Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

Effects of aging on the cerebral distribution of technetium-99m hexamethylpropylene amine oxime in healthy humans.

Some brain functions decline at a linear rate throughout adulthood. Others remain relatively stable until very late in the life cycle. This study characterized the effects of aging on the regional cerebral distribution of hexamethylpropylene amine oxime (HMPAO) in healthy human volunteers. The sample consisted of 26 men and 18 women with a mean age of 41.6+/-14.9 years (range: 19-73). Their past medical histories, physical examinations, and laboratory screening tests were normal. Single-photon emission tomography (SPET) scans of the brain were performed with a standardized acquisition and processing protocol on a triple-headed camera equipped with fan beam collimators. A 3-D restorative filter and a correction for uniform attenuation were applied before the images were reinterpolated in planes parallel to the line connecting the frontal and occipital poles. Mean counts per pixel were measured in multiple regions of interest (ROIs) within each hemisphere by custom fitting a set of templates to the images. The mean activity in each ROI was compared with the mean activity per pixel in the whole brain. Regression analyses were used to relate the activity ratios to age with both linear and nonlinear models. The relative concentration of radioactivity decreased significantly with age in most, but not all, gray matter structures. It increased in the white matter regions. The nonlinear model of aging fit the data significantly better than a straight line did. Most of the changes with age occurred during young adulthood. No further changes were detectable after the onset of middle age. The median breakpoint age at which the rate of change became negligible was 36.6 years. Aging significantly affects the relative uptake of HMPAO in healthy humans. It decreases in many gray matter regions and increases in most white matter regions. However, the changes do not appear to be linear. Most seem to occur during young adulthood before people reach their late thirties. The distribution then appears to remain relatively stable throughout middle age.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria.

The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO-cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed.

Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. Cytochromes aa3.

Using newer techniques of data collection that accumulate entire spectra at a series of discrete voltages and newer techniques of analysis that utilize the additional data, we have re-examined the redox behavior and corresponding difference spectra of redox centers responsible for the alpha absorbance features of cytochromes aa3 in beef heart mitochondria. Our analysis reveals three Nernstian components with Em values of 200, 260, and 340 mV with n values of 2, 2, and 1, respectively. The maximum alpha absorbance in the difference spectra for each of these species is located at 602, 605, and 607 nm respectively. Titrations in the presence of carbon monoxide led to the identification of the lowest voltage species as cytochrome a3. The Em of the carbon monoxide-liganded species was not raised. This is contrary to the result expected when a ligand has a much stronger affinity for the reduced form of a redox couple than the oxidized form. It is, however, consistent with a proton-pumping model of cytochrome oxidase in which the binding of ligand results in the dissociation of protons.

Thermodynamic and kinetic considerations of Q-cycle mechanisms and the oxidant-induced reduction of cytochromes b.

In coenzyme Q-cycles, it is proposed that one electron from the quinol reduces the Rieske iron sulfur center (Em approximately 280 mV) and the remaining electron on the semiquinone reduces cytochrome br (Em approximately -60 mV). The Em for the two-electron oxidation of the quinol is approximately 60 mV and therefore the reduction of cytochrome bT by quinol is not favorable. As the stability constant for the dismutation of the semiquinone decreases, the calculated Em for the Q/QH. couple is lowered to values below the Em of cytochrome bT. Contemporary coenzyme Q-cycles are based on the belief that the lower value for the Em of the Q/QH. couple compared to the Em for cytochrome bT means that the semiquinone is a spontaneous reducing agent for the b-cytochrome. The analysis in the paper shows that this is not necessarily so and that neither binding sites nor ionization of the semiquinone per se alters this situation. For a Q-cycle mechanism to function, ad hoc provisions must be made to drive the otherwise unfavorable reduction of cytochrome bT by the semiquinone or for the simultaneous transfer of both electrons to cytochrome bT and cytochrome c1 (or the iron sulfur protein). Q-cycle mechanisms with these additional provisions can explain the observation thus far accumulated. A linear path which is functionally altered by conformational changes may also explain the data.

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: passive membrane.

We describe the application of a popular and widely available electrical circuit simulation program called SPICE to modeling the electrical behavior of neurons with passive membrane properties and arbitrarily complex dendritic trees. Transient responses may be calculated at any location in the cell model following current, voltage or conductance perturbations at any point. A numbering method is described for binary trees which is helpful in transforming complex dendritic structures into a coded list of short cylindrical dendritic segments suitable for input to SPICE. Individual segments are modeled as isopotential compartments comprised of a parallel resistor and capacitor, representing the transmembrane impedance, in series with one or two core resistors. Synaptic current is modeled by a current source controlled by the local membrane potential and an "alpha-shaped" voltage, thus simulating a conductance change in series with a driving potential. Extensively branched test cell circuits were constructed which satisfied the equivalent cylinder constraints (Rall 1959). These model neurons were perturbed by independent current sources and by synaptic currents. Responses calculated by SPICE are compared with analytical results. With appropriately chosen model parameters, extremely accurate transient calculations may be obtained. Details of the SPICE circuit elements are presented, along with illustrative examples sufficient to allow implementation of passive nerve cell models on a number of common computers. Methods for modeling excitable membrane are presented in the companion paper (Bunow et al. 1985).

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: excitable membrane.

We present methods for using the general-purpose network analysis program, SPICE, to construct computer models of excitable membrane displaying Hodgkin-Huxley-like kinetics. The four non-linear partial differential equations of Hodgkin and Huxley (H-H; 1952) are implemented using electrical circuit elements. The H-H rate constants, alpha and beta, are approximated by polynomial functions rather than exponential functions, since the former are handled more efficiently by SPICE. The process of developing code to implement the H-H sodium conductance is described in detail. The Appendix contains a complete listing of the code required to simulate an H-H action potential. The behavior of models so constructed is validated by comparison with the space-clamped and propagating action potentials of Hodgkin and Huxley. SPICE models of multiply branched axons were tested and found to behave as predicted by previous numerical solutions for propagation in inhomogeneous axons. New results are presented for two cases. First, a detailed, anatomically based model is constructed of group Ia input to an alpha-motoneuron with an excitable soma, a myelinated axon and passive dendrites. Second, we simulate interactions among clusters of mixed excitable and passive dendritic spines on an idealized neuron. The methods presented in this paper and its companion (Segev et al. 1985) should permit neurobiologists to construct and explore models which simulate much more closely the real morphological and physiological characteristics of nerve cells.

Determination of the distribution of catalyst activity across a permeable membrane containing an immobilized enzyme. Indeterminacy of a functional approach to a structural problem.

Porous membranes were fabricated from collodion and impregnated with papain, inhomogeneously through the thickness of the membrane. These membranes were placed between reservoirs containing N-alpha-benzoyl arginineamide, a substrate for the enzyme papain. The progress of the reaction was monitored by sampling the reservoirs on each side for ammonia, a reaction product. From these data the diffusion coefficient, enzyme activity, and distribution of enzyme activity of the membrane were estimated. The limitations of this approach are discussed in the context of the analysis of biological transport systems.

Phase behavior of ganglioside-lecithin mixtures. Relation to dispersion of gangliosides in membranes.

Ganglioside GM1 and mixed brain gangliosides were mixed with 1-stearoyl-2-oleoyl lecithin (SOPC) and examined by differential scanning calorimetry as a function of ganglioside content and temperature. Low mole fractions of ganglioside GM1 and of mixed brain gangliosides are shown to be miscible with SOPC in the gel phase up to X = 0.3, with the possible exception of a small region of immiscibility for the mixed brain gangliosides system centered around X = 0.05. Above X = 0.3, the low-temperature phases demix into a (gel) phase of composition X = 0.3 and a (micellar) phase of composition X = 1.0. Above the endothermic phase transition temperature, no phase boundaries are discerned. It is pointed out that phase structures need to be determined in each domain delineated in the phase diagrams, and that cylindrical phases may exist at higher temperatures and intermediate compositions. The effects of addition of wheat germ agglutinin, which binds to ganglioside GM1, on a ganglioside GM1-SOPC mixture (X = 0.5), are described and interpreted in terms of partial demixing of ganglioside and lecithin. Behavior of the ganglioside-SOPC system is discussed with respect to the kinetics of cholera toxin action in lymphocytes, as well as to other physiological roles of gangliosides in membranes.

Regional ventilatory clearance by xenon scintigraphy: a critical evaluation of two estimation procedures.

Estimates of ventilatory clearance are usually made by inspecting xenon washout images. Quantitative computer procedures have been described that produce regional clearance rates, yet their accuracy is not well established. We define a mathematical model for scintigraphic ventilation data based on 96 clinical studies, and with this model we test the accuracy of two procedures used to estimate ventilatory clearance. The least-squares curve-fitting technique for both washin and washout data has the same accuracy as a modified Stewart-Hamilton method (A/H) that uses washout data alone. Both procedures demonstrate relative errors of less than 5% and coefficients of variation of 10-20% when regions with equilibrium count rates of 3 cps and clearance times between 10 and 90 sec are examined. Because the A/H procedure is preferred for its simplicity and speed, we analyze two of its main sources of error: early washin/washout termination and background activity. To measure regional ventilation by the A/H procedure, we recommend: (a) washin and washout periods at least three times the largest clearance time of clinical interest; b) a regional equilibrium count rate of at least 3cps; and c) a 25- to 50-sec average of the equilibrium count rate.

Hysteresis, oscillations, and pattern formation in realistic immobilized enzyme systems.

Hysteresis, oscillations, and pattern formation in realistic biochemical systems governed by P.D.E.s are considered from both numerical and mathematical points of view. Analysis of multiple steady states in the case of hysteresis, and bifurcation theory in the cases of oscillations and pattern formation, account for the observed numerical results. The possibility to realize these systems experimentally is their main interest, thus bringing further arguments in favor of theories explaining basic biological phenomena by diffusion and reaction.

Substrate inhibition kinetics in assemblages of cells.

The collective kinetic behavior of a linear array of cells containing a substrate inhibited enzyme is studied with a model in which each cell is considered a well-stirred compartment surrounded by a semipermeable membrane. At large values of a reaction-permeation modulus, wherein substrate access to interior cells is limited, plots of total reaction rate versus concentration of the external reservoir show sharp projections which correspond to dominant reaction occurring in a pair of symmetrically placed cells. Over prescribed ranges of reservoir concentration, multiple stable steady states can occur, some of which are characterized by asymmetric profiles of concentration and reaction rate across the array. A simple stability criterion is proposed and applied to arrays with arbitrary numbers of cells.