THE PHAGOCYTOSIS OF SOLID PARTICLES : III. CARBON AND QUARTZ.

1. The rates of ingestion of quartz and carbon particles by leucocytes, when both are in suspension in serum, was compared with the availability of the two particles as predicted from the calculated chances of collision with the leucocytes, and it was shown that carbon is ingested about 4 times as readily as quartz. 2. The greater ease of ingestion of carbon was verified by a new method of measuring phagocytosis, described as the film method in which the cells ingest particles as they creep about on a slide. 3. The relative rates of ingestion of carbon and quartz depend upon the condition of the cells, the difference increasing as the phagocytic activity of the cells decreases. 4. Sponge cells also ingest carbon about 3 times as readily as quartz. 5. The hypothesis is suggested that the cause of the more rapid ingestion of carbon may be identical with the cause of the greater instability of the carbon suspensions. 6. An inorganic analogy to this selective phagocytic action is offered. 7. The application to opsonins and agglutinins is discussed.     

THE PHAGOCYTOSIS OF SOLID PARTICLES : II. CARBON.

1. By measurements of the diameter and velocity of leucocytes and of the particles in two carbon suspensions, the relative rates of ingestion of the two suspensions by the leucocytes are predicted and the predictions verified experimentally. 2. The results indicate that 4.7µ particles of carbon are ingested as readily as 3.2µ particles. The more rapid apparent rate of ingestion of the 4.7µ particles is due to their greater availability rather than the greater capability of the leucocytes. 3. There is almost no phagocytosis of carbon in absence of serum or in heated serum. 4. The clumping of unwashed leucocytes is accllerated by serum and by the ingestion of carbon. 5. The available evidence indicates that the phagocytosis of bacteria does not follow the law for a monomolecular reaction, possibly because of the toxic effect upon the leucocytes of bacterial extracts.     

THE PHAGOCYTOSIS OF SOLID PARTICLES : I. QUARTZ.

1. A new quantitative method of measuring phagocytosis of solid particles is described. 2. A method of calculating the chances of collision between leucocytes and quartz particles of different sizes is developed. 3. The speed with which three suspensions of different sized quartz particles should be ingested by leucocytes is predicted from the calculated chances of collision, and the prediction is verified experimentally. 4. The formula for the chances of collision is also verified by varying the speed of rotation of the tubes in which the phagocytic mixtures are incubated.     

Bioconversion of Gelatin to Methane by a Coculture of Clostridium collagenovorans and Methanosarcina barkeri

A simple, stable, and transferable coculture of Clostridium collagenovorans and Methanosarcina barkeri that readily degraded gelatin into methane and carbon dioxide was developed. In monoculture, C. collagenovorans fermented all of the amino acids in gelatin except proline into acetate and carbon dioxide as the main products, with hydrogen, isovalerate, and isobutyrate detected in trace amounts (<1 mM). In coculture with M. barkeri, gelatin was transformed into methane and carbon dioxide, with varying levels of intermediary acetate formed as a function of incubation time. Various complex proteinaceous polymers could be readily transformed into methane and carbon dioxide at 30 to 40°C by a stable coculture which did not require exogenous growth factor additions. In addition, the coculture was readily transferable and preserved in the viable state for long periods, and methanogenesis could be initiated rapidly without the need for exogenous pH control.     

The sorting of food particles by Abra sp. (bivalvia: tellinacea)

The selection of food particles by Abra tenuis (Montagu), A. alba (Wood) and A. nitida (Müller) has been investigated.Material for ingestion smaller than 30μm is not selected according to size by A. tenius. Particles smaller than 0.5 μm are retained by the palliai organs and ingested but only particles larger than 1 μm appear to be retained with an efficiency approaching 100 %. The mesh size of the gill filter is found to be ≈ 3.0 × 0.5 μm.A. tenuis does not appear able to discriminate between particles smaller than 20 μm by their food value; however, relatively large silica particles which are devoid of food are partially rejected by the labial palps in favour of particles of similar size but having a bacterial coating. To a lesser extent the physical nature of particles seems to influence their selection by A. tenuis; clean angular particles are rejected in favour of clean rounded ones.Small, light particles appear to be transported on the gills directly to the mouth without coming into contact with the palps. Larger, heavier particles tend to drop from the gill to be caught by the palps which extend posteriorly to cup the entire ventral margin of the inner demibranch when the animal is feeding.The material ingested by A. alba is significantly finer than that taken into the mantle cavity indicating that the pallial organs actively select food by size. Selection of material for ingestion by size in A. nitida appears to be optional since only some of the animals examined had stomach contents significantly finer than material from the mantle cavity.     

OBSERVATIONS ON THE TRANSPORT OF CARBON DIOXIDE IN THE BLOOD OF SOME MARINE INVERTEBRATES

Although the results we have recorded merely serve to indicate the possibilities of this interesting field of investigation, we have sufficient data to enable us to draw certain general conclusions. In the first place it is evident that the bloods of the more highly developed marine invertebrates, such as the active Crustacia and the Cephalopods, are specially adapted for the carriage of carbon dioxide. The quantity of carbon dioxide taken up by the blood of Maia, Palinurus, or Octopus at any given tension of the gas is, in general, about twice or three times as great as that which is taken up by sea water under the same conditions. On the other hand, the blood of a slow, creeping form, such as Aplysia, or of a sessile animal such as the ascidian Phallusia shows no more adaptation for the carriage of carbon dioxide than does sea water. But our estimations of the CO₂ content of the blood as it circulates in the bodies of these more active invertebrates show that the conditions of transport of this gas differ considerably in some respects from those which obtain in mammals. For the invertebrate blood in the body contains only a relatively small quantity of carbon dioxide, averaging in the forms we examined from 3 to 10 cc. per 100 cc. of blood. This forms a marked contrast with the condition found in mammals where even the arterial blood contains about 50 cc. of CO₂ per 100 cc. of blood. The invertebrate, therefore, works at a very low CO₂ tension. There is a twofold significance in this circumstance. In the first place, it means that only the first portion of the carbon dioxide dissociation curve is in use in the respiratory mechanism. Now an inspection of our curves will show that at these low carbon dioxide tensions the dissociation curves tend to be steeper than at higher tensions. As we intend to show in a later paper it can be proved mathematically that, other things being equal, a blood with a carbon dissociation curve of moderate steepness, i.e. one in which the carbon dioxide content of the blood increases fairly rapidly with increase of carbon dioxide tension, is a more efficient carrier of the gas from the tissues to a respiratory surface than a blood in which the dissociation curve is either steeper or flatter. It would seem as if the active invertebrates avoid the use of too flat a part of their CO₂ dissociation curves by working over the initial steeper portion. Furthermore, it is seen that over the range of this initial steep portion of the curves the changes of reaction produced by the uptake of carbon dioxide are much smaller than at higher tensions of the gas; for these initial portions of the curves are more nearly parallel to the lines of constant reaction calculated for a temperature of 15°C. according to Hasselbalch''s method (10) on the assumption that the whole of the combined CO₂ is in the form of sodium bicarbonate. It is evident also that on this assumption the hydrogen ion concentration of the blood of invertebrates (with the exception of the tunicates) would appear to be practically the same as that of the warm-blooded vertebrates—a conclusion confirmed by the direct measurements of Quagliariello (9). On the other hand, our measurements do not lend support to the idea put forward by Collip (4) that in order to maintain an appropriate faintly alkaline reaction an invertebrate needs to retain carbon dioxide in its blood at a comparatively high tension. This idea was based on the observation that at comparatively high CO₂ tensions the blood of invertebrates contains considerably more sodium bicarbonate than does sea water. But our curves show that this is no longer true at the lower values of carbon dioxide tension, the amount of sodium bicarbonate falling off more rapidly in the blood than in the sea water with diminution of the carbon dioxide tension so that in order to maintain an appropriate reaction in the blood only a comparatively small tension of CO₂ is required. The largest amount of carbon dioxide that we found present in the circulating blood of any of the types examined was 9.7 cc. per 100 cc. of blood in the case of Maia, and in most cases the amount was considerably less. But even this lowest value corresponds to a tension of CO₂ of only about 3 mm., so that the tension gradient across the gill membrane must be even less than this. We would emphasize rather the circumstances that as the portion of the dissociation curve over which the reaction is approximately constant is of but small extent, it is necessary that in an active form like Octopus the carbon dioxide produced should be removed rapidly lest an accumulation of it should cause the limits of normal reaction to be exceeded; and this need is correlated with the extreme efficiency of the respiratory apparatus in this animal. It is interesting to notice that the mammal which, in order to obtain an appropriate reaction in the blood, has to work at relatively high carbon dioxide tensions where the dissociation curve is comparatively flat, secures a steeper physiological CO₂ dissociation curve in the body, and with it a more efficient carriage of carbon dioxide and a more constant reaction in the circulating fluid, in virtue of the effect of oxygenation on the carbon dioxide-combining power of its blood (3, 6). Returning now to the consideration of the actual form of the dissociation curves we have obtained—it is a significant fact that it is in those forms such as Maia, Palinurus, and Octopus whose bloods are rich in proteins—particularly hemocyanine—that the initial steep portion of the curve is observed. This suggests that in these forms the blood proteins act as weak acids and expel carbon dioxide from the blood at the low tensions which include the physiological range, just as in vertebrates the hemoglobin similarly displaces carbonic acid from its combination with alkali metal. On the other hand the cœlomic fluid of Aplysia contains no pigment and only 0.00672 per cent of protein nitrogen (Bottazzi (11)) and shows no initial rapidly ascending portion of the CO₂ dissociation curve. This is supported by the observation of Quagliariello (9) that the acid-neutralising power of the blood of an invertebrate is roughly proportional to its protein content. It seems as if the proteins of invertebrate blood like the blood proteins of vertebrates, exist in the form of sodium salts which are capable of giving up sodium for the transport of carbon dioxide as sodium bicarbonate. That this is so in the case of hemocyanine follows from the fact that the isoelectric point of this pigment occurs at a hydrogen ion concentration of 2.12 x 10^–5 N, i.e. at a pH of 4.67 (Quagliariello (12)) so that in the alkaline blood of the invertebrates possessing it, hemocyanine will act as a weak acid. It is probable that the initial steep portion of the carbon dioxide dissociation curves which we have found to be of such importance in the respiration physiology of Octopus, Palinurus, and Maia is produced by the competition of this acid with carbonic acid for the available sodium of the blood.     

ELECTROKINETIC PHENOMENA : X. ELECTRIC MOBILITY AND CHARGE OF PROTEINS IN ALCOHOL-WATER MIXTURES

1. The electrophoretic velocities of gelatin-, egg-albumin-, and gliadin-covered quartz particles in various alcohol-water solutions are, within the limits employed in usual experimental procedures, proportional to the field strength. 2. The electrophoretic mobilities of small, irregularly shaped quartz particles covered with an adsorbed film of protein in alcohol-water solutions are equal to the electroosmotic mobilities of the liquid past similarly coated flat surfaces. Hence the size and shape of such particles does not influence their mobilities, which depend entirely on the protein film. 3. The corrected mobility and hence presumably the charge of gelatin-covered quartz particles in solutions containing 35 per cent ethyl alcohol is proportional to the combining power of the gelatin; therefore the gelatin is adsorbed with the active groups oriented toward the liquid. The same is true in 60 per cent alcohol. 4. The charge calculated by means of the Debye-Henry approximation from the mobility of gelatin in solutions containing up to 35 per cent ethyl alcohol is, in the neighborhood of the isoelectric point, proportional to the combining power of the gelatin. Therefore the dielectric constant and the viscosity of the bulk of the medium may be used in the Debye-Henry approximation Q = 6 π η r v_m (1 + κ r) to predict changes in charge from mobility. 5. In the neighborhood of the isoelectric point gelatin is probably completely ionized in buffered ethyl alcohol-water mixtures up to 60 per cent alcohol. 6. In the presence of ethyl alcohol the isoelectric point of gelatin is shifted toward smaller hydrogen ion activities. This shift, like that caused by alcohol in the isoelectric points of certain amino acids, is approximately linearly related to the dielectric constant of the medium.     

Properties of a murine monocytic tumor cell line J-774 in vitro. I. Morphology and endocytosis.

A murine monocytic tumor cell line J-774 was maintained in culture in the presence or absence of endotoxin, in an attempt to induce differentiation similar to that found in activated peritoneal macrophages. The morphology and Fc and C₃ receptor functions of attachment and ingestion were compared in the treated and untreated cultures. J-774 cells maintained in culture for 72 h seemed to resemble endotoxin-activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffling was observed on the surface of the cells cultured for 3–4 days both in the presence and in the absence of endotoxin. As compared to the peritoneal macrophage where particles attached to the C₃ receptors are not ingested unless the cells are activated, J-744 cells attached and ingested particles via the C₃ receptor even without stimulation. The presence of endotoxin in the culture medium of these cells gave rise to more efficient phagocytosis but did not effect the temperature sensitivity of the phagocytic receptors. Both in treated and untreated cultures attachment to the Fc receptor was less dependent on the temperature than that of the C₃ receptor while ingestion was more sensitive in the Fc receptor as compared with the C₃ receptor.     

THE PERMEABILITY OF CELLS FOR OXYGEN AND ITS SIGNIFICANCE FOR THE THEORY OF STIMULATION

It can be demonstrated by an indicator method that living cells are as freely permeable to oxygen as dead cells, and that sudden admission of oxygen to the cell cannot account for increased oxidation as a result of stimulation. Oxygen penetrates as readily as carbon dioxide among the acids and ammonia among the alkalies. Exposure of living plant cells to high oxygen pressures does not initiate certain oxidations (except after some hours), which proceed readily in dead plant cells in the air. In the light of the preceding statement, about the permeability of cells for oxygen, this is interpreted to mean that more oxygen enters the cell at high pressure, but that the reacting substances (chromogen and oxidase) are kept apart by some phase boundary as long as the cell is alive. Increased oxygen concentration eventually produces injury to the cell.     

Experimental and calculated parameters on particle phagocytosis by alveolar macrophages.

Phagocytosis of three types of fluorescein-labeled test particles by rat alveolar macrophages (AM) were studied: spherical silica (3.2 microm), heat-killed Candida albicans (3.8 microm), and heat-killed Cryptococcus neoformans (6.1 microm) opsonized with specific IgG. These particles should attach to scavenger, mannose, and Fc receptors, respectively. Both control AM and AM pretreated for 20 h with interferon-gamma (12.5 or 50 U/ml) were studied. The sum of the number of attached and ingested particles per AM (accumulated attachment) was used as a measure of the attachment process, and the number of ingested particles per AM divided by the accumulated attachment (ingested fraction) was used as a measure of the ingestion process. The average ingestion time (IT), which is also a measure of the ingestion process, was calculated from the experimental data. The ingestion process was independent of the attachment process. IT increased with the time of observation. This is explained by the fact that IT determined from observation times shorter than the whole distribution of IT for a certain particle results in a shorter IT than the real average IT. C. albicans (mannose receptor) had the fastest ingestion process, C. neoformans opsonized with specific IgG (Fc receptor) had ingestion that was nearly as fast, and the silica particles (scavenger receptors) had the slowest ingestion process. Treatment with interferon-gamma markedly impaired the attachment process for all three types of particles (and three types of receptors) but clearly impaired the ingestion process only for silica particles (scavenger receptors).     

Organic coatings and ontogenetic particle selection in Streblospio benedicti Webster (Spionidae: Polychaeta)

Surface deposit feeders select food particles based upon characteristics including size, texture, specific gravity, and organic coatings. Spionid polychaetes feed at the sediment-water interface using a pair of ciliated palps and switch between surface deposit feeding and suspension feeding primarily as a function of water flow. Juveniles and adults of some spionid species have different stable isotopic carbon signals, indicating the ingestion of different food sources and potentially the ability to differentiate organic cues ontogenetically. In the present study, the feeding responses of juvenile and adult Streblospio benedicti Webster to seven organic coatings bound to glass microbeads were tested using five amino acids and two carbohydrates. Coated versus uncoated particles were presented in equal proportions based upon surface area. Juveniles and adults were highly selective for all seven types of organically coated beads—87.1% of all beads ingested were organically coated beads. For two of the organic coatings, there were ontogenetic differences; juveniles were more selective of threonine and adults were more selective of proline. These differences may result in ontogenetic diet shifts that allow maximization of energy and/or essential nutrients during critical early life-history stages. Particle selection in tentaculate surface deposit feeders is generally thought to occur primarily during particle contact and transport to the mouth, and is typically characterized as a passive process. Active particle selection at the site of the everted pharynx was observed and quantified for S. benedicti. Organically coated particles represented 50% of the ambient experimental treatment, 64.4% of the particles transported along the palp after contact, and 81.8% of the particles ingested after pharyngeal rejection behavior. Of the beads reaching the pharynx, 26.9% were rejected by ciliary sorting on the pharynx before ingestion, and 81.8% of the rejected beads at the site of the pharynx were uncoated. Our study demonstrates that microphagous feeders that generally handle food particles in bulk are capable of significant levels of active selection for organically coated particles.     

Dung feeding in adult scarabaeines (tunnellers and endocoprids): even large dung beetles eat small particles

Abstract 1. The maximum size of ingested particles was determined in 15 species of adult dung beetle (Scarabaeidae: Scarabaeinae) by mixing small latex or glass balls of known diameter into the dung used as food. Twelve species (tribes Coprini, Onitini, Oniticellini, and Onthophagini) were tunnellers (making dung stores for feeding and breeding in the soil below the pat) and three species (tribe Oniticellini) were endocoprids (feeding and breeding in the dung pat itself).
2. The test species, covering a wide range of body size (fresh weights 0.05–7.4 g), ingested minute particles only (maximum diameter 8–50 µm), and there was a statistically significant but numerically small increase in particle size with body weight.
3. When the effect of body size was taken into account, taxon (tribe), ecological group (tunneller/endocoprid), and dung preference (coarse/fine) had no significant effect on the size of ingested particles.
4. Tests using two tunnelling species did not indicate that beetles use their mandibles to grind dung particles prior to ingestion.
5. The results suggest essentially the same feeding mechanism in all adult tunnelling or endocoprid scarabaeines that eat fresh dung. Larger, indigestible plant fragments are avoided by filtration, and ingestion is confined to very small particles of higher nutritional value.     

Deposit- and suspension-feeding sea cucumbers (Echinodermata) ingest plastic fragments

Weathering of plastic bottles, bags, fishing line, and other products discarded in the ocean causes tiny fragments to break off. These plastic fragments may accumulate biofilms, sink, and become mixed with sediment, where benthic invertebrates may encounter and ingest them. Here we show that four species of deposit-feeding and suspension-feeding sea cucumbers (Echinodermata, Holothuroidea) not only ingest small (0.25 mm < maximum dimension < 15 mm) nylon and polyvinyl chloride (PVC) fragments along with sediment, but also ingest significantly more plastic fragments than predicted given the ratio of plastic to sand grains in the sediment. During four-hour feeding trials, holothurians ingested between 2- and 20-fold more plastic per individual than expected for PVC fragments, and between 2- and 138-fold more for nylon line. In addition, two species ingested 4 mm diameter PVC pellets. The ecological relevance of plastic ingestion was assessed in the laboratory by counting and characterizing small plastic particles discovered in sediment samples from the same field sites where our holothurians were collected. Substantial numbers of plastic fragments (105 to 214 fragments per liter of sediment) were found in samples from three different locations along the east coast of the U.S.A. In addition, plastic collected from the sediment from two of our field sites was analyzed for polychlorinated biphenyls (PCBs). Plastic from one site tested positive for Aroclor 1254 at a concentration of 0.0106 μg g^-1. While the negative effects of macroscopic marine plastic debris on a host of organisms are well documented, ingestion of small plastic debris by a wide range of benthic organisms, including both primary and secondary consumers, has received little attention. Given that plastics readily adsorb PCBs and other organic pollutants in marine environments, ingestion of plastic from sediment may provide a heretofore-undescribed pathway of exposure for benthic marine invertebrates.     

Effects of Cell-Bound Microcystins on Survival and Feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Are ball-rolling (Scarabaeini,Gymnopleurini, Sisyphini) and tunnelling scarabaeine dung beetles equally choosy about the size of ingested dung particles?

Abstract.  1. The maximum size of ingested particles was determined in 11 species of ball-rolling, adult dung beetle (Scarabaeidae: Scarabaeinae) by mixing small latex or glass balls of known diameter into their food. The tribes Scarabaeini, Gymnopleurini, and Sisyphini (four, four, and three species respectively) were represented, with mean body sizes ranging from 0.33 to 4.0 g fresh weight.
2. Only particles with maximum diameters of 4–85 µm were ingested. Hence rollers, like other known beetles feeding on fresh dung, filter out larger, indigestible plant fragments and confine ingestion to small particles of higher nutritional value.
3. The maximum diameter of ingested particles increased significantly with body weight, whereas taxon (tribe) had no additional effect. Because big rollers accept larger particles than do tunnellers (which make dung stores for feeding and breeding in the soil immediately below the pat) of similar weight, the slope of the diameter-against-weight regression for rollers was significantly higher than that found earlier for tunnellers.
4. An explanation could be that a typical food ball made by a roller is considerably smaller than the amount of dung available to a feeding tunneller of the same size. If the roller were as choosy about particle size as the tunneller, it might not get enough food. This applies to large rollers in particular because their food balls contain a higher proportion of coarse fibres than those made by small species.     

Particles and macrophages in murine Peyer's patches

Mice were given 1% suspensions of 5 insoluble particles (chrysotile asbestos, quartz, carmine, carbon, and iron oxide) in drinking water for 3 months. The particles were subsequently sought in intestinal Peyer's patches by light microscopy. Carbon and iron oxide particles were visible in Peyer's patch macrophages, particularly in the subepithelial region, but the other particles could not be detected. The findings suggest that particle surface properties as well as particle size govern accumulation in Peyer's patches. The cytochemistry of subepithelial, mid-dome, tingible-body, and serosal macrophages of control mice indicated diversity of macrophages within the patch. Macrophages of asbestos-fed mice contained more lysosomes than macrophages of controls. Macrophage abundance in the dome apex was not significantly altered by asbestos ingestion. The other particles did not produce detectable alterations in macrophage morphology.     

Exsheathment of the infective larva of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby Macropus eugenii.

The infective larva of L. eugenii is enveloped in two cuticles which are discarded when the larva exsheaths in the sacculated portion of the wallaby's stomach. In vitro larvae exsheathed in a 0·85% solution of sodium chloride at 37°C, buffered to pH 7 with bicarbonate ion and 40% carbon dioxide. Survival was enhanced if the liquid phase contained medium 199 and serum, and exsheathment was quicker if exposure to carbon dioxide was 1 h rather than 1 day or 7 days. As larvae exsheathed, contractions of the pharynx commenced, and medium was ingested, even when larvae were enveloped in both cuticles. The stimuli for exsheathment and the subsequent pattern of events are like those already recognised in some trichostrongyles.     

The Incorporation of 14C into the Protein of Particles Isolated from Plant Cells

The incorporation of ¹⁴C from labelled fructose, succinate,urea, and proline, by particulate preparations from dormantand tissue-cultured carrot cells, is examined. It is shown that¹⁴C is incorporated readily from proline, and less readily fromfructose. No significant incorporation occurs from succinateor urea. No differences are noted between the two kinds of preparation.It is concluded that the incorporation of ¹⁴C does not dependon prior transfer of the label to carbon dioxide followed byfixation of carbon dioxide, since the particles do not incorporate¹⁴C from supplied carbon dioxide. Incorporation of ¹⁴C by various fractions of dormant carrottissue is examined, and it is established that the greatestincorporation per mg. nitrogen occurs in particles isolatedat 10,000 g. A total cell homogenate fails completely to incorporate¹⁴C from proline into protein, and this may be due to suppressionof the activity of the particles by a constituent of the supernatantliquid. The presence of coconut milk reduces the incorporationof ¹⁴C from proline by particles sedimented at 10,000 g, andaddition of a protein hydrolysate reduces it further. Hydroxy-prolinedoes not appear to compete with proline for incorporation, andin this respect the paniculate preparations contrast with wholecells. Particles from carrot tissue are shown to be more active inincorporating ¹⁴C from proline than are particles extractedby the same procedure from red beet roots, potato tubers, andskunk cabbage inflorescences. They are, however, considerablyless active than a mitochondrial preparation from rat liver. It is demonstrated by paper chromatography that the bulk ofthe ¹⁴C incorporated in the particles from carrot cells remainsin proline and there is little or no conversion of proline tohydroxyproline in the preparations. The nature of the particlesemployed in this investigation is discussed, and their metabolismconsidered, in relation to the structure and activity of wholecells.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Penetration of soybean root systems by abscisic Acid isomers

The penetration of soybean (Glycine max L. cv. Ransom) root systems by exogenously applied isomers of abscisic acid was monitored by measuring the concentration of the chemical in the xylem exudate of root systems exposed to a three bar hydrostatic pressure difference. The cis-trans isomer penetrated more readily than the trans-trans isomer; however, up to 6 hours was needed to reach steady-state values. Exogenous abscisic acid also decreased volume flux through the root system and increased total carbon dioxide efflux from the vessel containing the root system.