Oxygen Perception and O2 Toxicity in the Freshwater Ciliated Protozoon Loxodes

ABSTRACT. Loxodes reached peak abundance close to the oxic-anoxic boundary (O₂ 5% atm) in two lakes, in test tube cultures, and in glass chambers with horizontal O₂ gradients. Vertical profiles of CO₂, pH, sulfide, and Fe²⁺ in a lake were not closely related to Loxodes abundance. In a laboratory experiment, Loxodes followed a retreating source of O₂ and was repelled by a high pO₂. This behavior was sustained when cells simultaneously swam up or down gradients of both CO₂ and pH. Aggregation of cells was abolished by KCN (10^-4-10^-6 M). Sodium azide (10^-1-10^-4 M) had no effect and 2,4-DNP sharpened the aggregation. Rotenone, Antimycin A, and HOQNO had no obvious effect. Cytochrome oxidase is probably the oxygen receptor. Loxodes striatus contained low activities of superoxide dismutase and catalase. Extracellular production of superoxide (O_-²) and hydrogen peroxide (H₂O₂) were probably not responsible for the exclusion of Loxodes from water with a high pO₂. Continuous exposure of Loxodes to oxygen at normal atmospheric pressure at 10°C led to 50% mortality in 10 days. Cells left free to swim in an oxygen gradient doubled their number in the same period. Light exacerbated the toxic effects of O₂. Behavioral responses to the dissolved oxygen tension probably controlled the spatial distribution of Loxodes.     

Photosensitivity in the Ciliated Protozoon Loxodes: Pigment Granules,Absorption and Action Spectra,Blue Light Perception,and Ecological Significance1

ABSTRACT. The ciliate Loxodes has a faint yellow-brown coloration due to large numbers of electron-dense granules located in the pellicle. Absorption and action spectra both indicate the presence of a blue-light receptor, which may be a flavin. Absorbance is minimal at wavelengths greater than 500 nm; there is a major peak at 360 nm and a pronounced shoulder at 435 nm. An action spectrum based on light-induced escape from oxic water shows a peak at 435 nm and a peak or shoulder at 360 nm. The pigment will generate superoxide when illuminated in the presence of oxygen. Loxodes living in an oxygen gradient in a spectrophotometer cell swims into and remains in anoxic water at light levels ≤10 Wm^-2. Loxodes can be exposed to light levels of 2–20 Wm^-2 in stratified lakes so its photobehavior can explain its periodic absence from oxic water. Photosensitivity in Loxodes may function as part of a predator-avoidance strategy.     

Developmental Polymorphism Induced by Intraspecific Predation in the Ciliated Protozoon Onychodromus quadricornutus1

ABSTRACT. The hypotrich ciliate Onychodromus quadricornutus is remarkable in its potential for voluminous size (up to 900 μm in length), its possession of a unique set of four dorsal spines or horns, and its capability to express two kinds of developmental polymorphism induced by intraspecific predation. Cell length frequencies in replicates of a well-fed clone show normal distributions; starvation followed by intraspecific predation, however, induces cells within a clone to transform into two size classes: small lanceolate cells and cannibal giants. Induction experiments indicate that a substance released by cannibal giants stimulates defensive spine growth in clonemates within 24 h. Giants can also induce spine growth in non-clonemates. Furthermore, O. quadricornutus cells exposed to the predacious ciliate Lembadion magnum also develop hypertrophied spines. Selection experiments show that conspecific giants prey on cells with undeveloped spines (< 20 μm in length) to a much greater extent than on cells with developed spines (>40 μm in length). Transformation of a population of similarly sized O. quadricornutus cells into two different size classes may function to increase the range of potential prey sizes available to the O. quadricornutus population; hypertrophied spines appear to function as an inducible defense against intermittent predators appearing in the system including conspecific giants. This is the first reported case of a defensive developmental polymorphism induced by intraspecific predation.     

The Fine Structure of the Cyst Wall of the Ciliated Protozoon Didinium nasutum1

SYNOPSIS. Electron microscope observations of the complex cyst wall of Didinium nasutum are reported. The cyst wall is composed of 3 major coats. The outermost coat, the ectocyst, consists of short strands of filamentous material which forms a diffuse, amorphous layer approximately 8–9 μ thick. Culture debris, bacteria and unidentified inclusions have been observed adhering to the outer coat. The mesocyst, approximately 2.5 μ thick, has 2 distinct regions. The outer region is differentiated into several slightly thicker layers which in face view have a honeycomb appearance. The deeper region of the mesocyst consists of compact lamellae lacking the obvious honeycomb appearance of the layers of the outer region. Finally, the endocyst (0.3 μ thick), which arises in the mature cyst in the space that develops between the pellicle and the mesocyst, consists of delicate fibrils in a compact matrix. Both mesocyst and endocyst may be undulant and folded. The structure, origin and possible relationships of the various coats composing the cyst wall are discussed. The present study also contributes information on the role and fate of mucocysts and other cytoplasmic structures during the formation of the cyst wall.     

Kinetic Behavior of Some Polyphosphate-Accumulating Bacteria Isolates in the Presence of Nitrate and Oxygen

This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen. It is shown that species were able to respire both electron acceptors for phosphate accumulation. A. tumefaciens and A. dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A. hydrophila and K. oxytoca eliminated overall phosphate only in oxic conditions. A. dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas. The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A. dispar have a denitrification rate superior when the cells were adapted to nitrate. Received: 15 December 1998 / Accepted: 21 January 1999     

Presence or Absence of Ras Dimerization Shows Distinct Kinetic Signature in Ras-Raf Interaction

Initiations of cell signaling pathways often occur through the formation of multiprotein complexes that form through protein-protein interactions. Therefore, detecting their presence is central to understanding the function of a cell signaling pathway, aberration of which often leads to fatal diseases, including cancers. However, the multiprotein complexes are often difficult to detect using microscopes due to their small sizes. Therefore, currently, their presence can be only detected through indirect means. In this article, we propose to investigate the presence or absence of protein complexes through some easily measurable kinetic parameters, such as activation rates. As a proof of concept, we investigate the Ras-Raf system, a well-characterized cell signaling system. It has been hypothesized that Ras dimerization is necessary to create activated Raf dimers. Although there are circumstantial evidences supporting the Ras dimerization hypothesis, direct proof of Ras dimerization is still inconclusive. In the absence of conclusive direct experimental proof, this hypothesis can only be examined through indirect evidences of Ras dimerization. In this article, using a multiscale simulation technique, we provide multiple criteria that distinguishes an activation mechanism involving Ras dimerization from another mechanism that does not involve Ras dimerization. The provided criteria will be useful in the investigation of not only Ras-Raf interaction but also other two-protein interactions.     

Phytochrome Responses to End-of-Day Irradiations in Light-grown Corn Grown in the Presence and Absence of Sandoz 9789

Corn seedlings were grown in white light in the absence and presence of the chlorosis-inducing herbicide San 9789. The resulting green and achlorophyllous seedlings were used to investigate phytochrome-mediated responses to end-of-day far red irradiation and reversal of these responses by subsequent red irradiation. Mesocotyl and coleoptile elongation increased in response to end-of-day far red irradiation, whereas the anthocyanin content of the coleoptiles was decreased. All three responses were reversible by red irradiation following the far red. Dose-response curves for far red induction and red reversal of these responses did not differ significantly for plants grown in the presence or absence of San 9789. Thus, San 9789 appears to affect neither phytochrome itself nor the response system involved. Chlorophyll screening likewise does not affect phytochrome relationships for these responses.     

Inward and Outward Movement of Ammonium in Root Systems: Transient Responses during Recovery from Nitrogen Deprivation in Presence of Ammonium

Morgan, M. A. and Jackson, W. A. 1988. Inward and outward movementof ammonium in root systems: transient responses during recoveryfrom nitrogen deprivation in presence of ammonium.— J.exp. Bot. 39: 179-191. Net ammonium uptake by 20-d-old wheat (Triticum aestivum cv.Kleiber) and oat (Avena sativa cv. Tarok) seedlings was increased5- to 10-fold when the seedlings were deprived of nitrate duringthe 14-20 d period. The effect of nitrogen deprivation was toincrease net ¹⁵N-ammonium influx and decrease net ¹⁴N-ammoniumefflux during a 1 h assay period. The sizeable rate of net ¹⁵N-ammoniuminflux resulting from nitrogen deprivation was stimulated furtherby prior exposure of the seedlings to ¹⁴N-ammonium for 5 h.Additional exposure to ¹⁴N-ammonium caused the stimulated rateof ¹⁵N-ammonium influx to decline. During the 1 h assays in¹⁵N-ammonium, net ¹⁴N-ammonium efflux increased after 2 h exposureto ¹⁴N-ammonium, peaked at 5–10 h, and then declined.The consequence of the differential response of the influx andefflux processes in wheat was a marked decrease in net ammoniumuptake in the initial 2–5 h, followed by a recovery which,in turn, was followed by a slow decline. In oat, there was norecovery in net ammonium uptake after 2–5 h. Interference in ammonium assimilation by presence of methioninesulphoximine after 5 h did not inhibit expression of the ammonium-stimulatednet ¹⁵N-ammonium influx at 10 h but did substantially increasenet ¹⁴N-ammonium efflux. In nitrogen depleted seedlings, andin those exposed to ¹⁴N-ammonium for 2 h, subsequent net ¹⁴N-ammoniumefflux during 1 h in ¹⁵N-ammonium exceeded the quantity of ¹⁴N-ammoniuminitially in the roots. The increase in ¹⁵N-ammonium influx upon nitrogen deprivation,its further stimulation with 5-10 h exposure to ammonium andits subsequent decline, are discussed as possibly resultingfrom (a) the operation of two ammonium influx systems (b) theinterplay of tissue ammonium and a product of its assimilationrespectively acting as positive and negative effectors of asingle influx system and (c) variations in energy supply fromthe shoots. Key words: Net ammonium uptake, stimulated ammonium influx, ammonium efflux, tissue ammonium     

Coordinated Flagellar and Ciliary Beating in the Protozoon Tritrichomonas foetus1

ABSTRACT. Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagelium is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.     

Structure of Ceramide-1-Phosphate at the Air-Water Solution Interface in the Absence and Presence of Ca

Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA₂α. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca²⁺ is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA₂α. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca²⁺ using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na⁺ concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca²⁺ is bound to the headgroup, ∼0.5 Ca²⁺ and ∼1.0 Ca²⁺ ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^- to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^-1s^-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^-1. It has also been demonstrated that in the absence of DNA O₂^-1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^? to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^?1s^?1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^?1. It has also been demonstrated that in the absence of DNA O₂^?1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^?1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.Oxygen is essential for aerobic respiration, but reactive byproducts of oxygen metabolism, such as the superoxide anion, can damage cellular molecules, including proteins, DNA, and lipids (1–3). SOD1s (copper- and zinc-containing superoxide dismutases) provide the primary defense against superoxide damage by catalytically removing it through a disproportionation reaction (4). This reaction involves redox cycling at the copper active site (5). SOD1s require several post-translational modifications to form an active molecule. Copper and zinc are bound by the enzyme, and an intramolecular disulfide bond is formed between two conserved cysteine residues. Although the zinc ion and disulfide bond are not directly involved in the disproportionation reaction, these modifications are required for proper stability and formation of the active site (6–10). The presence of an intramolecular disulfide bond is intriguing, given the fact that the cytosol favors reduced thiols.The activity of SOD1s in vivo is largely controlled through the aforementioned post-translational modifications. Most of what is currently known about activation of SOD1 in vivo has emerged through studies of the bakers'' yeast Saccharomyces cerevisiae SOD1. Here insertion of the catalytic copper requires the action of the copper chaperone for SOD³ (CCS) (11). CCS physically interacts with SOD1 to deliver the copper ion and catalyze the disulfide bond formation in an oxygen-dependent manner (12–15). In fact, S. cerevisiae SOD1 (ySOD1) is completely dependent on CCS for insertion of the catalytic copper and oxidation of the disulfide bond (11, 15, 16).Although ySOD1 is dependent on CCS for activity, other eukaryotic SOD1s are not. Mouse and human SOD1 (hSOD1), when expressed in CCS^−/− mouse fibroblasts and in ccs1Δ yeast, still retain some SOD1 activity (17–19). Moreover, the genome for the nematode Caenorhabditis elegans does not contain a CCS-like gene, yet harbors several Cu,Zn-SODs. Previous studies with C. elegans SOD-1 (wSOD-1) have shown that this SOD is activated completely independently of CCS (20). Together, these studies present a strong case for a second SOD1 activation mechanism independent of CCS.There must be inherent differences in SOD1 sequences that dictate whether the enzyme uses CCS or the CCS-independent pathway or both. Through targeted mutagenesis, sequences near the C terminus have been previously identified as being important (19). Yeast SOD1 contains dual prolines at positions 142 and 144, which when mutated in combination allow for CCS-independent activation. Conversely, hSOD1 and wSOD-1 contain non-proline residues at these positions, and if dual prolines are introduced, then CSS-independent activation is blocked (19, 20). How this pair of prolines influences SOD1 activation is not understood.It is interesting that nature has developed two activation mechanisms for such a key enzyme in oxidative stress protection, and these are not likely to be redundant. It was previously predicted that the two pathways draw upon distinct sources of copper (19), since the addition of the catalytic copper ion is limiting for enzyme activation. However, since disulfide oxidation is also limiting for enzyme activity, it is possible that the two pathways diverge at this level. In the current study, we investigate the requirements and regulation of the CCS-dependent and -independent SOD1 activation pathways. Our results strongly indicate that the two pathways do not diverge at the level of upstream copper transporter sources or the kinetics of copper incorporation into SOD1 but rather at the level of disulfide bond formation. Copper is required for CCS-mediated disulfide bond oxidation in yeast SOD1, whereas SOD1s that can be activated without CCS show no such requirement for copper in disulfide oxidation. Moreover, oxygen is required for enzyme activation through CCS, but the CCS-independent pathway is able to bypass the need for molecular oxygen. This allows for significant SOD1 activity to be found at a variety of oxygen concentrations by utilizing two activation pathways.     

Thermodynamic Parameters of Opioid Binding in the Presence and Absence of G-Protein Coupling

Abstract

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (K_a), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33°C. It was found that all the opioid agonists tested ([³H]dihydromorphine (DHM) μ alkaloid; [³H]DAMGO μ peptide; [³H]deltorphin-B δ peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their μ or δ receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na⁺ or Mg²⁺ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na⁺; or Gpp(NH)p + Na⁺ + Mg²⁺ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

Mechanism of Catechin Chemiluminescence in the Presence of Active Oxygen

The photon emission (chemiluminescence; CL) of catechin in the presence of active oxygen species (hydrogen peroxide, hydroxyl radical tert-butyl hydroperoxide and tert-butyl oxyl radical) and acetaldehyde was confirmed to occur non-enzymatically at room temperature in aqueous neutral conditions. The CL intensity [P] in the presence of active oxygen species (X), catalytic species (Y) and receptors (Z) is predicted by [P] = k [X] [Y] [Z]. The calculated photon constants (k) of 8 catechins and gallic acid were 8.23 × 10⁶ M⁻² s⁻¹ counts ((−)-epigallocatechin), 2.78 × 10⁶ ((−)-epigallocatechin gallate), 4.66 × 10⁵ ((−)-gallocatechin gallate), 4.36 × 10⁵ ((−)-gallocatechin), 2.70 × 10⁵ ((−)-epicatechin), 6.44 × 10⁴ ((−)-catechin), 5.85 × 10⁴ ((−)-epicatechin gallate), 4.78 × 10⁴ (gallic acid) and 3.54 × 10⁴ ((−)-catechin gallate), respectively. The system of active oxygen species, catalytic species and receptors is proposed to be a scavenging mechanism for active oxygen species. In the presence of acetaldehyde, (−)-epigallocatechin (maximum k value among catechins tested) reacted with tert-BuOOH to form tert-BuOH as determined by HPLC analysis.     

Rheology and Stability of Beverage Emulsions in the Presence and Absence of Weighting Agents: A Review

Cloudiness or opacity (cloudy appearance) is an important property in citrus beverages, since it enhances their juice-like appearance and gives it a natural fruit juice appeal. This property is achievable through the addition of oil-in-water emulsions known as clouding agents. These emulsions are thermodynamically unstable and tend to break down during storage. Moreover, product and legal constraints put severe limits on materials that can be used to insure emulsion stability, particularly the introduction of weighting agents into the oil phase. Weighing agents (density-adjusting agents) are lipophilic compounds with specific gravity higher than that of water and have a restricted use because of the perceived health risk disadvantage, undesirable taste, and oxidative instability. The stability of beverage emulsions is a problem of serious concern faced by the flavor and beverage industry. This paper provides an overview of research carried out by the authors on basic factors affecting the physical stability of beverage cloud emulsions having a bearing on droplet size/distribution, rheological properties of emulsion and phases components, and the stability of emulsion in concentrated and diluted forms with or without addition of weighting agents. Delivery of Functionality in Complex Food Systems: Physically-Inspired Approaches From Nanoscale To Microscale University of Massachusetts, Amherst, October 8–10, 2007