CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : IV. PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA FLEXILIS

Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance. Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue. When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

CHLOROPHYLL AMOUNT AS AN INDICATOR OF MATTER PRODUCTIVITY IN BIO-COMMUNITIES

The data of chlorophyll amounts in diverse bio-communities arecompiled and discussed with reference to matter production. The chlorophyll amount in euphotic zone of lakes and oceanswas less than 1 g/m², mostly less than 0.1 g/m². In phytoplanktonblooms it was ca. 0.1–1 g/m². Large values of 5–20g/m² were obtained in the outdoor mass cultures of Chlorella,in which the high population density and chlorophyll contentof the alga were observed. In terrestrial higher plant communitiesthe chlorophyll amount (ca. 1–10 g/m²) was usually higherthan in aquatic phytoplankton communities. The largest (13.3g/m²) was obtained with an evergreen gallery forest in Thailand.The chlorophyll amount of desert bio-community could be expectedto be as high as that in water blooms. The maximum chlorophyll amount in bio-communities seems to attainup to 20 g/m² when the conditions are favorable. ¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday. (Received December 25, 1962; )     

THE CYTOCHEMICAL REDUCTION OF NITRO BLUE TETRAZOLIUM AS AN INDEX OF POLLEN VIABILITY

A new technique for estimating pollen viability utilizing Nitro Blue Tetrazolium (Nitro-BT) is given. Pollen of 16 taxa was studied and scored for stainability. A comparison was made between Nitro-BT-stained pollen samples and aniline-blue-lactophenol-stained samples, because the validity of the latter stain has been questioned in recent years. It is demonstrated that Nitro-BT can discriminate between pollens which have a capacity for oxidative metabolism, representing potential viability, and those which do not. Suggestions for pollen stainability studies are given.     

INCORPORATION OF AMINO ACIDS INTO SEA URCHIN EGGS STIMULATED INSUFFICIENTLY WITH AN ACTIVATING REAGENT

Investigations were performed on the incorporation of valine-¹⁴C and leucine-³H into sea urchin eggs which were stimulated insufficiently with an activating reagent. Materials used were Pseudocentrotus, Hemicentrotus and Anthocidaris. An insufficient stimulation enhanced the incorporation of amino acids into the eggs, although it provoked no visible cortical changes. The amount of incorporation in this case was 1.6 to 2.7 times as much as that into untreated eggs. In fully activated eggs, the amount of incorporation was more than 4 times that in untreated eggs. The fact that the incorporation of amino acids is increased without accompanying breakdown of the cortical granules indicates that the increase may be linked to an invisible change, probably the fertilizationwave, which is caused by an insufficient stimulation.     

MICROINJECTION OF COLCHICINE INTO SEA URCHIN EGGS

Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two-cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid-stage of the cleavage in Clypeaster japonicus and 8 min before the mid-stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10⁻⁵ M to 3 × 10⁻⁶ M in final concentration in the cell.     

THE PENETRATION OF AMMONIA INTO FROG MUSCLE

1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH₄Cl. Analyses were made in the solution and in the muscles for K and NH₃, and the muscles were also analyzed for Cl, HCO₃, and Na. Control muscles were immersed in normal Ringer''s solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH₃ which penetrated. An increased loss of K due to the penetration of NH₃ was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH₃ ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH₃ is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH₄Cl into anesthetized cats causes an increase in the level of K in the blood plasma.     

THE PENETRATION OF CATIONS INTO LIVING CELLS

Direct tests of the cell sap of Nitella show that the protoplasm is normally permeable to Li, Cs, and Sr, and that penetration is more rapid in an unbalanced than in a balanced solution.     

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.     

METHODS OF STUDYING THE RESPIRATORY EXCHANGE IN SMALL AQUATIC ORGANISMS,WITH PARTICULAR REFERENCE TO THE USE OF FLAGELLATES AS AN INDICATOR FOR OXYGEN CONSUMPTION

1. Flagellates are positively chemotactic to a certain concentration of dissolved oxygen which is lower than that in water saturated with oxygen under atmospheric partial pressure. Consequently, when a small aquatic animal is held motionless between cover-slip and slide in a suspension of flagellates in water saturated with oxygen, the flagellates are attracted to those parts of the animal which are absorbing oxygen. The relative sizes of the flagellate aggregations then show the relative activities of the different surfaces of the animal in absorbing oxygen. 2. Applying this method to the red Chironomus larva it was found that the animal respires by the whole body surface except by the head and the "ventral gills" and that the relative intensity of oxygen intake by the different parts of the body varies in different individuals and in the same individual at different times. 3. The absence of oxygen intake by the "ventral gills" was confirmed with the microspectroscope. In oxygen-free water all the hemoglobin of the blood becomes reduced. When an air bubble is now introduced so that it touches the "ventral gills" oxyhemoglobin first appears in the nearest body segment to the bubble, not in the "gills." 4. When a small aquatic animal is held motionless between cover-slip and slide in a solution of an indicator which changes color about the neutral point of water the relative extent of color change at different surfaces of the animal''s body indicates the relative amounts of carbon dioxide given off by these surfaces. 5. Using this method with the red Chironomus larva similar conclusions were reached for carbon dioxide output as for oxygen intake.