Oscillations in cell morphology and redox state

Fluctuations in the intensity of light scattered and absorbed by cells in suspension have been analysed by smoothing, periodogram and power spectrum methods to reveal oscillations attributed to changes in cell morphology and the redox state of NADH and FAD (periods 10 s to 30 min). The rhythms are themselves periodically modulated in amplitude at a similar frequency and exhibit burst characteristics. The low frequency scatter dynamics are provisionally attributed to oscillations in gross morphology and the high frequency variation to changes at the cell surface. Agents, such as insulin and transferrin, affect the dynamics. The scatter results suggest that rhythmic changes in cell morphology associated with locomotion are largely inherent in the cell and not due to periodic attachment and detachment from a surface.     

ANGULAR LIGHT-SCATTERING STUDIES ON ISOLATED MITOCHONDRIA

Angular light-scattering studies have been carried out on suspensions of isolated rat liver mitochondria. The angular scatter pattern has a large forward component, typical of large particles. Changes in dissymmetry and in the intensity of light scattered at 90° have been correlated with changes in optical density during the course of mitochondrial swelling and contraction. Such changes can be measured at mitochondrial concentrations much below those required for optical density measurements. Changes in mitochondrial geometry caused by factors "leaking" from mitochondria, not detectable by optical density measurements, have been demonstrated by measuring changes in dissymmetry. Angular light-scattering measurements therefore offer the advantages of increased sensitivity and of added indices of changes in mitochondrial conformation.     

Dynamics of the Interactions of Basic Proteins with Polyribonucleotides

Pancreatic ribonuclease was irradiated in the dried state with electrons and then added to acetate buffer solutions that contained different concentrations of polyribonucleotides. Qualitatively similar results were obtained by adding a combination of unirradiated ribonuclease and lysozyme to such solutions. Such solutions scatter light strongly, and the intensity of the scattered light changes with time after mixing. The angular distribution of the scattered light was obtained as a function of time and compared with the rates at which hydrolysis products were formed. The turbidity of the solutions increases rapidly with time at the lower polyribonucleotide concentrations, and seems to result from a complex between inactive ribonuclease, or lysozyme, and oligonucleotides that appear during enzymic hydrolysis of the polynucleotides. The dissymmetry of the scattered light is approximately 5, indicating that the scattering centers are, if spherical, about 1500 A in diameter. The turbidities are remarkably high when one considers the low concentrations of protein and nucleic acid materials that are used.     

Understanding ion transport by quantification of mitochondrial swelling

Suspensions of mitochondria are turbid and scatter light. An increase in the matrix volume (swelling) due to the influx of permeable solutes results in a decrease in the amount of light scattered. This property can be used to study solute fluxes across the mitochondrial inner membrane. A rapid method for isolating mitochondria is presented along with three swelling experiments using energized and non-energized mitochondria to illustrate ion transport across energy transducing membranes.     

Determination of corneal gel dynamics

From observations of the dynamics of light scattered by the cornea, intensity autocorrelation func-tions that revealed two independent diffusion coefficients, D (fast) = 2.4±0.2×10^–7 cm²/s and D (slow) = 9.4±1.3× 10^–9 cm²/s, were obtained. The diffusion coefficients were found to be statistically independent of the position and depth on the lateral surface of the cornea from which the scattered light was sampled. The slow diffusion coefficients obtained from light sampled from within cross-sections of the cornea were, however, measurably different. Diffusion coefficients obtained independently from observations of the kinetics of corneal swelling for comparison were found to be several orders of magnitude greater than those obtained from light scattering. The large disparity in the diffusion coefficients obtained from the two independent methods invoked the possibility that the lamellar layers within the cornea behave as individual gel sheets. Irrespective of this additional hypothesis, divergent behavior in the measured total scattered light intensities and diffusion coefficients upon varying external conditions, such as temperature or pressure (stretching), was observed. Namely, a slowing down of the dynamic modes accompanied by increased “static” scattered light intensities was observed. Although the slowing down of the dynamic modes is possibly indicative of the reduced affinity of protein binding to the gel matrix that “softens” the gel, the divergent behavior in the scattered light intensities and diffusion coefficients is, however, more characteristic of a phase transition. In addition, the divergent behavior in the scattered light intensities and diffusion coefficients was reversible up to a critical temperature (∼55 °C) or stretching (∼16%). Received: 18 March 1998 / Revised version: 4 February 1999 / Accepted: 4 February 1999     

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.     

Evidence for the role of light intensity in the seasonal testicular cycle of a wild bird,black headed munia

Abstract

The effects of various light intensity levels (0.11, 0.48, 0.96 and 1.86 mW/cm²) of constant white light on the testicular cycle of a wild bird, Black headed munia were investigated. While in higher intensity (0.96 and 1.86 mW/cm²) treated birds precocious gonadal development was observed, low intensity illumination prevented gonadal regression, indicating a better maintenance of testicular activity under low intensity lighting in Black headed munia. Since the total hours of light exposure (24 hr/day) were the same for all the four experimental groups, and higher intensities could not prevent gonadal regression, it would seem that light intensity is more important than the hours of illumination in the reproduction of this bird.     

Effect of rapid freezing on the functional state of rat heart mitochondria

As evidenced by respiration, oxidative phosphorylation, ATPase and NADH-oxidase activities, mitochondria composing heart tissue slices are more damaged by freezing-thawing than isolated mitochondria. A change in the functional activity of mitochondria is manifested in an increased respiratory rate in the second metabolic state and decreased respiratory rate in the third metabolic state upon oxidation of succinate and alpha-ketoglutarate; the ability of mitochondria to synthetize ATP (inhibition of the respiratory control) varied and the ATPase and NADH-oxidase activities increased. These changes in the functional state of mitochondria appeared to be due to a rise of the proton conductivity of the inner mitochondrial membrane by freezing-thawing.     

温州蜜柑叶片光系统反应中心光能分配的变化

为深入了解果树光化学反应中心光能分配的状况,以柑橘为试材,采用调制荧光法对叶片光系统在高光强和低光强下的状态转换进行了研究．结果表明,光系统在100μmol·m^-2·s^-1的低光强下,由于QA的还原使PQ库处于还原状态,导致光能由PSⅡ转向PSⅠ分配,光系统处于状态2;在1000μmol·m^-2·s^-1的高光强下,PQ库无法得到电子而处于氧化状态,导致光能分配由PSⅠ转向PSⅡ,光系统处于状态1,叶片经磷酸酯酶抑制剂NaF处理后,光系统从高光强下状态2到状态1的转换受到抑制,高光强下过多的光能由PSⅠ向PSⅡ分配是导致PSⅡ光破坏的重要原因．     

温州蜜柑叶片光系统反应中心光能分配的变化

为深入了解果树光化学反应中心光能分配的状况,以柑橘为试材,采用调制荧光法对叶片光系统在高光强和低光强下的状态转换进行了研究.结果表明, 光系统在100 μmol·m^-2·s^-1的低光强下,由于QA的还原使PQ库处于还原状态,导致光能由PSⅡ转向PSⅠ分配,光系统处于状态2;在1 000 μmol·m^-2·s^-1 的高光强下, PQ库无法得到电子而处于氧化状态,导致光能分配由PSⅠ转向PSⅡ,光系统处于状态1.叶片经磷酸酯酶抑制剂NaF处理后,光系统从高光强下状态2到状态1的转换受到抑制.高光强下过多的光能由PSⅠ向PSⅡ分配是导致PSⅡ光破坏的重要原因.     

On the existence of two populations of mitochondria in a single organ. Respiration, calcium transport and enzyme activities.

Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome $\text{c}$ to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca²⁺ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome $\text{c}$ content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K<Superscript>+</Superscript> channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H₂O₂ release using different substrates and ATP-sensitive K⁺ transport activities are increased in mitochondria from animals on high fat diets. The increase in H₂O₂ release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K⁺ channels, indicating it was not related to an observed increase in K⁺ transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K⁺ transport in mitochondria can be modulated by diet.     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998     

Laser light-scattering analysis of protoplasmic streaming in the slime mold Physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 mum diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a "beating" pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

Laser light-scattering analysis of protoplasmic streaming in the slime mold physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 μm diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a “beating” pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

Dynamics of Redox Changes of Iron Caused by Light–dark Variations in Littoral Sediment of a Freshwater Lake

Depth profiles of oxygen concentration and the redox status of acid-extractable iron were measured in littoral sediment cores of Lake Constance incubated under a light–dark regimen of 12 h. While oxygen penetrated to 3.4±0.2 mm depth in the dark, photosynthetic oxygen production shifted the oxic–anoxic interface down to 4.0±0.2 mm or 5.9±1.6 mm depth, at low or high light intensity, respectively, and caused a net oxygen efflux into the water column. After a light–dark or dark–light transition, the oxygen concentration at the sediment surface reached a new steady state within about 20 min. The redox state of the bioavailable iron was determined in 1-mm slices of sediment subcores. After a dark period of 12 h, 85% of the acid-extractable iron (10.5 μmol cm⁻³ total) in the uppermost 8 mm was in the reduced state. Within 12 h at low or high light intensity, the proportion of ferrous iron decreased to 82 or 75%, respectively, corresponding to net rates of iron oxidation in the range of 244 and 732 nmol cm⁻³ h⁻¹, respectively. About 55 or 82% of the iron oxidation at low or high light intensity occurred in the respective oxic zone of the sediment; the remaining part was oxidized in the anoxic zone, probably coupled to nitrate reduction. The areal rates of iron oxidation in the respective oxic layer (21 or 123 nmol cm⁻² h⁻¹ at low or high light intensity, respectively) would account for 4 and 23% of the total electron flow to oxygen, respectively. Light changes caused a rapid migration of the oxic–anoxic interface in the sediment, followed by a slow redox reaction of biologically available iron, thus providing temporal niches for aerobic iron oxidizers and anaerobic iron reducers.     

Influence of Light Intensity on Plasma Melatonin and Locomotor Activity Rhythms in Tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm²), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm²) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm² range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm² light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).     

ADP requirement for prevention by a cytosolic factor of Mg2+ and Ca2+ release from rat liver mitochondria

One hundred micromolar Ca²⁺ added to rat liver mitochondria induces a transient uptake of Ca²⁺ plus a rapid efflux of the mitochondrial Mg²⁺. Addition of a cytosolic molecule, cytosolic metabolic factor, to mitochondria prevents the efflux of the two divalent cations. ADP is required for this cytosolic metabolic factor action. This requirement for ADP is specific as it is shown by experiments with traps for nucleotides and inhibitors of the translocase. The implication of cytosolic metabolic factor in the mitochondrial regulation process is discussed.