Principles of General Physiology

The present chapter was commenced by pointing out the necessity for some arrangement by which, in such organisms as the amoeba, sugar and other soluble food-stuffs are prevented from diffusing out and being lost to the protoplasm. From what we have learnt in the preceding section, it is plain that what is needed is a membrane with properties similar to those of Traube's copper ferrocyanide, but more perfectly semi-permeable. As we shall learn in more detail later, there is a remarkable similarity between the properties of the cell membrane and those of the artificial one, although it is not to be supposed that they have anything in common as regards their chemical nature. Both are permeable to ammonium chloride, impermeable to ammonium sulphate. It is usually stated that the cell membrane is impermeable to potassium chloride, while the copper ferrocyanide membrane, as we have seen, is freely permeable to it. But this statement needs qualification. Overton (1904, pp. 188-209) has shown that the muscle cell is not completely impermeable to potassium chloride and that, in fact, potassium salts fall into two groups, the first, typified by the sulphate and phosphate, to which complete semi-permeability exists, and the second, typified by the chloride. It will be noted that this behaviour is similar to that of the copper ferrocyanide membrane, which, according to Walden (1892), is permeable to chlorides, bromides, iodides, and thiocyanates, impermeable to sulphates, phosphates, and oxalates. The muscle cell, however, is only very slowly permeable by potassium chloride. Meigs (1913), moreover, finds that a celloidin membrane, impregnated with calcium phosphate, has most of the properties of the cell membrane, as regards permeability. It is impermeable to the chlorides of sodium, potassium, and calcium, to cane-sugar and alanine, somewhat permeable to glycerol and urea, freely permeable to alcohol. Although it seems scarcely likely that the cell membrane is actually composed of calcium phosphate, it is important that an artificial membrane of nearly perfect semi-permeability can be prepared.

Philippson (1913), again, shows that, if collodion membranes are impregnated with an ethereal extract of muscle, they become almost impermeable to inorganic acids, while retaining their permeability to organic acids, increasing in the series, formic-acetic-lactic-butyric. This result is of interest as a further step in the artificial production of membranes with properties similar to those of the cell membrane. That a membrane of some kind is actually formed on the surface of contact between protoplasm and water is shown by the observations of Kiihne and of Pfeffer referred to below (page 128). If any substances are present in the cell which lower surface energy, we know that they will be concentrated at the surface, and from Ramsden's experiments (page 55) we are prepared to find that a coherent membrane will probably be formed. It is not necessary, then, that an actual visible skin should be present, although in certain cases it appears to exist. Moreover, the kind of membrane contemplated in the statement just made forms, or may be regarded as, an integral part of the living protoplasm itself, and as long as this is living, will probably share its power of change and adaptation in response to changes in the environment. This point of view will require further treatment later.

When we come to the constituent cells of higher organisms, which are dependent for their food supply on substances in the blood or other liquid bathing them, we are at once met with a difficulty, if we assume the existence of such a semi-permeable membrane. If it prevents food-stuffs from being washed out of the cell, it must also prevent them from getting in. This difficulty has caused certain investigators to deny altogether the existence of a membrane impermeable to electrolytes and other crystalloids. Martin Fischer and Gertrude Moore (1907, p. 342), for example, appear to hold that imbibition by colloids is capable of explaining the phenomena for which a semi-permeable membrane was postulated.

In order to understand the nature of the evidence on this question, it is necessary to forestall somewhat a part of the subject matter properly belonging to the chapter on osmotic pressure. Suppose that we have a vesicle, say of copper ferrocyanide, containing a solution of sugar, and that we immerse it in water. Since the membrane is impermeable to sugar, but permeable to water, the sugar molecules inside exert a pull on water molecules, which enter and distend the

vesicle, by the process known as osmosis. This must for the present be taken as an experimental fact. If the water outside be replaced by a solution of sugar, but of a lower concentration than that within the membrane, water will enter until the concentration is equal on both sides ; if the solution outside is stronger than that inside, water will escape, until again the concentration is the same on both sides. It is not a necessity, moreover, that the two solutions, inside and outside, be of the same substance, so long as the membrane is impermeable to it. The amount of distension or collapse is clearly in exact proportion to the molecular concentration of the solutions, since on this depends the degree of dilution or concentration necessary to bring the inner and outer solutions into osmotic equilibrium. Now, careful investigations of the behaviour of the cells of the kidney by Siebeck (1912) and of the muscle cells by Beutner (1913, 1) have shown that living cells react in the same way as the semi-permeable membrane described above. The changes in volume are simply proportional to the molar concentration of the solutions used.

All the various members of the " Hofmeister series," in equal concentration, have the same effect. The process of imbibition, as we have seen (page 100 above), follows a different law. The series of electrolytes just referred to, in equal concentration, have different effects on imbibition according to their action on the properties of water, so causing it to be distributed between the two phases of the colloidal system in a different proportion. Moreover, sugar behaves, as regards its effect on the volume of cells, just as a salt of the same osmotic pressure, provided that the salt is one to which the membrane is impermeable, whereas, according to certain investigations, it is devoid of action on imbibition processes. Martin Fischer and G. Moore (1907, p. 339) find that non-electrolytes in general have no effect on the swelling of fibrin.

Further facts are, I think, unnecessary to show that the imbibition theory is insufficient to account for more than a small part of the behaviour of cells towards solutions of varying concentration. At the same time, there is no doubt that the power of changing the water content of cell constituents must play an important part in cell mechanics. We may now pass on to consider the nature and properties of the cell membrane. It will clear the way somewhat if I state the general conclusion which is forced upon us by consideration of the whole of the evidence on this disputed question, although, at first sight, it may seem rather a lame one. It is, in fact, that the cell membrane is sometimes permeable to crystalloids, sometimes not. This will seem more satisfactory when we find that the apparently capricious behaviour is in relation to functional changes in the cell, or dependent on the action of definite substances. As regards colloids, the membrane itself is probably always impermeable ; although in special cases, as the cells of secreting glands, there appear to be arrangements by which colloids can get in or out, probably by rupture of the membrane.

Impermeability to Crystalloids. — If a slice of living red beetroot be allowed to soak in tap water, it will be found that neither the red pigment nor the cane-sugar escapes from the cells. This fact can only be explained on two hypotheses : either the cell membrane is impermeable to these substances, or they are combined in an irreversible manner with the insoluble matter of the cells. Now, Moore and Roaf (1908, p. 80) appear to regard the existence of some kind of chemical combination between the proteins of cell protoplasm and electrolytes as sufficient to account for the difference of composition between cell and surrounding liquid, without the necessity of assuming the existence of a semi-permeable membrane. But, if this compound is reversible, as an adsorption process would be, there can be merely a quantitative difference between the cell contents and the outer solution, because an adsorption process is only in equilibrium with a finite concentration of adsorbed substance in the solution with which the surface is in contact. This is contradictory to experience in the case of the beetroot, and we shall find other instances as we proceed. If the hypothetical compound is a more strictly chemical one, it must be neither hydrolytically nor electrolytically dissociated, and, in fact, completely insoluble and inert. It is difficult to see of what value such a substance can be in the dynamics of the cell. Moreover, direct measurements by Hober (1912, 2) of the electrical conductivity of the interior of cells show that a part, at least, of the inorganic constituents &refree.

We are compelled, therefore, to assume the existence of a membrane of some kind, and the question to be answered is : Must the membrane be of necessity impermeable to electrolytes and other crystalloids, or is it sufficient if it is impermeable to colloids ? It is plain that if the latter alternative is found to be satisfactory, less difficulty will be found in imagining an adequate structure. It will be remembered that no artificial membrane is known as yet semi-permeable as regards potassium chloride, for example, to which the cell is usually semipermeable, but important steps have been taken already in this direction as mentioned above (page 115).

The chief evidence may be grouped conveniently under three heads : (1) The phenomena of changes in volume and internal pressure under the action of solutions of various concentrations. (2) The difference between the cell and the surrounding medium as regards presence and concentration of crystalloids. (3) The resistance of living cells to the passage of electrical currents through them. 1. When cells or blood corpuscles are placed in solutions of crystalloids of various concentration, it is found that in the case of most of these, provided that they do not injure the cell, there is a particular concentration in which no change of volume of the cell occurs. With solutions of a greater strength than this, a shrinking takes place, and with weaker solutions, a swelling. On the theory that these results are of osmotic origin, the solution which causes no change is called " isotoriie,*' and the others "hyper- and hypo-tonic" respectively. But the matter is not quite so simple as it might appear at first. The word "isotonic" implies that the solution which causes no change in the volume of the cells has the same osmotic pressure as the normal contents of the cell. How far this is true depends on the permeability of the membrane, as the following considerations will show. Suppose that we have a 5 per cent, solution of sugar enclosed in a bag of an elastic membrane, which is permeable to water, but impermeable to sugar, and that this is immersed in water. Water will enter the bag, which will be distended and probably ruptured, unless supported by an outer envelope, such as the cellulose wall of plant cells. The pressure developed when the cell is not allowed to increase in

volume is the full osmotic pressure of the sugar solution. The tense condition of the cell hereby produced is known as "turgor," and is the normal state of the plant cell, enabling the stems of the higher plants to remain rigid and erect, as long as the cell membranes retain their semi-permeable properties. That very considerable pressures do exist within plant cells is obvious from consideration of the growing cambium layer between the wood and the bark of a tree. Growth takes place at this situation, so that the wood is continually being increased in diameter ; it is clear, therefore, that the bark must have an enormous stretching force being continually applied to it, and that the growing cells must be exposed to great pressure, which would crush and kill them unless opposed by an equally great pressure within them. The stretched state of the bark can be seen by removing a ring of it, after cutting it through at one place. If it be then replaced in position, it will be found that the ends cannot be made to meet, represents this fact. From the tension required to stretch the bark to its original length, the pressure exerted on the cambium cells can be calculated I is common to find in plant cells pressures as high as 15 atmospheres. Now, pressures of this order can only be maintained either by osmotic forc.es or by imbibition. The construction of a plant cell, with its inner cavity of surrounded by a protoplasmic membrane, suggests at once an osmotic machine,

CELLS. — Cross section, slightly enlarged, of an internode of a Holly branch, from which the bark has first been removed and then replaced around the woody core. A very great tension is required to make the ends meet again at r. and we have already seen that imbibition is incapable of explaining the phenomena met with. From the molecular concentration of the cell sap, as determined by the depression of the freezing point, in the way explained in the next chapter, or in other ways, the maximum possible pressure that could be developed if the membrane were completely semi-permeable can be known. Although it is naturally a matter of difficulty to obtain the juice of one kind of coll alone, it appears from results obtained that, on the whole, the concentration of the cell sap is not greater than is necessary to give the turgor pressure known to exist.

A large number of measurements of depressions of freezing point will be found collected in the article by Bottazzi (1908); the usual figures correspond to pressures of about 11-15 atmospheres and would be given by a solution of potassium nitrate of nearly half molar strength (5 '05 per cent.). The shrinking of a cell placed in hypertonic solutions shows itself in plant cells by the protoplasmic layer retreating from the rigid cell wall, leaving a gap. between the two. This phenomenon is known as " plasmolysis," which was worked out mainly by de Vries (1884), and has played a large part in the investigation of the permeability of cells.

To interpret the facts observed when cells are exposed to solutions differing in osmotic pressure from that of the cell contents, let us return to the schema of the cell, viz., a solution of some substance contained within a membrane forming a vesicle, which can be immersed in water or solutions of various osmotic pressures. Suppose, first, that the membrane is impermeable to the solute, and that the vesicle is immersed in a slightly hypotonic solution of the same substance. The vesicle will at first absorb water, becoming distended, until its contents are diluted to such a degree that their concentration is equal to that of the outer solution. Nothing further will happen, but the cell remains permanently distended.

Next, let us imagine that the membrane is easily permeable both to water and to the solute, and that it is elastic as before. It is clear that, in this case also, the cell will be distended to begin with, because the osmotic pressure is greater inside than outside, while the solute cannot escape instantaneously. But subsequently, and contrary to the previous case, the original volume will be regained. As the solute gradually escapes, the internal osmotic pressure becomes equal to the external by free diffusion, and there can be no permanent force to keep the membrane stretched. In the previous case, the cell could return to its original volume only by escape of water ; but, since the solute could not escape, the original concentration would by this means be arrived at and equilibrium would no longer exist. Now, there may be numerous degrees of permeability between the two cases given, such that the solute may be able to escape at different rates. The result is that a longer or shorter time would elapse before the cell returned to its original size. In both cases, however, if no change of volume occurs at all, the conclusion may be drawn that tinouter solution is isotonic with the contents. If the change of volume is only temporary, while the membrane is elastic, it is to be concluded that this membrane is more or less permeable to the solute.

Another case to be considered is one that is met with in certain experiments on living cells or blood corpuscles, viz., when the membrane is permeable for the solute of the outer liquid, but impermeable for those of the cell contents. Suppose that the two solutions are isotonic. No immediate change will take place. But, presently, the cell will begin to swell. Why ? Because the solute of the outer solution passes into the cell, so that the osmotic pressure therein is now the original one plus that of the substance which has diffused in; while the outer solution remains the same as before, always assuming, as in all the cases discussed, that the volume of this solution is large compared with that of the cell. Ultimately, the state of affairs will be the same as if the outer liquid had been water only, since the concentration of the diffusible solute is equal on both sides of the membrane of the cell, while the latter retains the whole of the indiffusihle substance with its osmotic pressure.

It appears that, unless we know that the cell membrane is elastic, some uncertainty may arise aa to the conclusions to be drawn from the effect of a solution which is not isotonic with the cell contents. Suppose this solution to be hypotonic. The cell will at first increase in volume, as we have seen, whether the membrane is permeable to the solute or not. If it is impermeable to the solute, this increase in volume is permanent. But, if the increase in volume is not permanent, the cell must be more or less permeable. On the other hand, it seems possible, if the membrane is inelastic, that a permanent increase in volume might result from a hypotonic solution, even if the membrane is permeable to the solnte. The first effect having been to dilute the contents until their osmotic pressure is equal to that outside, while the membrane has allowed itself to be stretched without any elastic reaction, there does not seem to be any force capable of returning the cell to its original volume. This being so, caution is necessary in drawing conclusions, unless it is definitely known that the membrane is elastic.

Calculations made by Roaf (1912, i. p. 145) make it probable that equilibrium between diffusible substances inside and outside the cell takes place with great rapidity, so that it is possible that a process requiring seven days for equilibrium in an osmometer with parchment paper might be complete in O001 minute in the case of a cell, owing to the very large surface in proportion to volume in this latter case. It is justifiable to assume, then, that osmotic equilibrium of substances to which the membrane is permeable takes place practically almost instantaneously. But, at the same time, in the case of partial permeability, that is, if we regard the sieve as having only one hole in a thousand large enough to permit the passage of the molecules of a particular solute, the rate of diffusion of this solute through the membrane can be only about O'OOl of that of another solute, which can pass through all the pores.

Some experiments, made by Overton (1902) on the sartorius muscle of the frog, serve to show the impermeability of cells to crystalloids. When placed in ,0'7 per cent, sodium chloride, there was no change in weight, even in several hours ; hence this solution is isotonic with the muscle (Overton, p. 1 29). Suppose we add another substance to such a solution, if the muscle cells are impermeable to it they must shrink in order to increase -their osmotic pressure by loss of water. Overton adds methyl alcohol to the extent of 5 per cent. No effect is produced; hence the cells are permeable to methyl alcohol (p. 167), for this concentration of methyl alcohol raises the osmotic pressure of the salt solution very considerably. If the substance added is slowly permeable, a mixture of effects results. A muscle placed in a solution containing O35 per cent, sodium chloride, and 3 per cent, ethylene glycol, i.e., a solution whose osmotic pressure is equal to that of a 2 per cent, sodium chloride and therefore considerably hypertonic, loses weight at first, as if impermeable to glycol, but afterwards gains weight. The explanation is that the glycol can penetrate slowly, so that, after a time, its concentration within and without the cell becomes equal and the effect of 0'35 per cent, sodium chloride, which is hypotonic, remains alone (p. 195). As to the third possible case, glucose when added produces the same effect as sodium chloride of the same osmotic pressure, viz., permanent shrinking ; hence the membrane is impermeable to it (p.- 224).

There remains the possibility to be considered, whether the apparent impermeability to salts may not be sufficiently accounted for by the existence of a membrane semi-permeable as regards colloids only, but permeable to electrolytes, as appears to be the view taken by Roaf (1912, i. p. 145). Ostwald (1890) has pointed out that it is sufficient for a membrane to be impermeable to one ion only of an electrolytically dissociated salt in order that neither ion shall pass through. Suppose, therefore, that we have a salt of a protein present, which may be one with an acid to which the membrane is permeable, or a base of similar permeability. If this salt is not hydrolytically dissociated, the fact that the colloidal ion does not pass out will prevent the opposite diffusible ion from doing so. But in such a case the colloidal salt must be present without any colloidal salt of the other kind ; that is, we cannot have two colloidal salts, in one of which the anion is diffusible and in the other the cation.

For example, if there were a hydrochloride of a protein, and the sodium salt of a protein together, the positive and negative inorganic ions would escape together, or sodium chloride would diffuse out, without let or hindrance from electrostatic attraction on the part ( colloidal ion. The hypothesis of a membrane impermeable only to colloids will not, therefore explain the semi-permeability of the cell to neutral salts. We have seen above that there is no satisfactory evidence of combination between proteins and such salts, and, moreover, the hypothesis in question leaves the impermeability to glucose unaccounted for. Glucose does not form a compound with proteins of the kind required, and according to Asher (1912) exists free in the blood.

A further difficulty lies in the high osmotic pressure in certain cells ; to obtain a of 11 atmospheres, a half molar solution is necessary, and when we remember that tinmolecular weight of proteins is about 2,000, we see the impossibility of such a solution. The total solid content of cells is only about 20 per cent., ana of young, growing, cambium still less. Substances of small molecular weight only can give the observed osmotic pressure. The hsematocrite (Hedin, 1891), as applied to problems in permeability (Hober, 1910), is a practical use of the facts described in the preceding section.

2. We pass on to discuss some facts relating to the distribution of crystalloids between the cell and the surrounding medium, which necessitate the presence of a membrane impermeable to crystalloids. These facts are of interest in other ways. The red blood corpuscles of the rabbit contain much more potassium than the plasma which bathes them, and no sodium at all, according to the analyses of Abderhalden (1898, p. 100). Thus :— Such relations are impossible to account for except on the assumption of a membrane impermeable to sodium and potassium, unless these substances are combined with the colloids in an irreversible, non-dissociable, manner. It is easy to show, moreover, that the salts of blood serum readily pass through a membrane of parchment paper, which is impermeable to colloids, since they are frequently removed in this way. If the membrane of the rabbit's blood corpuscles were impermeable only as regards colloids, sodium salts from the serum must inevitably pass through.

It is true that, under certain conditions, as was found by Donnan (1911) and by myself (1911, ii. p. 249), independently, there may be different concentrations of a freely-diffusible salt in equilibrium within and without a membrane of parchment paper. This fact is brought by Roaf (1912, i. p. 145) in support of the opinion that a membrane impermeable to electrolytes is unnecessary, so that it must be considered briefly. Take the case of the sodium salt of a protein or of Congo-red, in solution inside a membrane of parchment paper. As long as water only is present on the other side of the membrane, the sodium ions cannot escape further than the position in which their osmotic pressure is balanced by electrostatic attraction to the opposite, colloidal, ion inside. A Helmholtz double layer is formed, the sodium ions being outside. Now it is not to be supposed that the same individual ions are always present in this double layer ; a perpetual interchange is going on between them and those present in the body of the solutions. Moreover, since their position is due solely to the fact of their possessing a positive charge, it is clear that if any other cations are in a position to interchange with them, the process will take place. This state of affairs will exist if any salt, say potassium chloride, is present in the outer solution. The external component of the double layer in such a case will consist of both K' and Na* ions in relative proportion, according to their respective concentrations in the solutions, and ultimately this same proportion will be established throughout both solutions, whatever the absolute concentration of the ions therein. This fact was pointed out by Ostwald (1890, p.- 714) as applying to the copper ferrocyanide membrane and found experimentally by W. A.

Osborne (1906) in the case of salts of caseinogen, or soaps within a parchment paper membrane, and by myself in that of Congo-red or of serum proteins in similar conditions. Although the ratio of the concentrations of the diffusible salts is the same on both sides of the membrane in such cases, as already remarked, the absolute concentration is greater on that side containing the colloidal solution. This fact seems to be due to the necessity that the concentration of non-dissociated salt must be equal on both sides ; there are, in fact, so far as one can see, no forces present capable of making possible a different concentration of electrically-neutral, freely-diffusible, substances. If, then, we have say sodium chloride in decimolar solution on the outside, and the sodium salt of Congo-red inside, assuming 10 per cent, of the sodium chloride undissociated, this concentration of undissociated moleeules must be the same inside ; this cannot lie the case if the total concentration of the chloride is the same on both sides, since that inside will be less dissociated than that outside, owing to the presence of the dye salt with an ion (Na~) common to both salts. This explanation of the unequal distribution of sodium chloride on the two sides of a membrane applies also if the diffusible salt placed outside has not, to begin with, an ion in common with the colloidal salt, say potassium chloride, because, as pointed out above, after equilibrium is attained, there will be present both inside and outside all the kinds of the diffusible ions of the system. This

I have shown experimentally to be the case, while Donnan (1911) has deduced it from thermodynamic considerations. We see, therefore, that the presence of a colloidal salt within a membrane, semi-permeable only as regards colloids, will not account for the unequal ratio of potassium to sodium in the plasma and corpuscles of the rabbit. Consider next the case of the muscle cell. The experiments of Katz (1896, p. 42) have shown that, in the rabbit, the ratio of the sodium to the potassium in these cells is as 0'46 to 4 ; whereas, as we have seen, the corresponding numbers for the blood plasma are as 4-44 to 0*259, and Fahr (1909) has made it practically certain that the sodium of frog's muscle is contained only in the intercellular lymph, etc., the muscle cells themselves containing no sodium at all. Such facts necessitate in this case also the existence of a membrane impermeable to salts.

According to Meigs and Ryan (1912, p. 411), however, the salts of smooth muscle are present in a non-diffusible form, and these authors do not admit the presence of a semipermeable membrane. The evidence given is, I think, not very convincing. Smooth muscles are stated, when immersed in hypotonic saline solution, to gain in weight according to a different time law from that of striated muscle in the same conditions. This fact is readily to be accounted for by a different amount of imbibition in the two cases. Imbibition may play a relatively important part in smooth muscle, although as we have seen above (page 116), it plays only an insignificant part in the case of striated muscle. Water taken in by imbibition is not, of course, active osmotically, so that in order to balance a given external osmotic pressure, more water must be taken in per unit time if part of it is inactive. Again, it is said that, if smooth muscle is immersed in an isotonic solution of cane-sugar, it gains weight much more rapidly than striated muscle does ; but we shall see presently that cane-sugar is by no means an innocuous substance for many cells, and the more rapid gain of weight is what would be expected if a certain amount of imbibition were taking place. It appears also that, when smooth muscle is cut across, its potassium content diffuses out very slowly ; the possibility of adsorption, or the formation of a new membrane on the cut surface, is not taken into due consideration. These observers also regard the loss of potassium phosphate by ordinary muscle in activity and its replacement as inconsistent with a semi-permeable membrane. But, admitting the loss of phosphate, we shall see later that there is an increase of permeability in the excited state and it may well be that the passage of salts takes place at this time.

There are many other facts, of interest also on their own account, which prove an impermeability to crystalloids. Bethe (1909) found that medusae, floating in sea water stained with neutral red, stored the dye in their cells with the orange-red colour which it has in a solution of neutral reaction. If hydrochloric acid were added to the water, so as to give the dye in it a cherry-red colour, it was found that no change was produced in the tint of the cells for several hours ; in fact, acid paralysis might be caused, but no change in the colour of the cells could be seen, until they were dead. The same thing was noticed with sodium hydroxide ; the cells did not become yellow, the colour of neutral red in alkaline solution.

From the experiments of O. Warburg (1910) on the eggs of a sea urchin, the same fact, amongst others, was clearly made out. In this case, it was shown that the absence of change of colour was really due to non-entrance of alkali, and not to some fixed state of the dye making it inert to alkali, by taking an alkali to which the cell membrane is known to be permeable, such as ammonium hydroxide, in which case the colour became yellow almost instantly.

The objection may be made that the chemical or adsorption compound of the dye with cell structures may be less sensitive to sodium hydroxide than to ammonium hydroxide. This has been dealt with by Newton Harvey (1913), who has shown that the adsorption compounds of neutral red with various proteins, with lecithin, etc., are affected by these two alkalies in exactly the same concentration. Moreover, when the sea urchin eggs are made actually permeable to sodium hydroxide, by the action of sea water saturated with chloroform, this alkali changes the neutral red in the cells just as readily as ammonium hydroxide does.