Apparent violations of the all-or-none law in relation to potassium in the protoplasm

The protoplasm of Nitella forms a thin layer surrounding a large central vacuole filled with sap. At the inner surface of the protoplasm is a non-aqueous layer called Y and at the outer surface is a similar layer called X. At each of these layers there is a potential due to the diffusion of KCl in contact with the layer. We thus have P = P_X + P_Y in which P is the total potential, P_X is the potential at X, and P_Y the potential at Y. We assume that when stimulation occurs P_Y disappears and P_X remains unaltered. The loss of part of the potential therefore involves no violation of the all-or-none law since the Y layer loses all of its potential and the X layer loses nothing. If the concentration of KCl in the external solution and in the sap is known the concentration in the protoplasm can be calculated at each stimulation by measuring the height of the spike.     

NATURE OF THE ACTION CURRENT IN NITELLA : VI. SIMPLE AND COMPLEX ACTION PATTERNS

The experiments indicate that the protoplasm of Nitella consists of an aqueous layer W with an outer non-aqueous surface layer X and an inner non-aqueous surface layer Y. The potential at Y is measured by the magnitude of the action curve and the potential at X by the distance from the top of the action curve to the zero line. These potentials appear to be due chiefly to diffusion potentials caused by the activity gradients of KCl across the non-aqueous layers X and Y. The relative mobilities of K⁺ and Cl^- in X and in Y can be computed and an estimate of the activity of KCl in W can be made. In the complete resting state the mobilities of K⁺ and Cl^- in X are not very different from those in Y. The action curve is due to changes in Y which suddenly becomes very permeable, allowing potassium to move from the sap across Y into W, and thus losing its potential. A gradual loss may be due to changes in ionic mobility in Y. When recovery is incomplete and Y has not yet regained its normal potential a stimulus may cause a loss of the potential at Y giving an action curve of small magnitude. The magnitude may vary in successive action curves giving what is called a complex pattern in contrast to the simple pattern observed when recovery is complete and all the action curves are alike. Complex patterns occur chiefly in cells treated with reagents. Untreated cells usually give simple patterns. A variety of complex action patterns is discussed. It is evident that the cells of Nitella show much more variation than such highly specialized cells as muscle and nerve which give stereotyped responses. In some cases it may be doubtful whether the all-or-none law holds.     

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.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS

The cells of Halicystis impaled on capillaries reach a steady P.D. of 60 to 80 millivolts across the protoplasm from sap to sea water. The outer surface of the protoplasm is positive in the electrometer to the inner surface. The P.D. is reduced by contact with sap and balanced NaCl-CaCl₂ mixtures; it is abolished completely in solutions of NaCl, CaCl₂, KCl, MgSO₄, and MgCl₂. There is prompt recovery of P.D. in sea water after these exposures.     

MECHANICAL RESTORATION OF IRRITABILITY AND OF THE POTASSIUM EFFECT

Treatment of Nitella with distilled water apparently removes from the cell something which is responsible for the normal irritability and the potassium effect, (i.e. the large P.D. between a spot in contact with 0.01 M KCl and one in contact with 0.01 M NaCl). Presumably this substance (called R) is partially removed from the protoplasm by the distilled water. When this has happened a pinch which forces sap out into the protoplasm can restore its normal behavior. The treatment with distilled water which removes the potassium effect from the outer protoplasmic surface does not seem to affect the inner protoplasmic surface in the same way since the latter retains the outwardly directed potential which is apparently due to the potassium in the sap. But the inner surface appears to be affected in such fashion as to prevent the increase in its permeability which is necessary for the production of an action current. The pinch restores its normal behavior, presumably by forcing R from the sap into the protoplasm.     

THE KINETICS OF PENETRATION : XVI. THE ACCUMULATION OF AMMONIA IN LIGHT AND DARKNESS

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH₄ X which is formed by the interaction of NH₃ or NH₄OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P'''''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO₂), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH₃ or NH₄OH in excess of the potassium lost as a base.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.     

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.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

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.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

STUDIES OF THE INNER AND OUTER PROTOPLASMIC SURFACES OF LARGE PLANT CELLS : II. MECHANICAL PROPERTIES OF THE VACUOLAR SURFACE

The vacuolar surface of Nitella is covered with a non-aqueous film too thin to be visible as a separate membrane. The motion of the protoplasm may subject this film to a good deal of mechanical disturbance. Apparently this does not rupture the film for no dye escapes into the protoplasm as the result of such disturbance when the vacuolar sap is deeply stained with neutral red or brilliant cresyl blue. When the deeply stained central vacuole breaks up into several smaller vacuoles, leaving the outer protoplasmic surface in its normal position, there is no evidence of the escape of dye into the protoplasm through the film surrounding the vacuole.     

The silkmoth cocoon as humidity trap and waterproof barrier

To better understand how silkmoth cocoons maintain the correct internal moisture levels for successful pupation, we examined cocoons from the long-domesticated mulberry silkmoth Bombyx mori as well as from two wild silkmoth species, Antheraea pernyi and Philosamia cynthia ricini. We determined fluid-independent values for the porosity, tortuosity and permeability of the inner and outer surfaces of cocoons. Permeabilities were low and, with the exception of A. pernyi cocoons, inner surfaces were less permeable than outer surfaces. B. mori cocoons exhibited the highest permeability overall, but only at the outer surface, while A. pernyi cocoons appeared to show different patterns from the other species tested. We discuss our findings in light of the ecophysiology of the various species and propose a ‘tortuous path’ model to help explain our results. The model describes how the structure of the inner and outer layers of the cocoon allows it to function as both a humidity trap and a waterproof barrier, providing optimum conditions for the successful development of the pupa.     

FORMATION AND STRUCTURE OF THE SPORE OF BACILLUS COAGULANS

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.     

Photosynthesis and biomass allocation of cotton as affected by deep-layer water and fertilizer application depth

Available water stored in deep soil layers could increase the photosynthetic capacity of cotton. It was hypothesized that the photosynthesis of cotton would be enhanced by changing the fertilizer application depth under different deep-layer water conditions. We examined two deep-layer water levels, i.e., well-watered (W₈₀) and not watered (W₀), combined with surface application (F₁₀) and deep application (F₃₀) of basal fertilizer. Compared to W₀, W₈₀ resulted in increased leaf area (LA), photosynthetic pigment contents, maximal PSII efficiency (F_v/F_m), effective quantum yield of PSII (Y_II) and PSI (Y_I), electron transport rate of PSII (ETR_II) and PSI (ETR_I). W₈₀ also increased the aboveground and root dry mass by 39 and 0.6%, respectively, and decreased the root/shoot ratio by 40–73%. Under the W₀ condition, higher values of F_v/F_m, Y_II, Y_I, ETR_II, and ETR_I were measured for F₁₀ compared to F₃₀ after 69 d from emergence. Under the W₈₀ condition, cotton plants with F₁₀ showed higher LA, F_v/F_m, Y_II, Y_I, ETR_II, and ETR_I, but there were no significant differences in the photosynthetic pigments compared to F₃₀. Our results suggest that sufficient water in deeper soil layers and the surface application of basal fertilizer could increase photosynthetic activity and efficiency, which promoted aboveground dry mass accumulation and partitioning towards reproductive organs.     

Variations in the Number of the Y Chromosomal Rrna Genes in DROSOPHILA HYDEI

The number of rRNA cistrons is measured by filter saturation hybridization in different stocks of D. hydei, where the wild-type X chromosome has one nucleolus organizer (NO) and the wild-type Y has two separated NO's. (see PDF) females having no X chromosomal NO show an rDNA content exceeding that of a Y chromosome. An even greater increase in the rRNA cistron number is measured in two translocation stocks where the (see PDF) is combined with one half of a Y and, therefore, each stock contains only one of the two Y chromosomal NO's. But when the same Y fragments are brought together with a wild-type X chromosome they lose about one-half of their rRNA cistrons within one generation. Males with two complementary Y fragments but having no X chromosomal NO show a considerably higher rDNA content than the (see PDF) females, although both are equal in respect of their NO number. Consideration is given to related phenomena in Drosophila melanogaster.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

Cytogenetic,cross-mating and molecular evidence of four cytological races of Anopheles crawfordi (Diptera: Culicidae) in Thailand and Cambodia

Twenty-nine isolines of Anopheles crawfordi were established from wild-caught females collected from cow-baited traps in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and four types of Y (Y₁, Y₂, Y₃, and Y₄) chromosomes were identified, according to differing amounts of extra heterochromatin. These sex chromosomes represent four metaphase karyotypes, i.e., Forms A (X₁, X₂, X₃, Y₁), B (X₁, X₂, X₃, Y₂), C (X₂, Y₃) and D (X₂, Y₄). Forms C and D are novel metaphase karyotypes confined to Thailand, whereas forms A and B appear to be common in both Thailand and Cambodia. Cross-mating experiments between the four karyotypic forms indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes. The results suggest that the forms are conspecific and A. crawfordi comprises four cytological races, which is further supported by very low intraspecific variation (mean genetic distance = 0.000–0.018) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA sequences (COI, COII).     

Estimating the capacity for improvement in risk prediction with a marker

Consider a set of baseline predictors X to predict a binaryoutcome D and let Y be a novel marker or predictor. This paperis concerned with evaluating the performance of the augmentedrisk model P(D = 1|Y,X) compared with the baseline model P(D= 1|X). The diagnostic likelihood ratio, DLR_X(y), quantifiesthe change in risk obtained with knowledge of Y = y for a subjectwith baseline risk factors X. The notion is commonly used inclinical medicine to quantify the increment in risk predictiondue to Y. It is contrasted here with the notion of covariate-adjustedeffect of Y in the augmented risk model. We also propose methodsfor making inference about DLR_X(y). Case–control studydesigns are accommodated. The methods provide a mechanism toinvestigate if the predictive information in Y varies with baselinecovariates. In addition, we show that when combined with a baselinerisk model and information about the population distributionof Y given X, covariate-specific predictiveness curves can beestimated. These curves are useful to an individual in decidingif ascertainment of Y is likely to be informative or not forhim. We illustrate with data from 2 studies: one is a studyof the performance of hearing screening tests for infants, andthe other concerns the value of serum creatinine in diagnosingrenal artery stenosis.