Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.     

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Osmotic stress as a mechanism of freezing injury

When red cells are suspended in solutions of nonpenetrating solutes of osmolality in excess of 1300 mosm, changes in membrane permeability are seen: there is an influx of extracellular solute, cell volume increases, and the cells will hemolyze on return to isotonic suspension. Furthermore, if cells suspended in such a solution are exposed to a downward temperature change (thermal shock), many will hemolyze.     

Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue

Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO3, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and α2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.Open in a separate windowClick here to view.^{(81M, flv)}     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

Effects of amiloride on gustatory neural responses to salts in the frog.

In frogs, the glossopharyngeal nerve (GL) innervates taste receptors on almost the entire tongue. The mandibular branch (MBF) and palatine branch (PN) of the facial nerve innervate taste receptors on a very small area at the base of the tongue and on the palate, respectively. In the present study, effects of amiloride, an epithelial sodium channel blocker, on the tonic responses of the GL, MBF and PN in frogs to NaCl, LiCl, KCl and CaCl(2) were investigated. In three nerves, amiloride at 0.5 mM, a relatively high concentration, did not affect the responses to 0.15 (concentration just above threshold)-0.5 M NaCl, 0.5 M LiCl and 0.3 M KCl, whereas it almost completely inhibited the response to 1.0 mM CaCl(2). Amiloride may exert an inhibitory action on the response to CaCl(2) by a competitive antagonism between Ca(2+) and a monovalent cation of amiloride, because the response to Ca(2+) is competitively inhibited by other cations such as Na(+) and Mg(2+). The lack of inhibitory effect of amiloride on the responses in the GL, MBF and PN to NaCl suggests that amiloride-sensitive sodium channels in the apical membrane of taste receptor cells are not involved in sodium taste transduction in frogs.     

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms.

The penicillin-binding proteins (PBPs) of Streptococcus pyogenes and two of its derived, stabilized (i.e., nonreverting) L forms, an osmotically fragile L form and a physiologic isotonic L form, were compared. The numbers of PBPs in the membranes of these organisms were 6, 4, and 2 for the coccus and the osmotically fragile and physiologic isotonic L forms, respectively. Likewise, the relative amounts of total PBPs were 1.00: 1.48:0.32 for this coccus and the osmotically fragile and physiologic isotonic L forms, respectively. The two largest PBPs (PBPs 1 and 2) of the coccus were absent in both L forms, while the smallest PBPs (PBPs 5 and 6) were found in all three membranes. Deacylation (half-life) of three of the four PBPs in the osmotically fragile L form membrane required a significantly longer time than did deacylation of these presumed identical enzymes in the parental coccal membrane. Conversely, there was no such difference between the only two PBPs of the physiologic isotonic L form and the same coccal membrane proteins. Intact cells of all three organisms secreted PBPs and what appeared to be penicilloic acid and a minimal amount of free penicillin. A greater amount of these PBPs was secreted by both L forms than by the coccus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and ratios of secreted PBPs were identical to those from labeled membrane preparations. These differences are correlated with some of our previous findings and are discussed in terms of inhibition of cell wall synthesis and resulting membrane changes in these two derived, stabilized coccal L forms.     

The radiation response of cultured mammalian V79-S171 cells exposed to a wide concentration range of sulphate salt solutions.

The radiation response of Chinese hamster cells (V79) exposed to a wide concentration range of Li2SO4, Na2SO4 or K2SO4 has been examined and compared with the radiation response of cells treated in an identical manner with LiCl, NaCl, or KCl solutions. At hypotonic salt concentrations, cells were radiosensitized by both the chloride and sulphate salts. At high salt concentrations, approximately greater than 0.9 M, a radioprotective effect was observed with both chloride and sulphate salts. At intermediate salt concentrations from about 0.2 to 0.9 M, the cells that were treated with the sulphate salt solutions were radioprotected; cells treated with chloride salt solutions were radiosensitized. The difference in radiation response was attributed to the difference in anions for the two types of salts used.     

Tonicity-volume relations in partially hemolyzed hypotonic systems

The linear relation between the red cell volume V and the reciprocal of the tonicity T of a hypotonic medium is not the linear one expected on the basis of the van't Hoff-Mariotte law, particularly when a fraction p of the cells are hemolyzed and the volume V/(1 – p) of the (1 – p) cells which remain intact is considered. In systems of relatively high tonicity in which p is zero, the relation is linear but its slope is usually too small; at lower tonicities in which p has a value between zero and 0.35, the volumes are larger than those expected on the basis of the van't Hoff-Mariotte law, while at still smaller tonicities they are much smaller than expected. The volume measurements referred to are made with a high speed hematocrit; the results obtained in systems containing relatively high and relatively low volume concentrations of cells are contrasted with each other, and allowance is made in the calculations for the volume of the hypotonic medium surrounding the cells being limited. Attempts are made at an explanation of the anomalous results in terms of incomplete packing, of a stepwise as opposed to an all-or-none loss of Hb from the cells, of a heterogeneity in the swelling properties of the cells of the population, of a loss of osmotically active substances from the intact cells, and of the red cell ghost having some degree of rigidity. These explanations are not satisfying, although some of them in combination may account for the phenomena observed. It seems likely that the structures involved in the hemolytic process (cells and ghosts) have different properties in different tonicity ranges and even when the same tonicity is established in systems which are dense, as opposed to dilute, with respect to cell concentration. The form of the tonicity-volume relation can be changed substantially, although not in the direction of greater linearity, by treating the cells with resorcinol, colloidal silicic acid, iodoacetate, or sodium oxalate. Small changes in pH, exposure to temperatures as high as 37°C., allowing the cells to stand for periods up to 24 hours, or the substitution of hypotonic plasma for hypotonic NaCl-buffer, produce, on the other hand, only minor changes in the tonicity-volume relation.     

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.     

Water exchange across red cell membranes: II. Measurement by nuclear magnetic resonanceT 1,T 2, andT 12 hybrid relaxation. The effects of osmolarity,cell volume,and medium

Summary We have used the nuclear magnetic relaxation of water protons to measure the diffusional permeability (P _w) of human red blood cells to water as a function of concentration of nonpermeable and permeable solutes. Measurements ofT ₁,T ₂, and a hybrid of the two were made and yielded the sameP _w. In the presence of the nonpermeable electrolyte NaCl, membrane permeability is constant between the volumes of 70 and 105m³ and increases both as the cells swell and shrink beyond these limits. Changes in both the internal and external osmolarity, using the permeable solutes urea and ammonium chloride, do not affect membrane permeability. The composition of the suspending medium also has a significant effect on membrane permeability. Cells suspended in plasma have a cell water lifetime about 30% longer than cells of the same volume suspended in serum, or isotonic saline with human serum albumin. Addition of a crude preparation of fibrinogen in physiological amounts to isotonic saline and human serum albumin restores the cell water lifetime to a value similar to that observed in plasma.     

PROLYTIC ION EXCHANGES PRODUCED IN HUMAN RED CELLS BY METHANOL,ETHANOL, GUAIACOL,AND RESORCINOL

When the washed red cells of heparinized human blood are exposed at 4°C. to methanol, ethanol, guaiacol, or resorcinol in hypolytic concentrations in isotonic NaCl, the prolytic loss of K at the end of 20 hours varies from about 25 per cent of the initial K content of the cells in the case of 3.1 M methanol to about 55 per cent of the initial K in the case of 0.04 M resorcinol. As in the case of the prolytic losses observed with other lysins, the K loss is rapid at first and then slows down so that what appears to be a new steady state is reached logarithmically. The K lost from the cells during the period of the prolytic loss is replaced by an approximately equivalent amount of Na, derived from the isotonic NaCl in which the cells are suspended. The Na which enters can be replaced by K by washing the cells in isotonic KCl, and this K can again be replaced by Na by washing the cells in isotonic NaCl. The remainder of the cell K., i.e. the K which was not lost during the period of the prolytic loss, is retained in the cell unaffected by these washing procedures. The capacity of red cells for undergoing disk-sphere transformations is scarcely affected by their having been exposed to hypolytic concentrations of methanol, ethanol, guaiacol, or resorcinol in isotonic NaCl, and their resistance to osmotic hemolysis and to lysis by saponin and digitonin is altered only in minor respects even when as much as 50 per cent of the cell K has been exchanged for Na. Some restriction to the movement of K between the cell and its environment is apparently modified irreversibly when the cell is exposed to hypolytic concentrations of lysins, and the modification is such that only a fraction of the cell K is affected, the fraction being a function of the lysin concentration, the duration of its action, and other factors. A modification of some part of the cell structure and of the properties dependent on its integrity is probably involved: K may be lost more readily from some cells than from others, from some parts of the cell more readily than from other parts, or the explanation may lie in changes in the extent to which Hb binds ions or in modifications of metabolic processes.     

Peripheral and central interactions between sugar,water, and salt receptors of the blowfly, Phormia regina

The peripheral and central nervous interactions between the sugar, water, and salt receptors of the blowfly were investigated electrophysiologically by simultaneously recording from the labellar chemoreceptors and the extensor muscle of the haustellum. Simultaneous stimulation of two water receptors with 10 mM LiCl resulted in a motor response even though stimulating the same two sensilla separately with 10 mM LiCl did not. There was a linear decrease in the motor response to two sensilla stimulation as the salt concentration in the stimulating solution increased. Although stimulating two sensilla simultaneously with 200 mM NaCl gave no motor response, simultaneously stimulating two sensilla with 10 mM LiCl and a third with 200 mM NaCl gave a greater response than did two sensilla stimulation with 10 mM LiCl alone, indicating cross-channel summation between the water and salt receptors. Similarly, simultaneously stimulating one sensillum with 100 mM sucrose and another with 10 mM LiCl or 500 mM NaCl gave a larger response than did 100 mM sucrose stimulation alone. The cross-channel summation between the sugar and water receptors was not affected by feeding the flies water. A central excitatory state (CES) which previously had been demonstrated behaviourally was investigated. A stimulation of one sensillum with 10 mM LiCl which previously had been ineffective gave a motor response if proceeded by a stimulation with 1 M sucrose on another sensillum. The effect of the time interval between the sugar and water stimuli was tested, but for intervals of 100 msec to 4 sec no definite correlation was found. In addition, CES with respect to the sugar receptor was demonstrated. The motor response to stimulation of a single sensillum with 100 mM sucrose was enhanced by preceding it with 1 M sucrose stimulation of another sensillum. The motor response to stimulation of two water receptors with 10 mM LiCl was partially inhibited by simultaneously stimulating a third sensillum with 4 M NaCl. Inhibition was also seen when a single sensillum was stimulated with a mixture of 10 mM LiCl and 10 mM sucrose and an adjacent sensillum was simultaneously stimulated with 1 M NaCl. Behavioural experiments showing inhibition of CES by salt were confirmed. Interposing a salt stimulus of 4 M NaCl between the 1 M sucrose and 10 mM LiCl stimuli reduced but did not totally eliminate the motor response to 10 mM LiCl. The basis for peripheral control of the relative activities of the water and salt receptors is discussed. A model is proposed to account for all the receptor interactions in the central nervous system.     

Inhibition of thirst when dehydrated rats drink water or saline

The present experiments sought to identify the physiological signals that inhibit thirst when dehydrated rats drink water or NaCl solution. Rats were deprived of drinking fluid but not food overnight. When allowed to drink again, the dehydrated animals consumed water or saline (0.05 M, 0.10 M, 0.15 M, or 0.20 M NaCl solution) almost continuously for 5-8 min before stopping. The volumes consumed were similar regardless of which fluid they ingested, but blood analyses indicated that increased plasma osmolality and decreased plasma volume, or both, still remained when drinking terminated. These results suggest that the composition of the ingested fluid is less significant than its volume in providing an early signal that inhibits thirst and fluid consumption by dehydrated rats. Analyses of the gastrointestinal tracts revealed that the cumulative volume in the stomach and small intestine correlated highly with the amount consumed regardless of which fluid was ingested. These and other results suggest that the volume of fluid ingested by dehydrated rats is sensed by stretch receptors detecting distension of the stomach and small intestine, which provide an early inhibitory stimulus of thirst.     

Anomalous features of the loss of K from human red cells; results of extended observations

1. The anomalous course of the curves relating K loss to time in systems containing human red cells in isotonic NaCl, and particularly the high positions of the asymptotes to which the curves apparently proceed, are due to the population of red cells consisting of at least two components, one of which loses K more readily than the other. 2. Since large K-Na exchanges can occur between red cells and an isotonic suspension medium without there being large volume changes, a restatement of the "dual mechanism of hemolysis" hypothesis, which takes account of the cell's being slowly permeable to cations, is required. If some approximations of minor consequence are allowed, the hypothesis can be restated in a quantitatively satisfactory way. 3. The general features of K-Na exchanges, including prolytic exchanges are summarized.     

A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium

A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.     

PLASMALEMMA POTENTIAL IN NITELLA

A considerable part of the response of the vacuolar potentialof Nitella flexilis to the change of external KCl, NaCl, RbCl,LiCl, or CaCl₂ concentration is caused by the response of thecell wall (a cation exchanger) to the external medium. The potentialswere measured on the internodes whose cell sap was exchangedfor simple salt solutions. The potential difference across theplasmalemma which is the internal potential measured againstthe cell wall phase changes largely with the change in concentrationof the external KCl, but also more or less with that of theexternal NaCl, LiCl or RbCl. CaCl₂ depolarizes the plasmalemmapotential by about 50 mv when the concentration is increasedfrom 10^–5 M to 10^–3 M, and hyperpolarizes it againby about 40 mv from 10^–3 M to 10^–1 M leaving thelevel of the peak of the action potential almost unchanged. ¹This work was supported by Research Grants from the Ministryof Education of Japan     

Isocratic separation of estrogen conjugates on DEAE-sephadex.

Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model $\text{y}\text{=}\text{a}\text{+}\text{b}\text{1}\text{x}$ where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. II. Disassembly and Reassembly of Proplastid-Nuclei Isolated from Cultured Cells

The effects of various concentrations of NaCl on the compactstructure of proplastid-nuclei (pp-nuclei) isolated from culturedtobacco cells were examined by fluorescence microscopy usinga DNA-specific fluorochrome, 4, 6-diamidino-2-phenylindole.Simultaneously, behavior of the four proplastid DNA-bindingproteins (mol wt: 69kDa, 31 kDa, 30kDa and 14kDa) identifiedpreviously (Nemoto et al. 1988) was investigated by SDS-polyacrylamidegel electrophoresis. When the concentration of NaCl was increased, the isolated pp-nucleiwere gradually dispersed, and at concentrations greater than0.4 M NaCl they were almost completely dispersed. During thisdisassembly process, the 31-kDa and 30-kDa proteins dissociatedfrom the pelletable pp-nuclear fraction to the supernatant at0.1 M NaCl, whereas the 69-kDa and 14-kDa proteins dissociatedto the supernatant only at 0.4 M NaCl. When the concentrationof NaCl was decreased again by dialysis, the pp-nuclei whichhad been dispersed by 2 M NaCl were gradually reassembled intocompact structures which were almost identical to the originalpp-nuclei. During this reassembly process, the 69-kDa and 14-kDaproteins first returned to the pellet fraction, and subsequentlythe 31-kDa and 30-kDa proteins moved into the pellet. This behavior of the proplastid DNA-binding proteins stronglysuggests that the association of these proteins with plastid-DNAis responsible for the compact organization of pp-nuclei. Inaddition, it was indicated that the 69-kDa and 14-kDa proteinsare more tightly bound to plastid-DNA than are the 31-kDa and30-kDa proteins. ⁴Present address: Biological Laboratory, Faculty of Science,Nara Women's University, Nara, 630 Japan. (Received August 24, 1988; Accepted February 28, 1989)