Cryosalts: suppression of ice formation in macromolecular crystallography.

Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

Location along the muscle's length is a determinant of myofibril size.

In a previous study of myofibril size in 'Pale' (fast-twitch-glycolytic) fibers of rabbit extraocular muscle (EOM), it was found that individual long Pale fibers demonstrate a substantial increase in the size of myofibril profiles from their proximal to their distal halves (Davidowitz et al., 1996b). That finding raised the question of whether such proximal-to-distal increase of myofibril size in the Pale fibers is determined by: (1) longitudinal position within the individual muscle fibers themselves or (2) location along the length of the muscle as a whole? This question was tested in the present study by comparing the original group of long Pale fibers, which extend the full length of the muscle, with two groups of short Pale fibers, which are respectively confined to the proximal and distal halves of the muscle. It was found that (a) in the proximal half of the muscle, the short fibers and the adjacent portions of the long fibers have the same smaller size of myofibrils, and (b) in the distal half of the muscle, the short fibers and the adjacent portions of the long fibers have the same larger size of myofibrils. This finding indicates that the proximal-to-distal increase of myofibril-profile size in these EOM Pale fibers is determined by location along the length of the muscle as a whole, and is not related to longitudinal position within the individual fibers themselves.

The polymer basis of kinetics and equilibria of enzymes: the accessible-volume origin of entropy changes in a class Abeta-lactamase.

The occurrence of enzymatic catalysis, as for any chemical reaction, depends critically upon close contact of the reactants, since making/breaking of bonds occurs over distances of about 0.2 A. Unlike small molecules, each enzyme molecule acts as an ordered solvent and reactant. Each group important to the enzyme reaction interacts with the substrate, then moves away, and subsequently binds another substrate. In other words, the group undergoes round trips in structure. For a round trip, the thermochemical state functions deltaG, deltaH, deltaS, etc., are zero. As a consequence, control of the binding of substrate must reside in the nonbinding conformations of the polymer since they govern the different fractions of time the macromolecule is in the correct conformation for bonding. Applying standard macromolecular models to the enzymes suggests that the majority of free energy for an enzyme reaction resides in the enzyme structure as an entropic contribution. Enthalpic contributions come from bond formation with the substrates and substrate structural changes. Further, it is shown that the molecular mechanisms that can effect binding and allosteric control fall into only three classes. Three x-ray structures of class A beta-lactamases (native, mutant, and with substrate) show the individual binding groups at the active site change their accessible volumes depending on substrate binding and mutant form. From these volume differences, the deltaS of reaction is calculated. The x-ray-derived deltaG = - TdeltaS matches the deltaG = -RT ln k1 from changes in rate constants for the same set of beta-penicillinases.

Cell growth patterns and lens geometry: a quantitative study from three-dimensional reconstructions.

The growth of the lens of the sea lamprey, Petromyzon marinus, was studied over the 5 years of larval development. Whole lenses (25) and Golgi-impregnated cells (393) were reconstructed with computer-assisted microscopy. Several cellular geometric parameters (length, width, curvature, surface, volume, shape) were correlated with the position of the cell's base on the lens capsular perimeter. Based on these correlations, the cells formed four groups that correspond to the central anterior, germinative, transitional and cortical fiber zones. A fifth zone, containing nuclear fiber cells, never stained. Lens growth is exponential during the 5 years. The anterior epithelium increases in size and in cell number by cell growth and division. The posterior mass increases in cell number by recruitment and increases in size by cell growth. A model is proposed to account for the size and shape of the lens based upon the coupling of anterior and posterior growth patterns. Four zonal boundaries are defined by changes in cell growth patterns. With growth, cells are subsumed into adjacent zones and zonal boundaries move away from the lens center. We find no support for the suggestion that cells migrate centrally.

Myofibril size variation along the length of extraocular muscle in rabbit and rat. I: orbital layer.

It is generally assumed that a muscle fiber is structurally uniform along its length. That assumption is not consistent with the observed variation of myofibrillar profile size along the length of both singly innervated fibers (SIFs) and multiply innervated fibers (MIFs) in the orbital (outer) layer of extraocular muscle (EOM). Muscle fibers were reconstructed in serial sections along the orbital layer of rabbit and rat EOM. For both the SIFs and MIFs, myofibril profile size was smallest (narrowest) near the endplate. In the SIFs of rat, for example, the myofibril profiles were 28% wider at a distance of 1.5 mm from the endplate than at the endplate itself. Measures of profile size included the mean intercept length and the mean shortest path from test points within the profile to the profile boundary. The possible effect of sarcomere length variation was controlled by normalizing the myofibrillar profile size data to a constant spacing of the myosin filament lattice. This morphometric approach was also used to quantify the further increase of profile size that occurs in the end portions of the orbital MIFs where the myobrillar organization is typically ill-defined.

Myofibril size variation along the length of extraocular muscle in rabbit and rat. II: global layer.

Systematic variation of myofibril profile size was observed along the length of both singly innervated fibers (SIFs) and multiply innervated fibers (MIFs) of the global (inner) layer of extraocular muscle (EOM). These findings contrast with the assumption that global layer fibers of EOM are structurally uniform along their length. Muscle fibers were reconstructed in serial sections along the global layer of rabbit and rat EOM. Long fibers of the 'Pale' SIF (fast twitch glycolytic) and the MIF (tonic) populations were sampled by EM in both proximal and distal portions of the muscle. In rabbit, myofibril size of the Pale SIFs showed a proximal-to-distal increase of 28% whereas the MIFs showed a proximal-to-distal decrease of 10%. In rat, these two fiber populations showed analogous smaller changes. Measures of profile size included the mean intercept length and the mean shortest path from test points within the profile to the profile boundary. The possible effect of sarcomere length variation was controlled by normalizing the measures to a constant spacing of the myosin filament lattice.

NMR imaging with shorted coaxial line probes.

At frequencies below 1 GHz, resonant sections of coaxial lines have long been used in CW-Electron Paramagnetic Resonance (EPR) with the sample placed at the position of maximum B1 at a short circuited end. Here, we show that because of the excellent separation of the B and E fields, the shield of the line can be removed in the region of the truncated end without greatly perturbing the RF properties of the line. The open region of the shield provides an aperture for local imaging in MRI. The B1 fields can be shaped by contouring the inner conductor and outer shield, and the image aperture is controlled by the shape of the shield cutout. The shield opening can range from a narrow longitudinal slit up to a full 360 degrees section that has only a few conducting strips of the shield remaining. Imaging with probes having shield diameters from 2 mm to 10 cm have been demonstrated. For imaging the useful depth is limited to approximately three to four times the probe's outer radius. Alternately, a relatively sharp cutoff at only a mm depth can be obtained by controlling the region of the shield removed, the RF power applied, and the probe diameter. The probes described here can be resonant or nonresonant. Because of the inherent broad bandwidth of the nonresonant truncated line probes, they have the potential for use in FT-EPR and FT-EPR imaging as well as other applications that require minimizing dead times.

A novel topical probe for MRI: the flat, truncated line probe.

The construction and imaging characteristics of flat, truncated line probes (FTLPs) are described here. These probes illustrate a novel design of local probes for magnetic resonance imaging, with four major differences from conventional loop surface probes: (1) The B1 fields are directed perpendicular to the usual loop probes' direction. (2) The RF fringe electric fields are inherently shielded, which allows reduced loading from electrically lossy samples. (3) The homogeneity across the plane of the probe can be adjusted locally. And (4) when not used with tuning and matching circuits, a probe's local impedance can be set to match the RF line impedance. The probes are, in essence, a single loop significantly flattened with the outside conductor (away from the imaged object) wider than the inside conductor (against the imaged object). The probes are shaped so as to provide a homogeneous signal across the plane area of the probe. The signal intensity drops off faster than a loop of the same size. With a minimally loading phantom, along the midline normal to the surface, the S/N at the surface region is approximately 20% greater than a commercial probe (Phillips R2) of the same area dimensions, while at 5 cm depth, the S/N is lower. However, when used for imaging a body--again along the midline normal to the probe--the S/N at 5 cm depth is equal, and rises to approximately twice that of the Philips R2 probe at the surface.

Steady-state kinetics of solitary batrachotoxin-treated sodium channels. Kinetics on a bounded continuum of polymer conformations.

The underlying principles of the kinetics and equilibrium of a solitary sodium channel in the steady state are examined. Both the open and closed kinetics are postulated to result from round-trip excursions from a transition region that separates the openable and closed forms. Exponential behavior of the kinetics can have origins different from small-molecule systems. These differences suggest that the probability density functions (PDFs) that describe the time dependences of the open and closed forms arise from a distribution of rate constants. The distribution is likely to arise from a thermal modulation of the channel structure, and this provides a physical basis for the following three-variable equation: [formula; see text] Here, A0 is a scaling term, k is the mean rate constant, and sigma quantifies the Gaussian spread for the contributions of a range of effective rate constants. The maximum contribution is made by k, with rates faster and slower contributing less. (When sigma, the standard deviation of the spread, goes to zero, then p(f) = A0 e-kt.) The equation is applied to the single-channel steady-state probability density functions for batrachotoxin-treated sodium channels (1986. Keller et al. J. Gen. Physiol. 88: 1-23). The following characteristics are found: (a) The data for both open and closed forms of the channel are fit well with the above equation, which represents a Gaussian distribution of first-order rate processes. (b) The simple relationship [formula; see text] holds for the mean effective rat constants. Or, equivalently stated, the values of P open calculated from the k values closely agree with the P open values found directly from the PDF data. (c) In agreement with the known behavior of voltage-dependent rate constants, the voltage dependences of the mean effective rate constants for the opening and closing of the channel are equal and opposite over the voltage range studied. That is, [formula; see text] "Bursts" are related to the well-known cage effect of solution chemistry.

The developing visual system and metamorphosis in the lamprey.

Metamorphosis of the sea lamprey, Petromyzon marinus, is a true metamorphosis. The larval lamprey is a filter-feeder who dwells in the silt of freshwater streams and the adult is an active predator found in large lakes or the sea. The transformation usually occurs in the fifth or sixth year of life. Enlargement of the eye has been long accepted as a distinctive indication of metamorphosis in the sea lamprey, but it had been thought that this was because eye development in the larva was arrested after the formation of only the small central region. Recent studies indicate that all of the retina begins its development in the larva and that ganglion, amacrine, and horizontal cells differentiate in the peripheral retina of the larva. Retinal development is arrested during the premetamorphic period, to be resumed during metamorphosis. Metamorphic contributions include the differentiation of photoreceptor and bipolar cells. With the early appearance of ganglion cells, retinal pathways to the thalamus and tectum are established in larvae, as is a centripetal pathway. Tectal development spans the larval period but a spurt in tectal growth and differentiation is correlated with the completion of the retinal circuitry late in metamorphosis. The metamorphic changes in retina and tectum complete the functional development of the visual system and provide for the adult lamprey's predatory and reproductive behavior.

Neural differentiation in the retina of the larval sea lamprey (Petromyzon marinus).

The peripheral retina of the sea lamprey develops in a 5-year-long process in which only certain neurons differentiate each year. The growth of cell layers, the differentiation of the neurons, and the morphology of their dendrites and axons were studied with normal, HRP, and Golgi preparations. Ganglion cells are differentiated in 3-year-old larvae, amacrine and horizontal cells in 4-year-old larvae, photoreceptor cells in stage I transformers, and bipolar cells in stage III transformers. Each new development is expressed as a radial gradient of differentiation. As a result of this protracted and stepped process, lamprey retinal neurons, particularly ganglion cells, differentiate in the absence of other cells to which they will ultimately be connected and may express their individual genetic programs more fully than in other vertebrate retinas. This could account for the unusual relationship of the ganglion cell, inner plexiform, and optic nerve layers and for the very high ratio of displaced to orthotopic ganglion cells.

The effects of n-pentane on voltage-clamped squid nerve sodium currents. A reinterpretation using kinetics of ordered systems.

The sodium-current voltage-clamp data of Haydon and Kimura obtained on squid nerves treated with n-pentane (J. Physiol. 312 (1981) 57) are fitted with a previously described model (K.A. Rubinson, J. Physiol. 281 (1978) 14P; Biophys. Chem. 15 (1982) 245). The apparently complex action of the perturbant can be interpreted as due to a shift in shielding of the applied potential jumps, a change in channel conductivity, and an increase in the rate constant of channel shutoff. The shift in shielding due to n-pentane is found to be quantitatively the same for variables describing both kinetic and equilibrium quantities, which are independent. The transmembrane sodium potential remains unchanged, however.

Closed channel-open channel equilibrium of the sodium channel of nerve. Simple models of macromolecular equilibria.

The consistency of an electrodiffusion kinetics to describe the time-dependent opening of sodium channels of nerve suggests that motions over relatively long distances (on the atomic scale) are involved in the equilibrium as well. As a result, it is expected that a relatively large fraction of possible macromolecular conformations are unreactive. An equilibrium constant between locally reactive forms and the unreactive conformations is introduced. The consequences of this formalism is investigated in a square well potential, a harmonic potential, and a system consisting of two harmonic potentials with different spatial extents. The limits of knowledge from Nernstian behavior are shown. As an alternative to the Nernstian analysis, the experimental data of the sodium channel's quasi-equilibrium - the probability of the channel's being open as a function of voltage - can be described as resulting from motion caused by an electric field on a charge which is confined by a harmonic potential. A force constant is found from this analysis. (Such Hookian force constants cannot be found from spectroscopic experiments in condensed systems where the large-displacement vibrations are overdamped and, hence, spectroscopically unobservable). From the force constant, an approximate value of the Young's modulus can be calculated. The modulus' value falls in the range for rubber. As for rubbers, the restoring force is, then, expected to be mostly entropic rather than enthalpic in origin. Using the appropriate theory for linear chains of rubber and the Young's modulus, the approximate length of the chain causing the rubber-like force is calculated. The result is found to be near the length suggested for the hydrophilic chains that connect transmembrane sections of the sodium channel.

Efferent projections of the torus semicircularis to the medulla of the tadpole, Rana catesbeiana.

Horseradish peroxidase injections into the medulla of tadpoles demonstrate, by back-filling, efferent neurons in the torus semicircularis which project to the ipsilateral superior olive. This projection, as well as one to the vicinity of the efferent neurons of the contralateral VIII and lateral line nerves, is confirmed by tracing anterograde transport following HRP injections into the torus semicircularis. These efferent neurons, located primarily in the principal nucleus of the torus within the terminal field of the projection from the superior olive, represent the only reported descending path to the superior olive and efferent nuclei in a non-mammalian vertebrate.

The acoustic and lateral line nuclei are distinct in the premetamorphic frog, Rana catesbeiana.

The transition from aquatic to terrestrial hearing in the frog occurs during metamorphosis and during the disappearance of the lateral line system. The coincidence in time of these two processes and morphological similarities between the acoustic and lateral line systems has led to the suggestion (Larsell, '34) that the lateral line nuclei are transformed into the acoustic nuclei. The relation between the acoustic and lateral line systems was investigated by studying the distribution of primary afferents, the dendritic patterns of the cells in the primary nuclei, and the development of the nuclei in the premetamorphic bullfrog, Rana catesbeiana. The posterior and anterior lateral line roots distribute to a neuropil located medial to the dorsal medullary nucleus. Horseradish peroxidase (HRP) injections into the contralateral tegmentum fill cells in the periventricular region whose dendrites ramify within the neuropil. These cells constitute the lateral line nuclei. The amphibian and basilar papillary roots of the acoustic system distribute to the more lateral nuclear region. The dendrites of these cells arborize within the nucleus and not in the lateral line neuropil. The dorsal medullary nucleus is, therefore, the acoustic nucleus (AcN). [3H]-thymidine labeling reveals that newly generated cells occupy the AcN within a few hours of their formation throughout the period when anatomical analysis shows the parallel growth and diminution of the lateral line neuropil and nuclei. This study indicates that the lateral line and acoustic systems are morphologically independent at the level of the primary afferents and primary nuclei throughout early development.

The transport and accumulation of oxyvanadium compounds in human erythrocytes in vitro.

Metavanadate, at physiologic pH the oxyanion form of pentavalent vanadium, is a potent reversible inhibitor of the sodium pump. Vanadium must enter cells to inhibit the sodium pump, and metavanadate may be converted to an inactive form inside of cells. Because of these factors and the complex inorganic chemistry of vanadium, we examined the kinetics of vanadium uptake and accumulation in normal erythrocytes in vitro at 37 degrees C. The kinetics of vanadium influx, efflux, and accumulation in erythrocytes in Tris-buffered, isotonic salt medium were fit closely by a model with vanadium in two possible oxidation states and with the vanadium permeating between two compartments. The equation for this model is: (formula: see text) in which subscripts i and o signify "inside" and "outside" the cells, respectively, and k1, k-1, and k2 are rate constants. 48V or EPR of vanadium(IV) gave similar estimates of the concentrations of the components. The k1 was 0.37 +/- 0.06 (S.E.M.) min-1 and k2 was 0.04 +/- 0.01 min-1. In control Tris-medium, k1 exceeded k-1 by a factor of 1.8. After 120 min of incubation in media with initial concentrations of 1, 10, or 100 microM vanadium(V), the total intracellular vanadium concentration exceeded that in the bath 4.5 to 18-fold. Vanadium influx was not appreciably changed by variations of external sodium or glucose levels. The k1 and k-1 were inversely related to external pH over the range 6.5 to 8.2. High O2 tension (95% to 100% O2) caused a decrease in k2, and the lipophilic oxidant, cumene hydroperoxide, accelerated the loss of accumulated vanadium from the cells, indicating that the k2 step represents reduction of vanadium(V) to vanadium(V) within the cells. On the basis of these studies we suggest that the intracellular concentration of vanadium(V), the inhibitor of the sodium pump, is determined by the combined effects of the rate of vanadium influx (dependent on the extracellular concentration of free metavanadate), the rate of vanadium efflux, and the rate of conversion of vanadium(V) to vanadium(IV).

The sodium currents of nerve under voltage clamp as heterogeneous kinetics. A model that is consistent with possible kinetic behavior.

A model is presented which explains in Na+ currents of voltage-clamped nerve as resulting from a heterogeneous initiation of a sequential kinetic process. This is in analogy with the heterogeneity of the kinetics of other dielectric relaxations. The results suggest that: (1) The kinetic processes responsible for the voltage response occur within the membrane rather than at the surface; (2) The heterogeneity is due to simultaneous thermal diffusion and electric field-induced charge migration: (3) The slow turnoff upon prolonged depolarization is a voltage-independent, thermally controlled process; (4) The fast turnoff upon instantaneous repolarization is the reverse of the turning-on process. All the kinetic parameters depend on the transmembrane potential in accord with the possible behavior expected from activated-state theory. The diffusion coefficient of the charged species in the membrane as found from the data agrees with that found by photobleaching experiments on general proteins in membranes. The charge on the molecule responsible for the heterogeneous "gating' can be calculated unambiguously from the data.

HPLC separation and comparative toxicity of saxitoxin and its reaction products.

A chromatographic method was developed that was used to purify saxitoxin and separate it from its chemically modified products and the reagents used in the reactions. The separation time is about 10 minutes. Using differential-refractive-index detection, quantitation of the products (+/- 10%) can be done on 30-100 microgram of toxin. A simple bioassay with crab leg nerves in vitro was used in conjunction with the chromatography to determine, within a factor of two, the inhibition binding constants of saxitoxin and its products. The binding constant for saxitoxin at ambient temperature, 18-21 degree C, is Ki approximately 80 nM. The acid-hydrolysis product has Ki approximately 8 microM under the same conditions. The chemistry of saxitoxin was investigated using the chemical and bioassays.

Concerning the form of biochemically active vanadium.

A survey has been made of the descriptive chemistry of vanadium as it pertains to physiological environments. Taking into account the vanadium concentration, pH, coordinating ligands and chelates, presence of other cations, oxidation-reduction potentials and the kinetics of the various vanadium-containing species, the following suggestions are made. Free vanadium ions will be monomeric. Monomeric vanadium(V) and vanadium(IV) will each exist in a specific hydrated form. Extracellular vanadium will be in the vanadate, Vv, form. Intracellular vanadium will most likely be predominantly in the vanadyl, Viv, form. Both extracellular vanadium(V) and intracellular vanadium(IV) will be bound to bi- or tridentate ligands if at all. If oxidation-reduction processes involving vanadium are fast relative to transmembrane transport, the transmembrane potential will not be coupled to the Vv/Viv Nernstian potential.

Voltage dependencies of slow (ms) chemical relaxations.

The exponential-relaxation rates (rate constant k) of relatively slow (ms) chemical reactions may change with applied potential. Three general cases are described which, by their dependencies on an externally applied voltage, should be experimentally separable. They are processes due to (1) permanent dipoles entirely in the field - - (d In k)/d V proportional, variant V(2); (2) surface charge change - - (d In k)/d V proportional, variant V; and (3) charge transport - - dk/d V proportional, variant V (or a delay proportional to 1/V. The general analysis presented may be useful in analyzing biological electrical processes.