An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

Cellular immunity in the mouse. I. In vitro lymphocyte reactivity

The mixed lymphocyte culture (MLC) and antigen-mediated proliferative response represent important correlates to the in vivo phenomena of allograft rejection and delayed hypersensitivity. This study defines an in vitro model to measure mouse lymphocyte responsiveness to allogeneic cells, antigen (tuberculoprotein), and nonspecific mitogens. Results describe optimal cells concentration, time and conditions of culture. Optimal conditions include the use of high cell concentration, flat-bottomed vials, RPMI-1640 medium, and fresh human serum. Peripheral blood lymphocytes demonstrated greater proliferation than lymph node lymphocytes, which in turn demonstrated greater activity than splenic lymphocytes. Significant proliferation occurred in serum-free media, dialyzed against fresh serum and supplemented with hydrocortisone and carrier protein. The MLC response in the mouse appears dependent on multiple subpopulations of cells and on soluble substances produced by them.     

An interactive computer system for the analysis of cell lineages.

We have developed an interactive computer system for analysing cell lineage data. It can be utilized in studies of cell motility, cell division, cell differentiation, and cell aging. It has enabled us to document the heterogeneity of human foreskin fibroblasts in culture and to propose that loss of proliferative potential may mean that cells enter a state of differentiation which makes them unable to respond to mitotic stimulation. Our method, which enables us to apply immunological and cytochemical probes after recording the history of a cell lineage, should allow us to define precisely features which uniquely distinguish cycling from noncycling cells on an individual cell basis.     

Cellular immunity in the mouse. IV. Altered thymic-dependent lymphocyte reactivity in the chronic graft vs host reaction and leukemia virus activation.

The lymphocytes obtained from several F₁ strains undergoing chronic GVH reactions were studied for in vitro alterations of thymic-dependent lymphoid function. Spontaneous blastogenesis was increased. The in vitro response to nonspecific mitogenic stimuli (PHA and CON-A) and specific antigenic challenge (SRBC and allogeneic cells) was initially increased and subsequently impaired. The degree of alteration was related to the severity of the observed disease and dependent upon the F₁-parental combination employed. Thymic-dependent lymphocytes obtained from animals with GVH disease possessed the ability to suppress actively the response of normal mouse cells in vitro to various T-cell mitogenic stimuli and this suppressive activity was present in the supernatant culture fluid from such cells. The mechanism of this altered in vitro T-cell reactivity is not yet completely understood, but may in part be related to the immunologic activation of murine leukemia virus from mouse cells undergoing allogeneic stimulation.     

FACTORS INVOLVED IN THE USE OF ORGANIC SOLVENTS AS PRECIPITATING AND DRYING AGENTS OF IMMUNE SERA

1. In concentrations of 70 to 75 per cent the organic solvents methyl, ethyl, and propyl alcohols, and acetone cause complete precipitation of serum proteins and produce maximum loss in solubility. We have referred to this concentration range as the critical concentration. 2. As the concentration of the solvents is increased from about 75 per cent precipitation continues complete but loss in solubility progressively decreases until at all concentrations above about 87 per cent the precipitates formed at room temperature are completely soluble. 3. The degree of resolubility of the precipitates formed even in these high concentrations of the organic solvent decreases as the temperature is raised and as the duration of exposure is increased. 4. At 5°C. the precipitates formed in all concentrations of these organic solvents are completely resoluble. Also these solvents exert maximum precipitating effect at lower temperature. 5. Maximum precipitating effect by these organic solvents occurs at about pH 6.0 precipitation becoming progressively less as the pH value is altered either way from this point. 6. The more concentrated the serum, the greater the proportion of protein present that will be precipitated by any given concentrations of organic solvent. 7. A method for preparing dry immune sera has been given. Such dried sera have been extracted with a number of organic compounds without loss in solubility or antibody activity.     

Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin

Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin (K_d ≈ 2 nm), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μm riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin (K_m ≈ 12 μm), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate (K₃ ≈ 5 nm) but less avidly complexes riboflavin (K_d ≈ 0.6 μm), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

Rheology of Human Blood, near and at Zero Flow: Effects of Temperature and Hematocrit Level

Static normal human blood possesses a distinctive yield stress. When the yield stress is exceeded, the same blood has a stress-shear rate function under creeping flow conditions closely following Casson's model, which implies reversible aggregation of red cells in rouleaux and flow dominated by movement of rouleaux. The yield stress is essentially independent of temperature and its cube root varies linearly with hematocrit value. The dynamic rheological properties in the creeping flow range are such that the relative viscosity of blood to water is almost independent of temperature. Questions raised by these data are discussed, including red cell aggregation promoted by elements in the plasma.