HEMOLYSIS OF CHICKEN BLOOD

1. The time-dilution curves are given for the hemolytic action of saponin, sodium taurocholate, and sodium oleate on nucleated chicken erythrocytes. 2. Saponin and sodium taurocholate cause hemolysis but leave the nuclei and ghosts in suspension, thereby making the end-point of hemolysis more arbitrary than the clear end-point for non-nucleated cell hemolysis. 3. The curves of hemolysis by saponin and taurocholate are shown to be of the same nature as are found in the hemolysis of non-nucleated cells. 4. Sodium oleate causes first hemolysis and then, in the stronger solutions, causes karyolysis. Two pairs of values for κ and c = ∞ are thus obtainable for the same reaction, one pair for the destruction of corpuscular membrane, the other pair for the destruction of the nucleus. 5. Viscosity changes are found in the lysin-cell system with strong concentrations of sodium taurocholate and sodium oleate. Time-viscosity curves are given for these changes. 6. Microscopically, the action of these lysins on the nucleated chicken red cell appears to be similar to their action on the non-nucleated erythrocytes.     

THE EFFECT OF HEMOLYTIC SUBSTANCES ON WHITE CELL RESPIRATION

Substances such as saponin, the bile salts, etc., which produce lysis of red cells also produce cytolysis of white cells from rabbit peritoneal exudates, the arbitrary criterion of their cytolytic effect being their ability to depress the O₂ consumption of the leucocytes. The amount of cytolysis increases regularly as the amount of the added lysin is increased, and sufficiently large quantities of saponin, sodium taurocholate, sodium glycocholate, or sodium oleate are capable of virtually abolishing the O₂ consumption altogether. At the same time, it can be shown that a lysin such as saponin is used up in combining with the white cells in much the same way as it is used up in combining with red cells, and the reduction in oxygen consumption appears to be roughly proportional to the amount so combined. The action of these lytic substances on white cells, in fact, is very similar to their action on red cells, due allowance being made for the fact that the cytolysis of the white cell is probably not an all-or-none process like hemolysis. White cell respiration is also depressed in hypotonic solutions, the respiration being virtually linear with the tonicity.     

THE PROLYTIC LOSS OF K FROM HUMAN RED CELLS

The prolytic loss of K., i.e. the loss of K which takes place from red cells exposed to hypolytic concentrations of lysins, has been measured in systems containing distearyl lecithin, sodium taurocholate, sodium tetradecyl sulfate, saponin, and digitonin, by means of the flame photometer. The lysins are added in various concentrations to washed red cells from heparinized human blood, and the K in the supernatant fluids is determined after various intervals of time and at various temperatures. The prolytic loss K_p is compared in every experiment with the loss K_s into standard systems containing isotonic NaCl alone, with no lysin. The losses K_s and K_p increase with time, so that new steady states are approached logarithmically. The values of K_p which correspond to the new steady states depend on the lysin used, being greatest with taurocholate and smallest with digitonin. The temperature coefficient of the loss is positive, and the extent and course of the losses have no apparent relation to the prolytic shape changes. In systems in which the loss of K is appreciable, it can be inhibited by the addition of plasma or of either cholesterol or serum albumin. Of these two substances, even when used in quantities which have an approximately equal effect in inhibiting hemolysis, serum albumin is much the more effective. Just as the prolytic loss of K occurs without the loss of any Hb, so in concentrations of lysin sufficient to produce hemolysis the loss of K, expressed as a percentage of the total red cell K, increases much more rapidly with lysin concentration than does the loss of Hb expressed as a percentage of the total Hb. The explanation of these relations depends on whether the loss of K is treated as being all-or-none in the case of the individual cell or as being the result of the loss of part of the K from all of the cells. This point has still to be decided.     

Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of G_m2 ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem. 55, 315–324; Hechtman & Leblanc (1977) Biochem. J. 167, 693–701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of G_m2 ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-β-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of G_m2 ganglioside decrease the K_m for G_m2 ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and G_m2 ganglioside was obtained by comparing gel-filtration profiles of ³H-labelled G_M2 ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and G_M2 ganglioside is the preferred substrate for enzymic hydrolysis.     

Changes from High Potassium (HK) to Low Potassium (LK) in Bovine Red Cells

Red cells of newborn calves contain 105–110 mmole K⁺ and 1–5 mmole Na⁺ per liter of cells. As the animals age the K⁺ content decreases to a value of 25–30 mmole/liter of cells after about 60 days. At approximately the same time, the sodium content reaches a value of 60–70 mmole/liter. The time required for half change (t_½) is 35–37 days for both Na⁺ and K⁺. The activity of (Na + K)-adenosine triphosphatase (ATPase) and the influx of K⁴² and Rb⁸⁶ into the red cells are high at birth and are reduced to 5 and 15% of their original values, respectively, in mature animals. t_½ for both is of the order of 30–35 days. The membrane Mg-ATPase activity is also high at birth and is reduced with a t_½ of 28–32 days to a final value of about 20% of its activity at birth. Separation of red cells according to their age showed that, in animals at the age of transition, newly formed red cells contain a higher K/Na ratio and a higher active transport capacity than older red cells of the same animal. It is suggested that the changes observed are a reflection of the average age of the red cell population as the animal grows.     

Regional Arterial Infusion with Lipoxin A4 Attenuates Experimental Severe Acute Pancreatitis

Objective

Investigate the therapeutic effect of regional arterial infusion (RAI) with Aspirin-Triggered Lipoxin A₄ (ATL) in experimental severe acute pancreatitis (SAP) in rats.

Materials and Methods

SAP was induced by injection of 5% sodium taurocholate into the pancreatic duct. Rats with SAP were treated with ATL (the ATL group) or physiological saline (the SAP group) infused via the left gastric artery 30 min after injection of sodium taurocholate. The sham group was subjected to the same surgical procedure, though without induction of SAP. Serum levels of amylase, phospholipase A2 (PLA2), interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured at 12 and 24 h after induction of SAP. Ascitic fluid, the pancreatic index (wet weight ratio) and myeloperoxidase (MPO) levels in the pancreas were determined and histopathological findings were evaluated. The expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), NF-κB p65, and heme oxygenase-1 (HO-1) in the pancreas were estimated by immunofluorescence and western blot, respectively.

Results

ATL rats had lower serum levels of TNF-α, IL-1β, and IL-6 (P<0.01), PLA₂ (P<0.05), and amylase levels (P<0.05) studied as compared with the SAP group. The pancreatic index in the ATL group decreased only at 24 h as compared with the SAP group (P<0.05). The histopathological findings and MPO levels in the pancreas significantly decreased in the ATL group as compared to the SAP group (P<0.05 and P<0.01, respectively). Immunofluorescence and western blot showed that ATL attenuated the expression of NF-κB p65, ICAM-1 and PECAM-1 in the pancreas, and increased the expression of HO-1 in SAP animals.

Conclusions

We demonstrated that RAI with ATL attenuated the severity of experimental SAP, maybe achieved by improving the expression of HO-1, and down-regulating the NF-κB signaling pathway, with decreased expression of ICAM-1 and PECAM-1 and reduced generation of pro-inflammatory cytokines.     

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be µ_H = 5 x 10^–5 cm./sec.     

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na⁺]_in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H⁺) or sodium ions (Na⁺), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H⁺- and Na⁺-driven components that functions as a sodium motor. Changing external sodium concentration ([Na⁺]_ex) in the range 1–85 mM resulted in changes in [Na⁺]_in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na⁺]_in and [Na⁺]_ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.     

Antioxidant,antimicrobial, toxicity and analgesic properties of ethanol extract of Solena amplexicaulis root

Background

This study was subjected to investigate different pharmacological properties of ethanol extract of Solena amplexicaulis root.

Results

The extract contains flavonoid, alkaloid, saponin and steroid compounds. The extract exhibited excellent antioxidant activity in DPPH radical scavenging activity. The extract also showed potent activity in brine shrimp lethality bioassay. The LC₅₀ value was found to 44.677 μg/ml. The extract showed better anti-bacterial activity against gram-negative bacteria. In antifungal assay, the maximum 79.31% of anti-mycotic activity was observed against Aspergillus ochraceus while minimum 44.2% against Rhizopus oryzae. MIC value ranged between 1500–3000 μg/ml. The extract was found moderately toxic with a 24-hr LD₅₀ value of 81.47 mg/kg in Swiss albino mice. The degree of inhibition by the ethanolic extract of the root was found less than that of standard analgesic drug diclofenac sodium. The extract also showed moderate anti-inflammatory and antinociceptive activity and anti-diabetic property. Reducing power of the extract was comparable with standard ascorbic acid. Moderate in vitro thrombolytic activity, lipid peroxidation inhibition property, metal chelating ability and stress-protective activity was also observed.

Conclusion

Ethanol extract of Solena amplexicaulis root can be valuable for treatment of different diseases.     

On the formation of calcium(II) taurocholate aggregate species in aqueous solution

Abstract

The influence of the presence of calcium(II) ions in solutions containing sodium and taurocholate ions at 25°C and in 0.5 mol dm^?3 N(CH₃)₄Cl as the constant ionic medium was studied. The composition and existence range of aggregates formed by taurocholate sodium and calcium(II) were investigated by means of two different procedures. First, the increasing calcium oxalate solubility due to the presence of taurocholate ions was studied as a function of the taurocholate, sodium and hydrogen ions. The free concentration of sodium and hydrogen ions was determined in solutions equilibrated with solid calcium(II) oxalate. After filtration, the concentration of calcium(II) (by atomic absorption spectro-photometry) and that of oxalate were also determined. In the second approach, electromotive force measurements carried out in solutions containing taurocholate, sodium and calcium(II) ions provided hydrogen and sodium ions free concentrations. The results from both procedures could be explained by assuming the presence of aggregates of different composition with the participation of sodium, calcium(II) and taurocholate ions, depending on the concentration of the reagents. No protonated species were present in appreciable concentrations. All the species found have even anion aggregation numbers. A strong analogy with the composition of sodium taurocholate and glycocholate is observed, while a comparison with sodium deoxycholate, glycodeoxycholate and taurodeoxycholate shows wide differences.     

Mass Transfer in the Cornea: II. Ion Transport and Electrical Properties of a Series Membrane Tissue

The electrical and active transport properties of isolated rabbit cornea are investigated by computer experimentation. The tissue is modeled as a series membrane system and the passive ion fluxes through it are described by the frictional formulation of irreversible thermodynamics. From short-circuit current (SCC) data, it is found that the epithelial sodium pump rate (P) is not appreciably changed when much of the sodium in the solution bathing the anterior corneal surface (concentration = c₁₁) is replaced by choline, with choline-free medium posteriorly. Simulations of open-circuited corneas, using the mean P computed from the SCC data, yield corneal and stromal potentials in agreement with experiment. The stromal fluid is calculated to become more hypotonic as c₁₁ is diminished, a result consistent with posttest measurements of the sodium content of experimental stromata. The apparent decrease in “bound sodium” which accompanies the reduction of c₁₁ is a result of the associated changes in steady stromal hydration; the epithelial sodium pump does not contribute to corneal deturgescence. The inclusion of a simple epithelial structure in the computations changes the value of P but affects neither its constancy nor the calculated behavior of the cornea under open-circuit conditions. A general algebraic relation among pump rates and ion fluxes in short-circuited series membrane systems bathed in complex media is derived and used to construct a relation between P and SCC for the cornea. This equation yields pump rates in good agreement with the computer results and is used to show that (a) P is independent of c₁₁ if d(SCC)/dc₁₁ is a constant related to the over-all corneal permeability to sodium, and (b) a Lineweaver-Burke plot of 1/SCC vs. 1/c₁₁ can appear to be linear at constant P.     

Two Subpopulations of Listeria monocytogenes Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (ΔpH), and the other consisted of cells that maintained ΔpH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate ΔpH for 90% of the cell population (ED₉₀) was predicted. ED₉₀ increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED₉₀ decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED₉₀. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.     

Cytosol-Membrane Interface of Human Erythrocytes: A Resonance Energy Transfer Study

The resonance energy transfer from donors embedded in the membrane of erythrocytes to the cytosol hemoglobin has been measured by comparing the donors' fluorescence decay in ghosts and in intact cells. A series of n - (9-anthroyloxy) stearic acids (n-AS) (n = 2, 6, 9, 12) and similar probes were used as donors, and their locations within the outer leaflet of the phospholipid bilayer were determined from their average efficiency of energy transfer, <T>. The energy transfer data for several membrane probes were analyzed according to a simple semiempirical model, in which the heme acceptors are assumed to form a semiinfinite continuum beyond a plane, whose normal distance (d) from particular donors may be determined if the heme density in the cytosol boundary layer is known. The hemoglobin concentration in the erythrocytes was varied by suspending the cells in buffers of different ionic strengths. This made it possible to study the ionic strength dependence of the heme concentration averaged over the cell (h_c), as well as that in the boundary layer (h_b). Both level off above approximately 600 mosM, as does the ratio h_b/h_c. By using the maximum heme concentration that can be obtained in osmotically shrunken cells as a limiting value, h_b is estimated to be 17 mM or less, under physiological conditions; and from the measured <T> for various probes, the distance d was found to range from 40 Å for 2-AS to 31 Å for 12-AS and 26 Å for 9-vinyl anthracene (9-VA). It is concluded that the hydrophobic probe 9-VA is located near the center of the phospholipid bilayer and that the cytosol hemoglobin is in contact with the inner membrane surface, or nearly so. This conclusion is valid for oxy- and deoxy-hemoglobin, and is shown to be independent of several systematic errors that might arise from the simple assumptions of the model used. The steady-state fluorescence anisotropy of the probes was found to decrease as they approach the bilayer's central plane. The methodology developed here may be used to extend studies of cytosol membrane interactions in ghost systems to intact cells, and is useful in the investigation of the morphology of normal and pathological intact erythrocytes.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

D,L-Sulforaphane Loaded Fe3O4@ Gold Core Shell Nanoparticles: A Potential Sulforaphane Delivery System

A novel design of gold-coated iron oxide nanoparticles was fabricated as a potential delivery system to improve the efficiency and stability of d, l-sulforaphane as an anticancer drug. To this purpose, the surface of gold-coated iron oxide nanoparticles was modified for sulforaphane delivery via furnishing its surface with thiolated polyethylene glycol-folic acid and thiolated polyethylene glycol-FITC. The synthesized nanoparticles were characterized by different techniques such as FTIR, energy dispersive X-ray spectroscopy, UV-visible spectroscopy, scanning and transmission electron microscopy. The average diameters of the synthesized nanoparticles before and after sulforaphane loading were obtained ∼ 33 nm and ∼ 38 nm, respectively, when ∼ 2.8 mmol/g of sulforaphane was loaded. The result of cell viability assay which was confirmed by apoptosis assay on the human breast cancer cells (MCF-7 line) as a model of in vitro-cancerous cells, proved that the bare nanoparticles showed little inherent cytotoxicity, whereas the sulforaphane-loaded nanoparticles were cytotoxic. The expression rate of the anti-apoptotic genes (bcl-2 and bcl-x_L), and the pro-apoptotic genes (bax and bak) were quantified, and it was found that the expression rate of bcl-2 and bcl-x_L genes significantly were decreased when MCF-7 cells were incubated by sulforaphane-loaded nanoparticles. The sulforaphane-loaded into the designed gold-coated iron oxide nanoparticles, acceptably induced apoptosis in MCF-7 cells.     

Spatial Distribution of Potential in a Flat Cell: Application to the Catfish Horizontal Cell Layers

An analytical solution is obtained for the three-dimensional spatial distribution of potential inside a flat cell, such as the layer of horizontal cells, as a function of its geometry and resistivity characteristics. It was found that, within a very large range of parameter values, the potential is given by [Formula: see text] where r = ρ/ρ₀, = z/ρ₀, ρ = (R_i/R_m)·ρ₀, δ = h/ρ₀; K is a constant; J is the assumed synaptic current; ρ, z are cylindrical coordinates; ρ₀ is the radius of the synaptic area of excitation; h is the cell thickness; and R_i, R_m are the intracellular and membrane resistivities, respectively. Formula A closely fits data for the spatial decay of potential which were obtained from the catfish internal and external horizontal cells. It predicts a decay which is exponential down to about 40% of the maximum potential but is much slower than exponential below that level, a characteristic also exhibited by the data. Such a feature in the decay mode allows signal integration over the large retinal areas which have been observed experimentally both at the horizontal and ganglion cell stages. The behavior of the potential distribution as a function of the flat cell parameters is investigated, and it is found that for the range of the horizontal cell thicknesses (10-50 μ) the decay rate depends solely on the ratio R_m/R_i. Data obtained from both types of horizontal cells by varying the diameter of the stimulating spot and for three widely different intensity levels were closely fitted by equation A. In the case of the external horizontal cell, the fit for different intensities was obtained by varying the ratio R_m/R_i; in the case of the internal horizontal cell it was found necessary, in order to fit the data for different intensities, to vary the assumed synaptic current J.     

Direct measurement of turgor and osmotic potential in individual epidermal cells : independent confirmation of leaf water potential as determined by in situ psychrometry

The pressure probe, which is routinely used to measure the turgor potential (Ψ_p) of individual epidermal cells in Tradescantia virginiana (L.), has also been used to sample small volumes of vacuolar fluid from these same cells (as low as 0.02 nl) for measurement of cellular solute (osmotic) potential (Ψ_s) in a micro freezing point osmometer. The water potential components Ψ_p and Ψ_o have been used to calculate the total water potential of individual epidermal cells (Ψ_cell) which has then been directly compared to the total leaf water potential (Ψ_leaf) measured psychrometrically. The relation of Ψ_leaf and Ψ_cell to leaf transpiration indicates that in T. virginiana, a relatively straightforward relation exists between the level of water flow through the leaf tissue, and the ΔΨ within the leaf, between two points along the water flow pathway. Substantial agreement was found between the two independent, in situ methods of measuring Ψ when extrapolated to zero transpiration conditions. These results are discussed with respect to the thermodynamics of water transport in plant tissues.     

COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S'' was delayed, and in this property, streptolysin S'' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S'' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S'', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase.