thyroid hormones.     

Biosensor design: microbial loading capacity of acetylcellulose membranes

Summary In the construction of biosensors incorporating probe oxygen electrodes as the biochemical signal transducer, acetylcellulose membranes have proved to be a popular but poorly characterized matrix for microbial cell immobilization. In this report the relationship between immobilized whole microbial cell concentration/mm³ of acetylcellulose membrane and the reduction in oxygen diffusion across the microbial electrode has been determined using enteric bacteria. Total inhibition of oxygen diffusion occurred when the cell concentration was raised from 1×10⁶ to 1×10⁷ cells mm³: equivalent to a microbial cell volume of 1.5% compared to the maximum theoretical matrix volume. To facilitate future biosensor design a biosensor immobilized cell concentration Bicc value and a zero oxygen diffusion ZO₂D value are suggested, and the parameters of immobilized cell concentration, reduction in oxygen diffusion by immobilized cells and microbial cell size are discussed.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Physiological effects of the presence and absence of gas vacuoles in the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin

Physiological evidence was obtained for a light shielding role for gas vacuoles inMicrocystis aeruginosa Kuetz. emend. Elenkin, by comparing photosynthetic oxygen evolution, growth behaviour and pigment composition of cells with intact or collapsed gas vacuoles. The oxygen evolution rates were strongly dependent on cell concentration, a maximum rate for cells with intact gas vacuoles occurring at about 1.4×10⁹ cells/ml and for cells with collapsed gas vacuoles at about 2.5×10⁹ cells/ml. By using light saturation curves for oxygen evolution, it was estimated that at low light intensities up to 30% of the photosynthetically useable light was shielded at a cell concentration of 6×10⁸ cells/ml. Collapsing the gas vacuoles twice daily did not alter the initial growth rate of the cultures, but enabled them to reach a higher final cell density. Collapsing of gas vacuoles during growth for about four generations resulted in a lower level of all acetone soluble pigments with a greater relative reduction in carotenoids than in chlorophyll a. Collapse of the gas vacuoles does not alter the cell volume. Various optical interactions which could account for light shielding are discussed.