ELECTRIC IMPEDANCE OF NITELLA DURING ACTIVITY

The changes in the alternating current impedance which occur during activity of cells of the fresh water plant Nitella have been measured with the current flow normal to the cell axis, at eight frequencies from 0.05 to 20 kilocycles per second, and with simultaneous records of the action potential under the impedance electrodes. At each frequency the resting cell was balanced in a Wheatstone bridge with a cathode ray oscillograph, and after electrical stimulation at one end of the cell, the changes in the complex impedance were determined from the bridge unbalance recorded by motion pictures of the oscillograph figure. An extension of the previous technique of interpretation of the transverse impedance shows that the normal membrane capacity of 0.9 µf./cm.² decreases about 15 per cent without change of phase angle, while the membrane resistance decreases from 10⁵ ohm cm.² to about 500 ohm cm.² during the passage of the excitation wave. This membrane change occurs during the latter part of the rising phase of the action potential, and it is shown that the membrane electromotive force remains unchanged until nearly the same time. The part of the action potential preceding these membrane changes is probably a passive fall of potential ahead of a partial short circuit.     

Culm strength of barley : correlation among maximum bending stress, cell wall dimensions, and cellulose content

Grass culms are known to differ in breaking strength, but there is little physicochemical data to explain the response. The fourth internode of four brittle and two nonbrittle barley (Hordeum vulgare L.) strains were used for physical and chemical studies of culm strength. Inner and outer culm diameters of brittle strains (3.6 ± 0.2 and 5.0 ± 0.1 millimeters) were not significantly different from those of nonbrittle strains (3.9 ± 0.2 and 5.2 ± 0.2 millimeters). Maximum bending stress, at which the culm was broken, was 192 ± 34 g/mm² for brittle and 490 ± 38 g/mm² for nonbrittle strains. Wall thickness and cell dimensions of epidermal, sclerenchyma, and parenchyma cells were measured in culm cross sections. The area of cell wall per unit cell area for each tissue was significantly correlated with the maximum bending stress (r = 0.93 for epidermis, 0.90 for sclerenchyma, and 0.84 for parenchyma). Cell walls of brittle culms had 6 to 64% as much cellulose content as those of nonbrittle culms. Maximum bending stress correlated significantly with cellulose content of the cell walls (r = 0.93), but not with the contents of noncellulosic compounds. The lower cellulose content of the brittle culm was significantly correlated with brittleness.     

ELECTRIC IMPEDANCE OF THE FROG EGG

Electrical impedance measurements were made upon unfertilized and fertilized eggs of the leopard frog, Rana pipiens, over a frequency range of 0.05 to 10 kc. Average values of 170 ohm cm.² were obtained for the plasma membrane resistance of the egg, 2.0 µf/cm.² for the plasma membrane capacity, 86° for the phase angle of the membrane, and 570 ohm cm. for the specific resistance of the interior. These values did not change upon fertilization. No spontaneous rhythmical impedance changes such as have been found by Hubbard and Rothschild in the trout egg were found in frog eggs.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

ELECTRIC IMPEDANCE OF ARBACIA EGGS

The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 10³ to 1.64 x 10⁷ cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.² which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.² at low frequencies which decreases to a value of 0.55 µf./cm.² at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.² capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.² for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization.     

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van''t Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10^–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.     

Sap6, a secreted aspartyl proteinase,participates in maintenance the cell surface integrity of Candida albicans

Background

The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases.

Results

After 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the β-mercaptoethanol (β-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains.

Conclusion

From these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of β-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.     

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [¹⁴C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.     

HYDROSTATIC PRESSURE AND TEMPERATURE IN RELATION TO STIMULATION AND CYCLOSIS IN NITELLA FLEXILIS

Nitella flexilis cells are not stimulated to "shock stoppage" of cyclosis by suddenly evacuating the air over the water or on sudden readmission of air, or on suddenly striking a piston in the water-filled chamber in which they are kept with a ball whose energy is 7.6 joules, provided the Nitella cell is not moved by currents against the side of the chamber. Sudden increases in hydrostatic pressure from zero to 1000 lbs. or 0 to 5000 lbs. per square inch or 5000 to 9000 lbs. per square inch usually do not stimulate to "shock stoppage" of cyclosis, but some cells are stimulated. Sudden decreases of pressure are more likely to stimulate, again with variation depending on the cell. In the absence of stimulation, the cyclosis velocity at 23°C. slows as the pressure is increased in steps of 1000 lbs. per square inch. In some cells a regular slowing is observed, in others there is little slowing until 4000 to 6000 lbs. per square inch, when a rapid slowing appears, with only 50 per cent to 30 per cent of the original velocity at 9000 lbs. per square inch. The cyclosis does not completely stop at 10000 lbs. per square inch. The pressure effect is reversible unless the cells have been kept too long at the high pressure. At low temperatures (10°C.) and at temperatures near and above (32°–38°C.) the optimum temperature for maximum cyclosis (35–36°C.) pressures of 3000 to 6000 lbs. per square inch cause only further slowing of cyclosis, with no reversal of the temperature effect, such as has been observed in pressure-temperature studies on the luminescence of luminous bacteria. Sudden increase in temperature may cause shock stoppage of cyclosis as well as sudden decrease in temperature.     

ELECTRON MICROSCOPICAL STUDY OF SKELETAL MUSCLE DURING ISOTONIC (AFTERLOAD) AND ISOMETRIC CONTRACTION

Bundles of the curarized semitendinosus muscle of the frog were fixed during isotonic (afterload) and isometric contraction and the length of the A and I bands investigated by electron microscopy. The sarcomere length, during afterload contraction initiated at 25 per cent stretch, varied depending on the afterload applied between 3.0 and 1.2 µ, i.e. the shortening amounted to 5 to 50 per cent. The shortening involved both the A and I bands. Between a sarcomere length of 3.0 to 1.7 µ (shortening 5 to 35 per cent) the A bands remained practically constant at about 1.5 µ (6 to 8 per cent shortening); the length of the I bands decreased from 1.4 to 0.3 µ (80 per cent shortening). Below a sarcomere length of 1.7 to 1.2 µ the A bands shortened from 1.5 to 1.0 µ (from 6 to 8 to 25 per cent). At sarcomere lengths 1.6 to 1.2 µ the I band was replaced by a contraction band. During isometric contraction the A bands shortened by about 8 to 10 per cent; the I bands were correspondingly elongated.     

Permanganate Fixation of Plant Cells

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO₄ at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO₄ at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO₄, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO₄ at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO₄. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO₄ or OsO₄.     

CONCENTRATION OF ACID PHOSPHATASE,RIBONUCLEASE, DESOXYRIBONUCLEASE, β-GLUCURONIDASE,AND CATHEPSIN IN "DROPLETS" ISOLATED FROM THE KIDNEY CELLS OF NORMAL RATS

1. Three fractions of "droplets" having diameters of 1 to 5 µ (fraction I), 0.5 to 1.5 µ (fraction II), and 0.1 to 1.0 µ (fraction III) were isolated from the kidney cells of normal rats. 2. All three "droplet" fractions showed 10 to 15 times higher activities of acid phosphatase, β-glucuronidase, ribonuclease, desoxyribonuclease, and cathepsin than the total homogenate and the mitochondrial fraction. 3. After a rough fractionation of the total homogenate, approximately 50 per cent of the 5 enzymes was found in the fractions which contained the "droplets" and approximately 30 per cent in the supernatant fluid. 4. The similarities between the enzymatic properties of the "droplets" from kidney cells and of the fractions isolated from liver cells by other investigators have been discussed.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

Efflux of Red Cell Water into Buffered Hypertonic Solutions

Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093°C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.     

A METHOD FOR INTRACELLULAR AUTORADIOGRAPHY IN THE ELECTRON MICROSCOPE

A technic is described for high resolution intracellular autoradiography in the electron microscope. Cultures of LLC-MK₂ monkey kidney cells were incubated for 72 hours in a medium containing 0.4 µcurie per ml of thymidine-H³. After labeling, the cells were fixed with osmium tetroxide and embedded in methacrylate. Ultrathin sections of the labeled tissue were taken up on Formvar-coated and carbon-stabilized electron microscope grids. A 150 to 450 A layer of silver metal was then evaporated onto the tissue. The coated grids were exposed to bromine vapor for 1.5 to 2 minutes under red light, allowed to dry for 1 minute, and then covered with a thin film of 1 per cent aqueous gelatin applied by means of a fine wire loop lowered over the grid supported on a glass peg. For autoradiographic exposure, the grids were stored 50 days in a light-proof container at 4°C with calcium chloride desiccant. Development was carried out for 5 minutes at 20°C in Promicrol (May and Baker, England) diluted 1:1 with water, followed by a 1 minute water wash and fixation for 2.5 minutes in 15 per cent aqueous sodium thiosulphate. After removal of the gelatin by immersion for 16 hours in water at 37°C, the autoradiograms were dried and examined in the electron microscope. Ultrastructural detail was fairly well defined and the cytoplasm of each labeled cell was covered with an electron opaque deposit of silver, suggesting that a polynucleotide containing thymidine may be synthesized in the cytoplasm. The matter is discussed.     

Developmental Changes in Cell and Tissue Water Relations Parameters in Storage Parenchyma of Sugarcane

The osmotic pressure of the cell sap of stalk storage parenchyma of sugarcane (Saccharum spp. hybrids) increases by an order of magnitude during ontogeny to reach molar concentrations of sucrose at maturity. Stalk parenchyma cells must either experience very high turgor at maturation or have an ability to regulate turgor. We tested this hypothesis by using pressure probe techniques to quantify parameters of cell and tissue water relations of sugarcane storage parenchyma during ontogeny. The largest developmental change was in the volumetric elastic modulus, which increased from 6 bars in immature tissue to 43 bars in mature tissue. Turgor was maintained relatively low during sucrose accumulation by the partitioning of solutes between the cell and wall compartments. Membrane hydraulic conductivity decreased from about 12 × 10⁻⁷ centimeters per second per bar down to 4.4 × 10⁻⁷ centimeters per second per bar. The 2.7-fold decrease in membrane hydraulic conductivity during tissue maturation was accompanied by a 7.8-fold increase in wall elasticity. Integration of the cell wall and membrane properties appears to be by the opposing effects of turgor on hydraulic conductivity and elastic modulus. The changes in these properties during development of sugarcane stalk tissue may be a way for parenchyma cells to develop a capacity for expansive growth and still serve as a strong sink for storing high concentrations of sucrose.     

THE INFLUENCE OF ENVIRONMENTAL TEMPERATURE ON THE UTILIZATION OF FOOD ENERGY IN BABY CHICKS

1. An optimum of environmental temperature is to be expected for the utilization of food energy in warm blooded animals if their food intake is determined by their appetite. 2. Baby chicks were kept in groups of five chicks in a climatic cabinet at environmental temperatures of 21°, 27°, 32°, 38°, and 40°C. during the period of 6 to 15 days of age. The intake of qualitatively complete food was determined by their appetite. Food intake, excretion, and respiratory exchange were measured. Control chicks from the same hatch as the experimental groups were raised in a brooder and were given the same food as the experimental chicks. The basal metabolism of each experimental group was determined from 24 to 36 hours without food at the age of 16 days. 3. The daily rate of growth increased with decreasing environmental temperature from 2.74 gm. at 40°C. to 4.88 gm. at 21°C. This was 4.2 to 6.5 per cent of their body weight. 4. The amount of food consumed increased in proportion to the decrease in temperature. 5. The availability of the food, used for birds instead of the digestibility and defined as See PDF for Structure showed an optimum at 38°C. 6. The CO₂ production increased from 2.95 liters CO₂ per day per chick at 40°C. to 6.25 liters at 21°C. Per unit of the 3/4 power of the body weight, 23.0 liters CO₂ per kilo^3/4 was produced at 40°C. and 43.4 liters per kilo^3/4 at 21°C. The CO₂ production per unit of 3/4 power of the weight increased at an average rate of approximately 1 per cent per day increase in age. The R.Q. was, on the average, 1.04 during the day and 0.92 during the night. 7. The net energy is calculated on the basis of C and N balances. A maximum of 11.8 Cal. net energy per chick per day was found at 32°C. At 21°C. only 6.9 Cal. net per day per chick was produced and at 40°C. an average of 6.7 Cal. 8. The composition of the gained body substance changed according to the environmental temperature. The protein stored per gram increase in body weight varied from 0.217 to 0.266 gm. protein and seemed unrelated to the temperature. The amount of fat per gram gain in weight dropped from a maximum of 0.153 gm. at 32°C. to 0.012 gm. at 21°C. and an average of 0.107 gm. at 40°C. The energy content per gram of gain in weight had its maximum of 2.95 Cal. per gm. at 38°C. and its minimum of 1.41 Cal. per gm. at 21°C. at which temperature the largest amount of water (0.763 gm. per gm. increase in body weight) was stored. 9. The basal metabolism increased from an average of 60 Cal. per kilo^3/4 at an environmental temperature of 40°C. to 128 Cal. per kilo^3/4 at 21°C. No indication of a critical temperature was found. 10. The partial efficiency, i.e. the increase in net energy per unit of the corresponding increase in food energy, seemed dependent on the environmental temperature, reaching a maximum of 72 per cent of the available energy at 38°C. and decreasing to 57 per cent at 21°C. and to an average of 60 per cent at 40°C. 11. The total efficiency, i.e. the total net energy produced per unit of food energy taken in, was maximum (34 per cent of the available energy) at 32°C., dropped to 16 per cent at 21°C., and to an average of 29 per cent at 40°C.     

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using ¹⁵N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.     

ELECTRON MICROSCOPE REPLICAS FROM THE SURFACE OF A FRACTURE THROUGH FROZEN CELLS

A technique is described, which is new in certain details, for fracturing frozen cells and taking replicas from the fracture surface. This technique has been developed as a cytological preparative technique complementary to those currently used in electron microscopy. The present paper describes results for human red cells, chosen for preliminary work to facilitate recognition of artifacts. The cells are suspended in 20 per cent glycerol, 0.9 per cent NaCl solution, allowed time to equilibrate, and rapidly frozen in a small chamber plunged into propane at -196°C. The chamber is kept at -196°C., and fractured in vacuo, in such a way that the fracture plane runs through the frozen suspension, and through the cells in its path. After fracture, the chamber is brought to -105°C., still in vacuo, to etch the fracture surface, and a platinum-carbon replica is deposited on the surface from a carbon arc. The replica is subsequently cleaned in concentrated alkaline solution and examined in the electron microscope. The outlines of the fractured cells can be recognised. There are indications that in areas of the replica which correspond to the interior of a cell, individual haemoglobin molecules can be seen, and in favourable cases the arrangement of some of the four molecular subunits.     

REVIVAL OF MAMMALIAN SPERM AFTER IMMERSION IN LIQUID NITROGEN

1. A wide variety of procedures was used to test the motility of mammalian sperm after plunging them into liquid nitrogen at –195°C. and later rapidly warming them to 35°C. by plunging them into a suitable balanced and isotonic medium. 2. Using seminal fluid sperm from the same human donor, maximal numbers of motile sperm survived vitrification when the samples were (a) very fresh, (b) untreated with plasmolysing solutions, (c) plunged into the refrigerant in the form of a foam. The maximum yield of motile human sperm recoverable from the liquid nitrogen was 50 per cent. Since in this sample only 75 per cent of the sperm were alive before immersion, 67 per cent of the living sperm survived vitrification. 3. Experiments with sperm from 31 rabbits were made with a variety of conditions of pretreatment to obtain maximal yields of recoverable, motile sperm after vitrification by liquid nitrogen. (a) A consistent recoverable yield of about 0.5 per cent was obtained when the untreated suspension of sperm was smeared on cellophane and partially dried in air before immersing in liquid nitrogen. (b) On a few out of many occasions plasmolysis for several minutes with hypertonic Ringer solution gave a recoverable yield of 0.1 per cent as did (c) pretreatment with hypertonic Ringer and butyric acid.