SPECIAL ARTICLES AMBOID MOVEMENT, TISSUE FORMATION AND CONSISTENCY OF PROTOPLASM1

1. IN 1901 we found through testing by direct means the consistency of the protoplasm that in the blood cells of Limulus amoeboid movement depends primarily upon alternating changes in the consistency of the protoplasm, a phase of liquefaction being followed by a phase of hardening. There may be added to these changes in consistency changes in surface tension.1 Subsequently we showed in experiments in which we likewise tested the consistency of the cells directly that the consistency varies under different conditions, that these variations correspond not only to the pseudopodial activity, but also to the agglutinability of the blood cells and to the formation of tissue like structures from

previously isolated cells,2,3 that these changes due to stimulation explained the stereotropism of tissue cells,2,4 that they played a part in the processes of inflammation,1, 2 phagocytosis, and thrombosis.5, 6, 7, 8 More recent observations in 1919 showed that it is possible to vary greatly the character of the amoeboid movements and that the changes in the amoboid movements correspond to the changes in the consistency of the protoplasm; these observations suggested that the taking up of fluid from the surrounding fluid on the part of the cells is an important factor in these

1 From the Department of Comparative Pathology, Washington University, and from the Marine Biological Laboratory, Woods Holl.

1 Leo Loeb, Jour. Med. Research, 1902, VII., 145. 3 Leo Loeb, Biological Bulletin, 1903, IV., 301. 2 Leo Loeb, Virchow's Archiv., 1903, CLXXIII., 35.

♦ Leo Loeb, Anatomical Record, 1912, VI.

5 Leo Loeb, Virchow's Archiv, 1905, CLXXXV., 160.

Leo Loeb, Hofmeister's Beitraege z. Chem. Physiol. u. Pathol., 1904, V., 191.

7 Leo Loeb, Pflueger's Archiv, 1910, CXXXI., 465.

processes. We furthermore showed that certain phenomena of wound healing can be imitated in this experimental amabocytetissue and that the formation of giant cells which takes place in sensitive cells in contact with a foreign body represents an application of the same principle." Here we have to assume that the process of liquefaction may proceed so far that two cells may flow together. We also pointed out that variations in the hydrogen ion content of the cells under the influence of stimulation might explain these changes.

2. A continuation of these experiments in Woods Hole last summer showed that in the blood cells of Limulus it is possible to produce graded variations in the character of the pseudopods and amoeboid movements through graded changes in the osmotic pressure in the surrounding medium. Again we find correspondence between the consistency of the protoplasm and the character of the pseudopods and amoeboid movement. A particularly great fluidity of the protoplasm could be produced through the use of a slightly hypotonic solution of KCl. In this case the change in consistency became so marked that it affected not only the ectoplasmic layer of the cell, but extended to the whole of the granuloplasm. There is reason to assume that these changes are associated with the taking up of fluid from the surrounding medium. Under those conditions a very peculiar phenomenon which we described previously, a circus movement of the whole cell exoplasm as well as of the granuloplasm, can be produced regularly. These movements, however, take place only if the temperature of the surrounding fluid is sufficiently high. It does not occur in cells kept at a temperature of 10°.

3. Exposure of the blood cells to a temperature of approximately 40°-42° for a short period of time produces in the periphery of

/9 Leo Loeb, "The movements of the Amœbocytes and the experimental production of amoebocyte (cellfibrin) tissue," Washington University Stud

478.

(Here a general discussion of the subject is given.)

the blood cells the appearance of multiple drops into which the granuloplasm moves subsequently as it does into typical pseudopods. Transition can be observed between these drops and the typical pseudopods.

It is also possible to produce experimentally in the amœbocytes structures which very closely resemble ova in which maturation membranes have formed. Jacques Loeb has formerly shown that this formation depends upon a proces of cytolysis. In the blood cells these structures appear under conditions in which the cell has taken up fluid from the surrounding medium and the consistency of the protoplasm resembles that of a liquid. All kinds of transition between these structures, drop pseudopods and the typical tonguelike pseudopods can be found. These and other observations very strongly suggest that the formation of pseudopodia, the appearance of drops at the surface of the cells and the formation of fertilization membranes related phenomena and that the latter two conditions represent extremes in a process which, when acting in medium intensity, leads to the formation of the typical pseudopodia. 4. Through changes in the consistency of the protoplasm in the blood cells of Limulus it is possible to imitate the structures corresponding to different tissues. Especially did we obtain in certain cases through an increase in the consistency of the cells tissues which resembled those composed of ganglia and glia cells. It may thus be possible to obtain indications as to some of the conditions which induce the cells of different tissues to assume different forms.

WASHINGTON UNIVERSITY

LEO LOEB

THE RELATIVE NUMBERS OF TWINS AND TRIPLETS1

are

Ir may be of interest to call attention to a simple relation between the number of human twin and triplet births. The relation was noticed a number of years ago and I supposed

1 Contribution from the Zoological Laboratory of the University of Illinois, No. 172.

it had been recorded, but a search has failed to reveal any published statement.

If 1/n is the proportion of twin births to all births in a large population during any period, then the proportion of triplet births during the same period is very near to 1/n2. The agreement of the data is often startling. Thus in 13,360,557 births in Prussia during the years 1826-1849 as recorded by Veit2 the number of twin births is one in 89.1 and the number of triplet births one in (88.9). In 1,339,975 births in the United States registration area in 19173 the number of twin births is one in 93.1 and the number of triplet births one in (93.0)2.

From the statistical relations it would appear that triplets are produced by the coincidence of two independent processes occurring with equal frequencies. One of these processes by itself gives rise to twins. This relation would apply to any mode of origin of multiple births or to different combinations of them provided that each followed the rule. The principle might be applied to the two chief explanations of multiple births as follows:

1. Multiple Ovulation.-Normally a single ovum is discharged from the ovaries. There is some coordinating mechanism which prevents the ripening of other ova at the same time. Suppose that as a result of a purely intrinsic factor, once in n times an ovum appears which fails to respond to this mechanism. The chance that two such extra ova will appear at the same time is once in n squared. Obviously this presupposes that the failure to respond is due to independent processes in the two ova. To put the case more concretely, suppose that the approach to maturation of an ovum is accompanied by an internal secretion which acts upon other ova and keeps them from completing the process at the same time. The overwhelming major

2 Veit, G., 1855, Monatsschrift für Geburtskunde und Frauenkrankheiten, 6: 127.

3 Birth statistics for the birth registration area of the United States, 1917, U. S. Bureau of the Census, Washington, 1919.

ity of the ova would be properly inhibited but occasionally an ovum would fail to respond because of some peculiarity in its organization. Suppose that such peculiarities are due to local factors appearing with a frequency of 1/n. Then the chance that two such independent local factors will act at the same time and thereby cause the simultaneous discharge of two supernumerary ova is 1/n2.

2. Monozygotic Twins and Triplets.-Normally a single embryonic area appears in the blastodermic vesicle and through some coordinating mechanism inhibits the formation of additional embryonic areas. Suppose that once in n cases a cell or group of cells acquires physiological independence as the result of an intrinsic factor and forms a second embryonic area. The chance that two such cells or groups of cells will arise at the same time is once in n squared if it is supposed as in the previous case that the two events are independent of each other.

In order that the stated numerical relations may ensue, the important consideration in either mode of origin of multiple births is the independence of the two events which give rise to triplets. If, in the fluctuations of the general physiological state of the mother, the condition is sometimes such as to result in twins and sometimes in triplets, it is hard to see why the square" relation should exist. For instance if it is postulated that additional ova are stimulated to complete the maturation process as a result of an unusual amount of an internal secretion and that the number of extra ova depends on the quantity of the secretion there is no reason for expecting the observed relation between one extra and two extra embryos. This difficulty seems to apply to all general agents that may be postulated as acting upon the ovaries as a whole in the cases of multiple ovulation or upon the developing embryo as a whole in the case of monozygotic twins and triplets. If, however, each supernumerary ovum is due to an independent local action and such local actions have a certain average frequency the coincidence of two such actions would give the observed numerical relation of triplets to twins.

If the explanation as stated applies to the relation between triplet and twin births it is to be expected that it will apply to quadruplets as well. In that case the expected number of quadruplets is one in n3. Unfortunately the numbers are too small for a reliable conclusion. In the largest available collection of data, the one mentioned above, there are 36 quadruplets in 13,360,557 births or one in (71.9)3 which is somewhat greater than the expected number, one in (89.1)3.

As in other statistical relations the biological significance in the present instance can not be proved directly from the mass of data. When one considers the vicissitudes of fertilization, the chances of death of individual embryos, the demonstrated influence of the spermatozoon in certain cases of twinning and numerous other biological factors, to say nothing of faulty registration statistics, it is hard to believe that the simple numerical relation of triplets to twins can be more than the result of the combination of numerous and as yet unanalyzed forces. A knowledge of the fact may, however, aid in the analysis.

UNIVERSITY OF ILLINOIS

CHARLES ZELENY

THE AMERICAN CHEMICAL SOCIETY SECTION OF SUGAR CHEMISTRY

C. A. Browne, chairman Fred. J. Bates, secretary (Concluded)

The sugar industry of Peru: CHAS. A. GAMBLE. Electric oven for rapid moisture tests: G. L. SPENCER. This oven (patented August 3, 1920) is a convenient arrangement for passing a rapid current of heated air through a sample. The air is drawn over a heating element, composed of a spiraled nichrome wire coiled around a suitable core, and thence through the sample contained in a capsule, fitted with a gauze or metal filter-cloth bottom. The temperature of the air is controlled by a rheostat. Any substance through which a current of hot air may be passed, without melting, may be dried in this oven. Raw sugar may be approximately dried in 3 minutes and to constant weight in 10 minutes; 100 gram samples of cane bagasse are dried in less than 60 minutes; cotton saturated with water is dried in 10 minutes.

Refining raw sugars without boneblack: C. E. COATES. Raw sugars from the tropics have been refined off and on in Louisiana for a number of years. This was profitable on occasions when the margin between raws and granulated was high. During the past two years this margin has been so high as to offer exceptional profits to the refiner. A number of Louisiana sugar houses purchased raw sugars during the last six months and refined them by several different methods: Phosphoric acid and lime; sulphur-dioxide and lime; filtercel alone; activated wood charcoals. At the beginning of the campaign, the yields were several per cent. below those obtained in the standard bone black refinery process, but as the season went on this difference was diminished until at the present time the yield is about one per cent. short of good refinery practise. The yields by the various methods are nearly the same. There is no reason why melting tropical raw sugars can not become a part of the routine practise in both cane and beet sugar houses. The quality of the sugars obtained is excellent and in the case of the activated charcoals the sugars were beyond any criticism of any sort.

Recent advances in defecation: W. D. HORNE. The Dorr Clarifier has been devised to remove the insoluble impurities from cane juice. Hot limed juice continuously enters the central well of the superimposed settling compartment equipped with slowly revolving scraper mechanisms. Clear juice flows from the periphery, and a thick mud is constantly withdrawn from the bottom. The Williamson Clarifier aerates a warm defecated raw sugar solution and then passes it through a long shallow heating tank provided with suitable baffles, causing all insoluble matters to rise in a scum, which is mechanically skimmed off, while clear liquor is continuously syphoned out below.

Comparative analysis of refined sugars: FREDERICK BATES and associates at the Bureau of Standards. A résumé was given of the results of an elaborate and exhaustive study of about 250 samples of refined sugars produced in the United States. One of the principal objects of this work was to determine the feasibility of preparing accurate specifications and definitions of the refined sugars. The work included the determination of the direct polarization, invert sugar, moisture and ash. The acidity and alkalinity were determined by developing a new method, using the hydrogen ion concentration with gratifying results. The average acidity and alkalinity was found to be

small and indications are that the departure from neutral is a powerful factor in determining the properties of the sugar. In addition, the sugars were screened to determine average size of grain. A preliminary report was given on the importance of improving and applying the so-called candy test to refined sugars.

A graphic method for estimating reducing sugars in presence of sucrose: C. A. BROWNE. The slight reducing action of sucrose upon Fehling's copper solution introduces a considerable error in determining reducing sugars in presence of large amounts of sucrose. The author proposes a general graphic method which consists of a chart containing the reduction curves for dextrose in amounts from 0 to 250 mgs. in presence of sucrose from 0 to 5gs. The correct amount of dextrose, corresponding to the amount found, is determined by finding the curve which passes through the intersection of the coordinates for grams sucrose present (as determined by Clerget) and mgs. dextrose found. The starting point of this curve on the base line indicates the correct amount of dextrose.

Commercial production of d-Mannite: W. B. NEWKIRK and C. F. SNYDER. At the request of the Army, the manufacture on a commercial scale of d-Mannite from manna was undertaken. The crude manna was dissolved in water in a melter heated by steam coils. The liquor was 17° Brix. It was heated to boiling and defecated. Three methods of defecation were employed. Method 1: .005 per cent. phosphoric acid added to hot liquor and let stand for thirty minutes, neutralized with lime, allowed to settle and the clear liquid decanted and filtered. The filtered liquor was boiled in open pan to 30° Brix (hot), placed in tank and crystallized. The crystals were separated in a large centrifugal. Method II.: The raw liquor was treated with 0.10 per cent. Kieselghur and filtered and the filtrate concentrated as above. Method III.: The raw liquor was treated with 0.5 per cent. commercial vegetable carbon and filtered and the filtrate concentrated as above. Method I. was the most satisfactory. The mother liquors from the first crystallization were concentrated to 40° Brix and allowed to crystallize; the mother liquors from the second crystallization were concentrated at 60° Brix and a third crop of crystals obtained. The mother liquor from the third crystallization was concentrated at 80° Brix and a fourth crop of crystals obtained. The final mother liquors were concentrated to 80° Brix and a fifth crop of crystals obtained. A tabulation is given of the

Preservation of bagasse in sugar cane mill control: GUILFORD L. SPENCER. Formaldehyde has been generally used in preserving sub-samples of cane bagasse in preparing a composite sample, representative of several hours grinding. This has been ascertained to be only moderately efficient under modern milling conditions. The late M. Henri Pellet, in Egypt, suggested and used ammonia in protecting the samples. The author found this usually efficient but occasionally there is loss of sugar. This suggested the addition of chloroform and preservation of the bagasse in an atmosphere of ammonia and chloroform. This mixture is apparently very efficient. Refinery press-cake holds its polarization during several weeks in the presence of this preservative. The solution for polarization must be acidulated with acetic acid before clarification with lead subacetate.

Glass vacuum pan for laboratory use: M. J. PROFFITT.

Changes in the polarizing constants of sugars during refining: A. F. BLAKE. The Clerget sucrose value for sugars, as pointed out by Browne at the Cleveland meeting, normally exceeds the polarization by about one third the percentage of invert. This is true of raw sugars as shown by numerous analyses, but in the products of a refinery, soft sugars and syrup, the value of the ratio (S-P)/I is very low. Analyses of sugars at all intermediary stages of refining are given, in order to determine where the change takes place. It is concluded that some change takes place during defecation and filtration of low test material and in the handling of the muds and scums due to action of lime on the invert sugar, but that by all means the principal cause of the reduction of the value of this ratio is bone-black filtration. The factor is strongly negative for the first material coming off the boneblack, but increases in following portions until in the last portion it is about equal or slightly exceeds material going on. The average value of the factor for all material going on is much higher than the average coming off. Since boneblack absorbs invert from first material and gives it up to later material it is supposed that by selective action it might absorb more

levulose than dextrose. This is proved by tests on invert sugar. Another cause is the molecular rearrangement of dextrose and levulose into glutose, etc. A high value of the ratio in refined products indicates inversion during refining. Losses of sucrose figured upon Clerget values exceed those figured on polarization, while losses of organic material are much less.

A report on the sugar industry of France since the war: T. H. MURPHY. The French sugar industry, born of the Napoleonic wars, almost perished in the World War, being 66 per cent. destroyed. Formerly, the 213 sugar factories supplied all French consumption and 78,739 tons per year for export. Now the 60 small factories remaining can supply only a small portion of the nation's requirements, and about 400,000 tons per year are imported. One hundred and forty-two factories were 85 per cent. destroyed, and all copper coils, bronze tubing, copper and brass screens, in fact everything made of copper, bronze or brass, and all electrical equipment, was stripped out of the war-wrecked factories and taken to Germany. The plants where sugar machinery and equipment was made, suffered the same fate. The damage to sugar factories was over $89,000,000. Reconstruction in France has made enormous strides, but on account of the highly specialized machinery and equipment required, the sugar industry, has, as yet, been able to accomplish very little. Photographs of destroyed sugar factories shown.

The composition and preparation of a sugar syrup of maximum solubility: R. F. JACKSON and C. L. GILLIS. One of the large branches of the sugar industry is the manufacture of syrups for direct consumption. If the syrup consists only of sucrose, the saturated solution may contain only 38.7 per cent. of the sugar. Such a solution is too thin for a desirable product and is susceptible to fermentation. If concentrated to a denser consistency, it becomes supersaturated and deposits sugar crystals. If, however, the sucrose is partially inverted, the density may be considerably increased, but if the inversion is carried too far, the relatively low solubility of dextrose limits the density to which the syrup may be concentrated. A study was made of the mutual solubilities of the three constituent sugars, namely, sucrose, dextrose and levulose in the presence of each other. The solubility of sucrose in varying proportions of invert sugar was determined to very high concentrations of the latter. Similarly the solubility of su