Role of calcium in the initiation of fast axonal transport of protein: Effects of divalent cations

Fast axonal transport of [³H]protein has been examined in bullfrog primary afferent neurons incubated in media supplemented with divalent cations that can act as agonists or antagonists of calcium ions. Incubation in calcium-free medium (CFM) had no effect on the rate of transport, but reduced the amount of transported [³H]protein by 40–60% relative to transport in the contralateral preparation maintained in normal medium. Preparations incubated in CFM supplemented with 1.8 mM SrCl₂ (equimolar to the CaCl₂ concentration in normal medium) carried out transport at control levels. Incubation conditions in which primary afferent somata were exposed to the Sr²⁺-medium while nerve trunks were maintained in CFM also supported normal transport. By contrast, selective exposure of nerve trunks to Sr²⁺-medium, and somata to CFM resulted in a reduced level of transport similar to that observed when the whole preparation was incubated in CFM. The depression of transport resulting from incubation in CFM was shown to be reversible when preparations were transferred from CFM to either Sr²⁺-supplemented CFM or to normal medium. By contrast to the effects of Sr²⁺, Ba²⁺ (up to 18 mM) did not substitute for Ca²⁺ in the transport process. When normal medium was supplemented with calciumantagonist cations, the amount of transport was depressed (Co²⁺ > Mn²⁺ >> Mg²⁺), with no concomitant effect on the rate of transport. Results of studies with Co²⁺, as well as those with Sr²⁺, suggest that a major locus of action of these cations is within the neuronal soma at a step subsequent to protein synthesis, and prior to the onset of protein translocation via the transport system. Thus, it is inferred that these divalent cations affect a calcium-dependent step that occurs during the initiation phase of fast axonal transport.     

Legionella pneumophila cell envelope: Permeability to hydrophobic molecules

Legionella pneumophila is sensitive to a number of toxic hydrophobic compounds. Suspensions of cells bound large amounts of the dye crystal violet, and disk agar diffusion assays confirmed the marked sensitivity to this compound. Fatty acids were also inhibitory to the growth ofL. pneumophila in liquid media, and growth inhibition increased with increasing chain length to a maximum with myristic acid. Oxygen uptake by respiring cells was inhibited by similar concentrations of fatty acids.L. pneumophila was also sensitive to low concentrations of progesterone. These results indicated thatL. pneumophila has an outer membrane with unusual permeability to hydrophobic compounds. This characteristic was accompanied by a measurable cell surface hydrophobicity as determined by adherence of the bacterium to the hydrocarbon hexadecane.     

Altered metabolic incorporation of fucose and leucine into PNS myelin of 25-week-old diabetic (C57BL/Ks〈db/db〉) mice: Effects of untreated diabetes on nerve metabolism

Sciatic nerves of 25-week-old genetically diabetic (C57BL/Ks 〈db/db〉) mice and their litter-mate controls were removed, and their metabolic incorporation of [³H]fucose and [¹⁴C]leucine into myelin was studied in vitro. Untreated diabetic animals showed significant increases (p<0.05) in the fucose/leucine incorporation into myelin when compared to values found for their litter-mates. These results correlated well with previous experiments performed on alloxan or streptozotocindiabetic rats and thus show the in vitro incubation procedure to be a good indicator of altered metabolic conditions in peripheral nerves due to diabetes mellitus. The resulting ratio increases seen in diabetic animals is at variance with the decrease in ratios found in animals undergoing typical Wallerian degeneration. These results suggest that different metabolic processes operate in untreated diabetics than in normals or in those undergoing other degenerative nerve processes.     

Behavioral development in okapis and giraffes

Observations on the behavioral development of two okapi calves and one giraffe calf were made at Brookfield Zoo. The following behaviors were monitored for 4 to 6 mo after birth; nursing duration and nursing attempts, mother-infant distance, bunting the mother's udder, lying, moving, maternal grooming, mother and infant autogrooming, object licking, tail chewing, and contact by others in the herd. Behaviors in both species showed oscillating patterns with high levels of mother-infant contact behaviors at 3–4 wk, 9–11 wk, and 14–15 wk in okapis. Giraffe infants showed similar oscillations with high periods of contact about 2–5 wk later than those in okapis. Other behaviors oscillated in concert with these, with specific correlations occurring between nursing behaviors and grooming behaviors. A main difference between okapi and giraffe development centered around maternal motivation during the high contact (regressive) periods. In okapis, after 10–12 wk there was a low rate of nursing success, whereas in giraffes the percentage of success in nursing rose with later behavioral oscillations. The regressive periods became conflict periods in okapis, whereas in the giraffe, the mother initiated the periods. This difference was in accordance with the unique strategy of infant rearing in wild giraffes in which there is an extended “hider” period when older calves are left together in shaded areas with an adult sentry. Field studies also indicated probable oscillations of mother-infant contact and a prolonged period of the mother initiating contact with her calf.     

Seasonal Changes in the Content of Urotensin II in the Urophysis of the Goby,Gillichthys mirabilis

A seasonal study of urotensin II content of the urophysis of the goby, Gillichthys mirabilis. was conducted from March 1979 to June 1980 in relation to certain internal and environmental changes. Urotensin II content (lowest in November–January) is inversely correlated with female gonadosomatic index and to some extent with rainfall (and hence dilution of the environmental salinity). In addition, there appears to be a direct correlation between UII content and daylength and temperature.     

A radioactive assay for acetylcholinesterase using anion-exchange disk

A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [¹⁴C]acetate from [¹⁴C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.     

Regulation of Pi,uptake by Acer pseudoplatanus cells

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by Acer pseudoplatanus cells grown as cell suspensions. At low external P_i concentrations up to 10 mm, sycamore cells incorporate phosphate against a concentration gradient, by a process which is energy dependent. Under these conditions the intracellular P_i concentration is maintained constant (2–3 mm). On the contrary at high external P_i concentrations, higher than that which counterpoises the cytoplasmic P_i concentration (approximately 10 mm), P_i enters the cell by slow diffusion and the intracellular P_i concentration increases continuously as the extracellular P_i concentration increases from 15 to 50 mm. When sycamore cells are transferred to a phosphate-deficient medium, growth slows down considerably and ceases after 4–5 days. During this time, intracellular P_i concentration falls from 3 to 0.1 mm and phosphate esters from 8 to 2 mm. Phosphate starvation stimulates the uptake indicating that phosphate uptake depends on the intracellular phosphate and/or cytoplasmic ester-P pool. P_i uptake by P_i-starved cells is strongly dependent on the pH of the medium.