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Isolated dog hearts were perfused for 24 hours at 5° C with one of three solutions: plain Krebs solution, Krebs solution containing 167 mg/L of procaine hydrochloride, or Krebs-procaine solution with washed red cells (hematocrit 2%). Coronary vascular resistance (CVR) of hearts perfused with Krebs solution alone increased sharply between the eighth and the twelfth hours of perfusion and then stabilized. Increase of myocardial firmness paralleled the increase of CVR. The addition of procaine to Krebs perfusate kept the heart flaccid and suppressed the steplike increase in CVR but failed to improve significantly the preservation of the hearts. The addition of washed red cells to Krebs perfusate kept CVR near baseline level, improved coronary perfusion flow, doubled myocardial oxygen consumption, reduced edema formation, and improved left ventricular stroke work (LVSW) of the preserved heart. This improvement seems mostly related to a mechanical effect of red blood cells on capillary flow distribution of the heart.

A STRIKING feature of aggregation in large species of Dictyosteliumis the appearance of repeated waves of centripetal movement propagated outwards from the centres1,2. I accounted for them as follows3–8. The cells are not only oriented by the chemotactic agent, acrasin9, but induced to secrete it; they thus relay the orienting signals. The detailed aggregation pattern, before contact modifies it, records the transmission of these signals. To avoid an infinite regress, some secreted factor must induce its own secretion; this was tentatively assumed to be the attractant itself. There would then be separate thresholds for attraction and secretion. The dual responsiveness develops during the preaggregation phase. Aggregation centres are started by the first cells to secrete signals supraliminal for their neighbours. That separate cells, having oriented to the signal and transmitted it outwards, do not reorient to the signal now coming from behind them and transmit it centripetally, was ascribed to some combination of reduced receptor, motor, and secretory activities, expressed as an absolute or relative refractory period, and to changes in cell shape, spacing, orientation, and polarity, once the cells have responded. The precise signal profile may be significant. After an interval, the cells can transmit a new pulse in any direction. Commonly the same cells as before initiate the later pulses and thus remain dominant, but pulses from other pacemakers can recruit and reorient those cells they reach first. Secreted acrasin is rapidly inactivated enzymically. It was argued that this is functionally useful in maintaining the detectability of the signal even with a single source, and vastly more so with literally millions of sources. Enzymic breakdown could also yield a metabolite for recycling. Later work10–12has very largely confirmed this. The attractant in D. discoideumis cyclic AMP13, and the inactivating enzymes phosphodiesterases (PDE)14–16. Cyclic AMP pulsed from a microelectrode induces propagated waves of movement17. A mathematical model has incorporated most of the above features18. The present work demonstrates directly that cyclic AMP does induce aggregating cells to secrete cyclic AMP.

A factor diffuses from Escherichia colt that temporarily or permanently prevents separate pre-aggregation cells of Dictyostelium discoideum from forming aggregation centres or streams. Centres are inhibited at a greater distance from the bacteria than are streams. The inhibitor does not support the slime-mould’s growth, and it has different properties from previously discovered aggregation-centre inhibitors released by the amoebae themselves. The factor, as produced, does not inhibit Polysphondylium violaceum cells from forming centres or streams. Yet although E. coli itself attracts pre-aggregation P. violaceum cells, it does not mimic a P. violaceum aggregate in inducing them to form streams.

1. A description is given of the development of certain structures--centre, stream, and ring--formed by the amoebae of four species of Dictyostelium and Polysphondyliumduring aggregation, and of the outcome of experimentally cutting a stream, recombining lengths of stream in simple geometrical relationships, and excising a centre. 2. In all cases there is a high degree of intraspecific variation in behaviour, and it is not possible to predict, except in terms of rough probability, what the future course of development will be. 3. An amoeba at this stage of the life cycle can exist in one of four main states. In the first, it is unaggregated and cannot react chemotactically; in the second, it is unaggregated but can react; in the third, it is in a stream moving towards a centre or has properties similar to those of a stream cell; and in the fourth, it is in a centre and nearly stationary. Though an enormous variety of patterns may be produced by large numbers of interacting amoebae, their behavioural repertory may be considered, without too much simplification, to be restricted to approaching these four states in various sequences and at various rates. 4. Most of these sequences can be observed over a wide range of environmental conditions. Changes in humidity produce the greatest effect: the drier the culture, the stronger the drift from the first state to the fourth. 5. It is suggested that variability is occasioned by the simplicity of the mechanisms controlling development.

1. When all available food has been consumed, the amoebae of Dictyostelium and Polysphondylium aggregate towards collecting centres; this movement involves chemotaxis. 2. Just after they have finished feeding, the amoebae adhere little or not at all to one another if they come in contact; they do not secrete the chemotactic agent acrasin, and cannot orient in an acrasin gradient. 3.Later they become able to respond to an acrasin gradient, if this is great enough. In the absence of one, they may be sufficiently adhesive to form loose amorphous associations, or, depending on the species and the environment, still be quite separate. Amoebae with similar properties may be released by the spontaneous break-up of aggregated masses. In some species, amoebae that are unaggregated may lose their sensitivity to acrasin after a few hours. 4.The elongated amoebae attracted to a source of acrasin may condense into streams. Stream amoebae move at varying speeds; they adhere strongly to others of the same species and to a variable extent to those of different species. They secrete the acrasin characteristic of their own species. Along a stream, there is often no continuous centrifugal decrement in secretion, as far as can be detected. The amoebae in it can be guided by the direction in which their adherent neighbours are flowing, though if they are exposed to an adequate acrasin gradient they will respond to it. Commonly, an adjacent stream or a centre planted beside them is not strong enough to attract them, unless they are freed to some extent from the influence of their neighbours, especially those ahead of them. 5. Except for their being effectively non-motile and more nearly isodiametric, the amoebae in a centre are similar to those in a stream and may secrete acrasin at much the same concentrations as these do. With both types, there may be short-term fluctuations in secretion, and in at least one species a long-lasting decrease. 6. The dimensions and gross appearance of an aggregation at the population densities used are partly determined by the time intervals over which the amoebae become acrasin-sensitive and centres are initiated, and by the relationship between these.

Cellular-slime-mould amoebae that adhere poorly to one another on contact and effectively do not secrete the chemotactic agent acrasin may become strongly adhesive and start to secrete it. This change, which is particularly important for their aggregation, has been called integration, and the reverse change, disintegration. In Polysphondylium violaceum a cell may receive the integrative stimulus at some distance from the acrasin source that attracts it, but usually it does not change till it has moved close to it or actually reached it; so either an aggregation is simply a heap without any tributary cell-streams, or virtually continuous streams are slowly built out from the centre. In Dictyostelium discoideum the spread of integration, in comparison with cell velocity, may be so rapid that the inflowing streams very soon reach their ultimate extent, though if the population density is not too high their cells only slowly establish contact with one another. As the peripheral cells become integrated, they ‘relay’ the centre’s influence. Whether the streams are uninterrupted or ‘stippled’, there need be no continuous and finely graded centrifugal decrement in acrasin secretion: orderly aggregation is ensured by the sequence in which secretion is induced, which results in the centrifugal propagation of one or more comparatively narrow zones in which the gradient is adequate for orientation. A spontaneous or an experimentally produced decrease in the strength of the integrative stimulus, or adaptation to the stimulus, or both, may induce some integrated cells to revert to the unintegrated state. The spread of disintegration, too, in comparison with cell velocity, tends to be more rapid in D. discoideum than in P. violaceum: in the former species all the cells in a considerable length of stream may begin to separate at almost the same time ; in the latter they may detach themselves in succession. As disintegration may affect any part of an aggregation, various patterns result therefrom. If it slowly spreads from the centre of a P. violaceum aggregation while the streams are still growing at their outer ends, a ‘fairy ring’ is formed, in which cell movement remains polar. Populations of a D. mucoroides strain, if not too sparse, aggregate at a comparatively fixed time after consuming the available food ; but those of D. discoideum at widely different densities and degrees of starvation produce synchronous outbursts of aggregation when transferred from darkness to light. The development and distinctive properties of the initiators of aggregation centres are considered. These cells can release acrasin into the medium without there being any there already or without there being sufficient to induce the remaining cells to secrete. Much evidence is against aggregation being basically a sexual phenomenon. [Quarterly Journal of Microscopical Science, Vol. 99, part 1, pp. 103-121, Jan. 1958.]

The movement of slime-mould amoebae towards certain collecting centers, which have been started by a few of the amoebae, depends basically on their orientation to the maximum gradient of a chemical, acrasin. Because the gradient is produced by diffusion, the direct range of a center must be limited by the size and maximum sensitivity of the responding amoebae, the amount of chemical that can practicably be produced, and the time needed for orientation to occur after secretion has begun. In fact, its range is rapidly and economically extended by a relay system, as acrasin not only orients an amoeba but induces it to become sticky and secrete acrasin. But as a result, there may be a hundred-thousand sources within an aggregation; and the organism meets the problem of maintaining detectable gradients by inactivating secreted acrasin with an extracellular protein. However, this further reduces the importance of any influence the chemical secreted by the centre could have at the periphery. The amoebae move towards the center not because of a sustained gradient of secretion outwards from it, but because of the time sequence in which the relay operates. In one type of aggregation, the delay before an oriented amoeba begins to secrete is sufficient for it first to reach the amoebae that attracted it; and as it can then depend on contact guidance, it is not disturbed by the acrasin production of the amoebae on its flanks and at its rear. The result is that the amoebae, stuck together in radial streams, converge on the point where acrasin secretion began. In another type of aggregation, the delay is less, and the amoebae start to make acrasin before they reach their primary attractors; nevertheless, they too condense into radial streams, because, once the field has been polarized, there is a greater probability that an amoeba will continue to be attracted in the direction in which it has been oriented than in any other. The center owes its position not to constantly secreting more than the peripheral amoebae but to secreting before them; and this is primarily due to the ability of its initiator to release acrasin into the medium without their being any there already. If the time relationships of the relay are seriously disturbed, aggregation becomes chaotic. The secretion of a center may fluctuate, and this may lead to the periodic re-excitation of the relay system. The resultant pulses spreading out over an aggregation may reinforce flow guidance in its streams and are especially useful in repetitively reasserting the authority of the center in an aggregation in which the oriented amoebae are not in contact. Also, acrasin is used more economically if an adequate gradient is maintained for only a fraction of the total time. Various reactions with neighboring relay systems are possible: and peculiarities in the spread of a relay may lead to an aggregation's being circularly polarized. Specificity in aggregation may depend on differences in cellular adhesiveness, the acrasin, or the pulse pattern. There are many resemblances between the orientation of aggregating amoebae and of growing nerve fibers.

In an ordinary agar-plate culture of the collective amoeba, or cellular slime mould, Polysphondylium violaceum, the solitary phase ends when compact and increasingly refractive cell clumps develop among the unoriented cells and secrete the chemotactic factor acrasin. Neighbouring cells may crawl towards them, either singly or aggregated in streams, which relay chemotactic and contact guidance centrifugally (Shaffer, 1957a, 1958). As already briefly reported (Shaffer, 1959), aggregation centres may be started by single, differentiated cells ; these have been called founders.

The surface of a separate Dictyostelium discoideum cell exposed to fluorescent antiserum is initially uniformly stained. When the cell moves, labelled material is left on the glass over the area occupied by the cell while in the antiserum. The surface of new pseudopods is fluorescent. The bulk of the label soon accumulates on the rear of the cell and is towed along behind it. The rest of the surface may remain faintly fluorescent for long periods, but this residual label is immediately left behind if the cell advances while strongly compressed between agar and glass. This evidence does not clearly decide between the hypotheses of a permanent, advancing, lateral cell surface and a temporary, stationary one. The fact that the lateral surface of an advancing cell does not drag forward viscous medium in contact with it does not favour the former hypothesis.

Earlier workers examined the behaviour of foreign particles placed as markers on aggregates of D. discoideum that were migrating over the surface of the culture plate (Bonner, 1959; Francis, 1959, 1962). Comparable observations, made on aggregates in other conditions and at other stages, have now provided further information about the movement of individual cells within the aggregates. Before reporting them, the course of development must be described in some detail.

The Xenopus tail preparation described here was initially developed for studying the action of substances on the vertebrate nervous system. For this both physiological preparations and whole animals have their disadvantages. The former are short-lived ; the latter may die before any major chemical or structural changes have had time to occur. It was therefore thought desirable to seek a relatively long-lived preparation that possessed a functional central nervous system, but yet was not dependent on the conduction of nerve impulses for its survival. The tail of a Xenopus laevis tadpole seemed to be highly suitable for this purpose, as it contained complete reflex arcs and was sufficiently small to carry out its necessary exchanges by diffusion. Moreover, it was so transparent that nerves and muscle striations could be studied microscopically during life. Although agents to which the skin was impermeable could easily be injected into the tail, it was thought best to seek media that would be suitable for partially flayed tails.

The four-bar mechanism is analyzed, and a differential equation is derived which tells whether or not it is possible to find a particular design in which the position of the driver and follower are related by a prescribed function during the entire range of operation. If there is such a mechanism, the length required for each of the links may be found by substituting into the appropriate equations, also derived in this paper.

The recently developed methods of analyzing stress and strain distributions in the plane plastic flow of an ideally plastic material are applied to the problem of machining. It is shown that the idealized stress-strain relationship involves justifiable assumptions for this application. Analytical expressions are obtained for orthogonal machining which give the machining force, the chip thickness, and the chip deformation in terms of the tool geometry, the relevant coefficients of friction, and the appropriate yield stress of the work. With increasing friction at the tool face or decreasing rake angle the development of a built-up nose arises naturally as a consequence of the analysis. The theory includes this phenomenon. The results of this theory are compared with published experimental results, and with other theoretical analyses. Satisfactory agreement with experiment is obtained.

Polysphondylium violaceum and Dictyostelium discoideum myxamoebae have simple double-layered nuclear membranes, a cytoplasmic reticulum of particle-covered membranes, and small mitochondria consisting of convoluted tubules tightly packed in double membranes. In addition to objects still recognisable as bacteria, their food vacuoles contain concentric (or spiral) membranes, apparently formed secondarily from undigested material; these are ultimately ejected. Where the triple-layered plasma membranes (~70 A wide) of cells in the early aggregates are apposed to one another, they run parallel but separated by a layer of rather constant thickness (~200 A), as in many unspecialised metazoan tissues. Thus studies on slime moulds may well increase our understanding of cell adhesion and tissue formation in metazoa.

A study has been made of acrasin, the agent inducing chemotaxis in the amoebae of cellular slime moulds. A method has been developed for subjecting sensitive amoebae to a fluctuating gradient set up by an artificial source that can be renewed at intervals of as little as a few seconds with fresh test solution. Amoebae orient to a gradient maintained with the cell-free liquid freshly obtained from the immediate surroundings of a natural source. Acrasin solution as secreted loses its activity very rapidly at room temperature. A highly active stable solid is obtained by drying methanolic culture extracts ; it resists boiling and exposure to acids and alkalis. Its solubility decreases rapidly in passing up the alcohol series. The original instability has been shown to be due to the presence of another extracellular slime-mould product, possibly an enzyme; it, unlike acrasin, cannot pass rapidly across a dialysis membrane, is heat labile, and can be precipitated by ammonium sulphate. The advantages of the organism’s itself inactivating acrasin are considered. Some of the advantages of a source’s releasing acrasin in pulses are discussed ; but it is not essential for orientation for it to do so. Sensitive amoebae not only are oriented by an acrasin solution but are caused to secrete acrasin: this is the basis of a chemotactic relay system.

Haiti suffers from both the worst poverty and highest child mortality rates in the Western Hemisphere. The purpose of this investigation was to assess the relationship of child mortality to relative degrees of poverty within Haiti.Method: We conducted a survey of 656 women, 15–45 years of age, living in a particularly poor rural area of southern Haiti. Data was collected regarding living conditions (dwelling size, occupants, construction characteristics, presence of a latrine, and water access) and pregnancy outcome (number of pregnancies, pregnancy losses, stillbirths, and child deaths). Relative poverty, as indicated by living conditions, was compared with pregnancy outcome using pooled and individual variables.Results: Using a scoring system for pooled variables, a significant correlation was found between overall living conditions and overall pregnancy outcome, i.e. women living in a thatched house with a dirt floor and no latrine were more likely to have a pregnancy loss, stillbirth, or child death when compared to mothers living in a house with a cement floor, sheet metal roof, and access to a latrine (p<0.01). Among individual descriptors of poverty, we noted that women living in a dwelling with a dirt floor reported 71 child deaths following 817 live births (9%). Whereas, women living in a dwellings with a cement floor reported 63 child deaths following 1101 live births (6%), p = 0.01, relative risk of child mortality = 1.5.Conclusion: Child mortality risk is heterogeneous. Even within a poor neighborhood in a poor country, stratifications of poverty and health outcome are discernable. Certain living conditions may be practical indicators of risk and thereby useful to direct interventions and resources.

We tested the effect of 2 exposure methods and 2 environmental factors, temperature and humidity, on Beauveria bassiana (Deuteromycotina: Hyphomycetes) mycosis in Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae). We found that the standard EPA bioassay method of exposing nontarget insects by immersion resulted in a per-insect dose that was 5 times greater than that from spray applications. However, this difference did not significantly affect mycosis levels in a simulation of field applications conducted in the greenhouse. In contrast, differences in environmental conditions between the laboratory and greenhouse had a large effect on mycosis. Constant temperatures between 15 and 35°C had a significant effect on both germination rate and vegetative growth of B. bassiana, with the fastest germination occurring at 25-32°C and the fastest growth occurring at 30°C. Although temperature affected the rate of conidial germination, total germination eventually reached 97-100% at all temperatures. Contrary to these results, levels of mycosis in lady beetles decreased as temperatures increased over this same range. Although high levels of humidity are required for conidial germination, we found no well-defined threshold period of high humidity exposure required for mycosis in this insect. In other words, the effects of temperature and humidity on free-living stages of the pathogen are not sufficient to explain the effect of these factors on mycosis. We suggest that temperature and humidity also affect physiological interactions between the host and pathogen.

THE α-toxin of the gas-gangrene organism Clostridium welchii type A is one of the few lethal toxins of known enzymic action; it is a lecithinase C, which splits lecithin into phosphoryl choline and a diglyceride1. It affects the enzymic activity of isolated mitochondria and lyses red blood cells3. Unfractionated welchii toxin caused histological changes in fragments of tissues and organs, including the release of fat globules, lysis of nuclei, and general break-up, that agreed more or less with the clinical picture4, and destroyed guinea pig spleen in culture5.

IN Dictyostelium and Polysphondylium, members of the Acrasieæ, there is an aggregation stage in which previously independent amœboid cells stream towards certain cell clumps, which thus act as collecting centres, and there form communal fruiting bodies1. Communication between the centres and the reacting cells is through the external medium. Bonner2produced indirect evidence that a chemical diffused outwards from the centres, setting up a concentration gradient by which the separate cells oriented themselves; pointing out that isolation in vitro would be the final proof of the chemical's existence, he named it ‘acrasin’.