THE REACTIONS OF HALICYSTIS AND OF VALONIA TO INJECTIONS OF CERTAIN PROTEINS

It is shown (1) that Valonia and Halicystis cells exhibit varying degrees of tolerance to injections of animal peptone, animal proteose, crystallized egg albumen, and diphtheria toxin; (2) that Valonia cells display decreased tolerance to egg albumen in increasing dosages, although Halicystis is completely tolerant of the highest dosage used; (3) that the mortality curves of Valonia injected with egg albumen and of both Valonia and Halicystis injected with diphtheria toxin show the delayed effect characteristic of laboratory mammals when treated similarly; (4) that Valonia cells injected twice with egg albumen exhibit no change in susceptibility to its effects; and (5) that neither species of algae gives evidence of having formed antibodies against the antigens used.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : IV. INFLUENCE OF GUAIACOL ON THE EFFECTS OF SODIUM AND POTASSIUM IN NITELLA

In Nitella, as in Halicystis, guaiacol increases the mobility of Na⁺ in the outer protoplasmic surface but leaves the mobility of K⁺ unaffected. This differs from the situation in Valonia where the mobility of Na⁺ is increased and that of K⁺ is decreased. The partition coefficient of Na⁺ in the outer protoplasmic surface is increased and that of K⁺ left unchanged. Recovery after the action current is delayed in the presence of guaiacol and the action curves are "square topped."     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : III. SOME EFFECTS OF GUAIACOL ON HALICYSTIS

Lowering the pH of sea water from 8.2 to 6.4 lowers the positive P.D. of Halicystis reversibly (this does not happen with Valonia). Exposure to sea water at pH 6.4 does not affect the apparent mobility of Na⁺ or of K⁺ (this agrees with Valonia). Guaiacol makes the P.D. of Halicystis less positive (in Valonia it has the opposite effect). Exposure to guaiacol does not reverse the effect of KCl in Halicystis which in this respect differs from Valonia. The P.D. can be changed from 66 mv. positive to 23 mv. negative by the combined action of KCl and guaiacol. Exposure to guaiacol affects Halicystis and Valonia similarly in respect to their behavior with dilute sea water. Normally the dilute sea water makes the P.D. more negative but after sufficient exposure to guaiacol dilute sea water either produces no change in P.D. or makes it more positive. In the latter case we may assume that the apparent mobility of Na⁺ has become greater than that of Cl^- as the result of the action of guaiacol. (Normally the apparent mobility of Cl^- is greater than that of Na⁺.) In Halicystis, as in Valonia and in Nitella, an organic substance can greatly change the apparent mobilities of certain inorganic ions (K⁺ or Na⁺).     

EFFECTS OF POTASSIUM ON THE POTENTIAL OF HALICYSTIS

Sea water in which sodium has been replaced by potassium produces about the same degree of negativity in Halicystis and in Valonia. With increasing dilution of this sea water up to 1 ÷ 16 the degree of negativity steadily falls off in Halicystis. This differs from the situation in Valonia where Damon finds that with increasing dilution the negativity passes through a minimum after which increasing dilution produces increasing negativity. But conditions in the two organisms differ so greatly that a comparison is of rather doubtful significance.     

THE KINETICS OF PENETRATION : XIX. ENTRANCE OF ELECTROLYTES AND OF WATER INTO IMPALED HALICYSTIS

When cells of Halicystis are impaled on a capillary so that space is provided into which the sap can migrate, the rate of entrance of water and of electrolyte is increased about 10-fold. In impaled Valonia cells the rate is increased about 15-fold. After a relatively rapid non-linear rate of increase of sap volume immediately after impalement (which may possibly represent the partial dissipation of the difference of the osmotic energy between intact and impaled cells) the volume increases at a linear rate, apparently indefinitely. Since the halide concentration of the sap at the end of the experiment is (within the limits of natural variation) the same as in the intact cell, we conclude that electrolyte also enters the sap about 10 times as fast as in the intact cell. As in the case of Valonia we conclude that there is a mechanism whereby in the intact cell the osmotic concentration of the sap is prevented from greatly exceeding that of the sea water. This may be associated with the state of hydration of the non-aqueous protoplasmic surfaces.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : V. POTENTIALS IN HALICYSTIS

Interest in the study of Halicystis and of Valonia has been stimulated by discoveries of marked contrasts and striking similarities existing side by side. This is illustrated by new experiments with the alkali metals and alkaline earths. In Halicystis the apparent mobilities of K⁺, Rb⁺, Cs⁺, and Li⁺ (calculated by means of Henderson''s equation from changes in P.D. produced by replacing sea water by a mixture of equal parts of sea water and 0.6 M of various chlorides) are as follows, u _K, = 16, u _Rb = 16, u _Cs = 4.4, and u _Li = 0.2; u _Na is taken as 0.2. These values resemble those in Valonia except that in the latter u _Cs is about 0.2. No calculation is made for u _NHNH4, because in these experiments even at low pH so much NH₃ is present that the sign of the P.D. may reverse. This does not happen with Valonia. According to Blinks, NH₄ ⁺ at pH 5 in low concentrations acts like K⁺. The calculation gives u _Mg = 1.9 which is similar to the value found for Valonia. No calculation can be made for CaCl₂ since it produces protoplasmic alterations and in consequence Henderson''s equation does not apply. This differs from Valonia. Evidently these plants agree closely in some aspects of electrical behavior but differ widely in others.     

The penetration of radioactive sodium intoValonia andHalicystis

Conclusions In comparing the uptake of Na^* byValonia and that byHalicystis, it was found that the greater proportion was taken up by the protoplasm in the former case, and by the sap in the latter case.In former experiments only the sap was tested for penetration and found to contain negligible concentrations of Na inValonia and comparatively larger concentrations inHalicystis. It is therefore of interest to show that Na^* does penetrateValonia but is taken up by the protoplasm considerable concentrations; but that it does not pass into the sap under normal conditions, thereby showing that the semi-permeable membrane between the sap and the protoplasm is the region of non-penetration. InHalicystis this is not the case since Na^* was found in both the sap and the protoplasm of this cell.Further work on the difference between these two membranes would be of interest in elucidating the movement of K and Na ions through membranes.This paper was assembled by Matilda M. Brooks.     

Characterization of production processes for tetanus and diphtheria anatoxins

Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.     

Studies of microbial toxins in Xenopus laevis oocytes

Xenopus laevis oocytes have been incubated or microinjected with cholera and diphtheria holotoxins or their respective isolated fragments A and B. Effects on progesterone-induced maturation, protein synthesis and cAMP levels were observed. Xenopus laevis oocytes were highly susceptible to cholera toxin upon incubation as evidenced by the increase of cAMP (two-fold increase in cAMP with 0.1 nM cholera toxin) and the blockade of progesterone-induced maturation. When isolated cholera toxin fragments A or B were incubated with oocytes, no activity could be detected. However, microinjection of cholera toxin fragment A into oocyte was able to mimic the effects of incubated holotoxin. Microinjection of cholera toxin B fragment was only effective at very high concentrations, probably due to trace contaminations by the A fragment. On the other hand, Xenopus laevis oocytes were very resistant to diphtheria toxin action upon incubation, a result attributable to lack of specific membrane receptors since, after microinjection of diphtheria toxin A fragment into oocytes, inhibition of protein synthesis was demonstrated. By simultaneous microinjection of highly radioactive adenine-labelled NAD and diphtheria toxin fragment A into oocytes, radioactive ADP ribosylation of the elongation factor 2 (EF₂) was observed. It is proposed that Xenopus laevis oocytes provide a new experimental approach for studying the mechanisms of action of microbial toxins.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA. II

In measurements of P.D. across the protoplasm in single cells, the presence of parallel circuits along the cell wall may cause serious difficulty. This is particularly the case with marine algae, such as Valonia, where the cell wall is imbibed with a highly conducting solution (sea water), and hence has low electrical resistance. In potential measurements on such material, it is undesirable to use methods in which the surface of the cell is brought in contact with more than one solution at a time. The effect of a second solution wetting a part of the cell surface is discussed, and demonstrated by experiment. From further measurements with improved technique, we find that the value previously reported for the P.D. of the chain Valonia sap | Valonia protoplasm | Valonia sap is too low, and also that the P.D. undergoes characteristic changes during experiments lasting several hours. The maximum P.D. observed is usually between 25 and 35 mv., but occasionally higher values (up to 82 mv.) are found. The appearance of the cells several days after the experiment, and the P.D.''s which they give with sea water, indicate that no permanent injury has been received as a result of exposure to artificial sap. If such cells are used in a second measurement with artificial sap, however, the form of the P.D.-time curve indicates that the cells have undergone an alteration which persists for a long time. On the basis of the theory of protoplasmic layers, an attempt has been made to explain the observed changes in P.D. with time, assuming that these changes are due to penetration of KCl into the main body of the protoplasm.     

Optimization of diphtheria toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> using a casein-based medium in a fermenter

The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of K _La in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal K _La value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production.     

Chronic mucocutaneous candidiasis: T cell deficiency associated with B cell dysfunction in man

Routine immunologic screening of four patients with chronic mucocutaneous candidiasis (CMC) revealed that they manifested positive Schick tests in vivo despite adequate diphtheria toxoid immunization and the presence of circulating hemagglutinating antibody to diphtheria. Plasma from these individuals was found to neutralize Schick toxin in rabbits. Unlike normal individuals who preferentially make IgG neutralizing antibody to diphtheria toxin when immunized, these patients with CMC have neutralizing activity in plasma fractions containing IgM. IgM is predominantly an intravascular protein which would account for the failure of our patients to neutralize Schick toxin in vivo. These findings suggest that T cell deficiency as it occurs in CMC may lead to B cell dysfunction in man.     

PROTOPLASMIC ASYMMETRY IN NITELLA AS SHOWN BY BIOELECTRIC MEASUREMENTS

Using multinucleate cells of Nitella 2 or 3 inches in length it is possible to kill one end with chloroform without producing at the other any immediate alteration which can be detected by our present methods. When a spot in external contact with sap is killed its potential difference falls approximately to zero and it is therefore possible to measure the potential difference across the protoplasm at any desired point merely by leading off from that point to the one where the protoplasm has been killed. The results indicate that the inner and outer protoplasmic surfaces differ, for when both surfaces are in contact with the same solution (cell sap) there is an electromotive force of about 15.9 millivolts, the inner surface being positive to the outer (i.e. the positive current tends to flow from the inner surface through the electrometer to the outer surface). The situation resembles that in Valonia where the corresponding value (with Valonia sap applied to the outside) has been reported as about 14.5 millivolt (the inner surface being positive to the outer). It would seem appropriate to designate this as radial polarity.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : I. EFFECTS OF GUAIACOL ON VALONIA

In normal cells of Valonia the order of the apparent mobilities of the ions in the non-aqueous protoplasmic surface is K > Cl > Na. After treatment with 0.01 M guaiacol (which does not injure the cell) the order becomes Na > Cl > K. As it does not seem probable that such a reversal could occur with simple ions we may assume provisionally that in the protoplasmic surface we have to do with charged complexes of the type (KX _I)⁺, (KX _II)⁺, where X _I and X _II are elements or radicals, or with chemical compounds formed in the protoplasm. When 0.01 M guaiacol is added to sea water or to 0.6 M NaCl (both at pH 6.4, where the concentration of the guaiacol ion is negligible) the P.D. of the cell changes (after a short latent period) from about 10 mv. negative to about 28 mv. positive and then slowly returns approximately to its original value (Fig. 1, p. 14). This appears to depend chiefly on changes in the apparent mobilities of organic ions in the protoplasm. The protoplasmic surface is capable of so much change that it does not seem probable that it is a monomolecular layer. It does not behave like a collodion nor a protein film since the apparent mobility of Na⁺ can increase while that of K⁺ is decreasing under the influence of guaiacol.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA. III

Evidence that the inner and outer protoplasmic surfaces in Valonia are unlike is found in the high P.D. across the protoplasm when the external solution has the same composition as the vacuolar sap. Earlier experiments with artificial sap have been repeated, using natural as well as artificial sap. Good agreement between the data with the natural and the artificial solution was found both in the magnitude of the P.D.''s observed and in the shape of the P.D.-time curves. The P.D.''s, however, were considerably higher than the values formerly reported as usual, while the cells proved much less liable to alteration produced by exposure to sap. It is suggested that the cells used in the recent experiments were in a more vigorous condition, perhaps as a result of exposure to stronger illumination. The interpretation of the shape of the P.D.-time curves, proposed in an earlier report, and based on the theory of protoplasmic layers, is further discussed. It is assumed that the fluctuations in P.D. are due to an increase in the concentration of K in the main body of the protoplasm.     

Expression of DNA coding for diphtheria toxin chain a is toxic to plant cells

DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. The introduction of this chimeric molecule into tobacco cells by electroporation lowered the level of the transient expression of the coelectroporated chloramphenicol acetyltransferase reporter gene indicating that expression of diphtheria toxin chain A in plant cells is toxic. We have developed a binary vector pGA987 which can be used for probing a variety of plant promoters.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : VI. SOME EFFECTS OF GUAIACOL ON NITELLA

Values have been calculated for apparent mobilities and partition coefficients in the outer non-aqueous layer of the protoplasm of Nitella. Among the alkali metals (with the exception of cesium) the order of mobilities resembles that in water and the partition coefficients (except for cesium) follow the rule of Shedlovsky and Uhlig, according to which the partition coefficient increases with the ionic radius. Taking the mobility of the chloride ion as unity, we obtain the following: lithium 2.04, sodium 2.33, potassium 8.76, rubidium 8.76, cesium 1.72, ammonium 4.05, ½ magnesium 20.7, and ½ calcium 7.52. After exposure to guaiacol these values become: lithium 5.83, sodium 7.30, potassium 8.76, rubidium 8,76, cesium 3.38, ammonium 4.91, ½ magnesium 20.7, and ½ calcium 14.46. The partition coefficients of the chlorides are as follows, when that of potassium chloride is taken as unity: lithium 0.0133, sodium 0.0263, rubidium 1.0, cesium 0.0152, ammonium 0.0182, magnesium 0.0017, and calcium 0.02. These are raised by guaiacol to the following: lithium 0.149, sodium 0.426, rubidium 1.0, cesium 0.82, ammonium 0.935, magnesium 0.0263, and calcium 0.323 (that of potassium is not changed). The effect of guaiacol on the mobilities of the sodium and potassium ions resembles that seen in Halicystis but differs from that found in Valonia where guaiacol increases the mobility of the sodium ion but decreases that of the potassium ion.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

THE PENETRATION OF STRONG ELECTROLYTES

The entrance of strong electrolytes into Valonia is very slow unless the cells are injured. This, together with the very high electrical resistance of the protoplasm, suggests that they may penetrate largely as undissociated molecules formed at the surface of the protoplasm by the collision of ions. Under favorable circumstances KCl may be absorbed to the extent of 3 x 10^–8 mols per hour per sq. cm. of surface together with about 0.17 as much NaCl. Other substances which seem to penetrate to some extent are Li, Rb, Br, BrO₃, I, IO₃, and selenite. Little or no penetration is shown by SCN, ferricyanide, ferrocyanide, formate, salicylate, tungstate, seleniate, NO₂, SO₃, Sb, glycerophosphate, and many heavy metals and the alkaline earths. In sea water whose specific gravity had been increased by CsCl cells of Valonia floated for over a year and there was little or no penetration of Cs except as the result of injury. The penetration of NH₄Cl decreases the specific gravity of the sap and causes the cells to float: under these circumstances they live indefinitely. It is probable that NH₃ or NH₄OH penetrates and is subsequently changed to NH₄Cl. It would seem that if the sea contained a little more ammonia this would be a floating organism.