Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

The authors examined the safety and pharmacokinetics of FK506, a new hepatically metabolized immunosuppressant, after single-dose intravenous (i.v.) infusion (20 micrograms.kg(-1) x 4 hours-1) and oral (80 micrograms/kg) administration in six nondialysis patients, aged 27 to 53 years, with chronic renal failure awaiting transplantation. A two-period, randomized, crossover study protocol was used with blood samples drawn for 72 hours after each dose and a washout period of 4 days. Whole-blood FK506 levels were determined using a standard, two-step, nonspecific enzyme immunoassay. There were no significant changes in vital signs, EKG, or complete laboratory test battery for any patient during the entire study period. No side effects were noted after i.v. or oral FK506 dosing. Mean +/- SD distribution half life was 0.9 +/- 0.2 hours, elimination half life (t1/2 beta) 33 +/- 8 hours, total body clearance (CL) 2.4 +/- 1.1 L/hour, and bioavailability 14 +/- 12%. There was no significant correlation between serum creatinine (Cr) and CL (r = 0.36) or between Cr and t1/2 beta (r = -0.30). It was found that FK506 is incompletely and erratically absorbed after oral administration and is rapidly distributed outside the blood compartment after IV dosing. An extended sampling period seems necessary to accurately characterize the slow elimination phase of FK506.

Sodium fluxes were measured in erythrocytes from three species of mammals. Unidirectional fluxes were slowest in swine RBCs (low sodium cells), fastest in dog RBCs (high sodium cells), and between these extremes in ox cells (intermediate level of internal sodium). In addition, effiux and influx in swine cells both correlated positively with intracellular sodium concentration between 12 to 49 #eq/ml. Tracer effiuxes in swine and beef cells were separated into three components: active transport, diffusion, and exchange diffusion. The last two also contributed to influx. Transport was greater in swine cells than in beef, while the leak was similar in both. Pump to leak ratios were about 91 for swine and 3 for beef, a difference that probably accounts for the higher cell sodium in the latter. Exchange diffusion was faster in beef ceils than in swine resulting in a larger tracer movement in beef. The exchange mechanism was temperature-sensitive, but was not inhibited by strophanthin. The unidirectional fluxes in canine cells were inhibited by low temperature, but they were sensibly unaffected by strophanthin. When placed in magnesium Ringer's solution (inhibits exchange diffusion in beef and swine cells) dog RBCs lost more than half of their internal sodium at a rate approximating the isotope flux in plasma or normal Ringer's solution. It was, however, not possible to separate the total tracer movement into pump, leak, and exchange.

The effects of verapamil and its optical isomers on the electrical and mechanical characteristics of single muscle fibers of Callinectes danae were studied. Verapamil (10-20 microng/ml) blocked the procaine- and TEA-induced spikes; the blockade was preceded by reduction in the rate of rise of the upstroke and increase in the duration of the action potentials. Inhibition of Ba-spikes required higher concentrations of verapamil (greater than 50 microng/ml). These concentrations reduced the amplitude of the normally occurring graded electrogenic membrane responses and reduced the rate of development of the current-induced tensions. With lower concentrations (10-30 microng/ml) verapamil enhanced the negative afterpotentials and the peak amplitude of the local contractions elicited by depolarizing current pulses, while the graded membrane responses were not markedly modified. Verapamil (1-100 microng/ml) did not affect the resting membrane potential but increased the effective membrane resistance. Determination of the cable characteristics by DC pulses indicated that verapamil (1-10 microng/ml) shortens the membrane length constant, increases the specific resistivity of the sarcoplasm and, in most cases, increases the membrane time constant. Verapamil (10 microng/ml) induced tension in these crab fibers. The contractions were potentiated in Na-deficient media, by increase in [Ca]0, and by membrane depolarization; "Ca-free" salines depressed, and procaine abolished these contractions. The results suggest that verapamil affects both Ca and K conductances and interferes with the Ca-sequestering mechanisms of these fibers. The (-)-isomer of verapamil was more effective than the (+)-isomer with respect to tension development, prolongation and subsequent blockade of procaine-spikes and enhancement of current-induced after-potentials and contractions.

This report deals with the diffusional and nondiffusional water fluxes of muscle fibers of the crab, Chionoecetes bairdi. Graphical analysis of the deuterium exchange indicates that two fiber compartments exist for water. The first, comprising about 60-70 % of the fiber water, probably represents the sarcoplasm which is bounded externally by the plasma membrane. The second compartment might represent intracellular organelles. The ratio between the nondiffusional and diffusional fluxes is very much larger than that found earlier for erythrocytes and for the giant axon of the squid. A ratio of such size is unlikely to be caused by unstirred layers and more accurate determinations of the water flux must include study of the influence of the complex morphology of these muscle fibers.