Identification and partial characterization of the high-affinity choline carrier from rat brain striatum.

[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.

Impact of Soil Texture on the Reproductive and Damage Potentials of Rotylenchulus reniformis and Meloidogyne incognita on Cotton.

The effects of soil type and initial inoculum density (Pi) on the reproductive and damage potentials of Meloidogyne incognita and Rotylenchulus reniformis on cotton were evaluated in microplot experiments from 1991 to 1993. The equilibrium nematode population density for R. reniformis on cotton was much greater than that of M. incognita, indicating that cotton is a better host for R. reniformis than M. incognita. Reproduction of M. incognita was greater in coarse-textured soils than in fine-textured soils, whereas R. reniformis reproduction was greatest in a Portsmouth loamy sand with intermediate percentages of clay plus silt. Population densities of M. incognita were inversely related to the percentage of silt and clay, but R. reniformis was favored by moderate levels of clay plus silt (ca. 28%). Both M. incognita races 3 and 4 and R. reniformis effected suppression of seed-cotton yield in all soil types evaluated. Cotton-yield suppression was greatest in response to R. reniformis at high Pi. Cotton maturity, measured as percentage of open bolls at different dates, was affected by the presence of nematodes in all 3 years.

Efficacy of Paecilomyces lilacinus in Suppressing Rotylenchulus reniformis on Tomato.

Effects of rice-cultured Paecilomyces lilacinus on Rotylenchulus reniformis were studied in both greenhouse and field microplot tests with 'Rutgers' tomato. Numbers of R. reniformis were reduced (P

Modulation of high-affinity choline carrier activity following incubation of rat hippocampal synaptosomes with hemicholinium-3.

Membrane carriers display structural and functional asymmetry with a substrate binding site which can be oriented alternately, but not simultaneously, to the extracellular and intracellular environment. Hemicholinium-3 is an inhibitor of the high-affinity choline carrier in cholinergic nerve terminals which binds to the transporter at the outer membrane surface but is not taken up into the cell. In the present study, we investigated the decline in choline transport which occurs during the first few minutes cholinergic nerve terminals are incubated in physiological salt solutions. Following incubation of rat hippocampal synaptosomes with hemicholinium-3, samples were washed free of the inhibitor and high-affinity choline uptake was measured. Choline uptake into hemicholinium-treated nerve terminals was significantly greater than control (132 +/- 4%). This effect appeared not to be due to an increase in uptake of choline above initial values in the hemicholinium-treated synaptosomes, but to a decrease in choline carrier activity in control samples by more than 25% during the first few minutes of incubation. Addition of hemicholinium-3 to samples after the preincubation induced decrease in choline uptake, followed by a wash period to remove the inhibitor resulted in elevation of choline uptake levels to initial levels. The effect of hemicholinium-3 was concentration-dependent, requiring near saturating concentrations of the inhibitor to elicit the effect. Measurement of acetylcholine content of synaptosomes at different points during the incubation procedure revealed that there was a trend for transmitter levels to vary inversely compared to choline uptake activity, but the differences were not statistically significant during treatments when significant changes in transport activity were measured.

Comparison of Two Inoculum Preparation Methods for Rotylenchulus reniformis.

Three greenhouse experiments were conducted to determine whether NaOCl-extracted eggs would provide an acceptable inoculum source for Rotylenchulus reniformis. Two tests (one each on loamy sand and sandy clay) were designed to compare eggs extracted from roots with sodium hypochlorite (NaOCl) with mechanically extracted vermiform males, females, and juveniles from soil as inoculum sources. Infection rates for both inoculum types were low (< 1-3%) on roots of 'Ransom' soybean 14 days (loamy sand soil) or 30 days (sandy clay soil) after inoculation. A larger number of infective females from the mechanically extracted than from NaOCl-extracted inoculum penetrated the roots in the loamy sand; however, in the heavier soil (sandy clay), NaOCl-extracted eggs were the better inoculum source. Significant reproduction occurred on infected plants, regardless of inoculum preparation method or soil type. Extraction of eggs by the NaOCl method is much easier and quicker than mechanical extraction of vermiform nematodes from soil. A third test was conducted to determine the infectivity of R. reniformis from eggs extracted at different NaOCl concentrations. Five initial inocnlum levels (0, 500, 2,500, 5,000, and 10,000) and four NaOCl concentrations (0.25, 0.50, 0.75, and 1.0%) were compared on 'Rutgers' tomato harvested on two dates, 17 and 23 days after inoculation. Again, infection rates of roots were low (

Reproductive and Damage Potentials of Two Populations of Rotylenchulus reniformis on Sweetpotato and Related Comparisons with Meloidogyne javanica on Tomato.

Two Rotylenchulus reniformis populations (North Carolina and Georgia) were compared on sweetpotato and tomato. 'Beauregard' sweetpotato and 'Better Boy' and 'Marion' tomato were excellent hosts for both R. reniformis populations. On Beauregard sweetpotato, the two populations did not differ in fecundity; however, on both tomato cultivars, the Georgia population reproduced at a higher rate than the North Carolina population (P

Effects of Root Decay on the Relationship between Meloidogyne spp. Gall Index and Egg Mass Number in Cucumber and Horned Cucumber.

A greenhouse study was conducted to determine if root necrosis had an effect on the relationship between root-knot nematode gall index and egg mass number. Thirty-four cultigens of Cucumis (14 accessions, 12 cultivars, and six breeding lines of C. sativus, and two accessions of C. metuliferus) were evaluated against four root-knot species (Meloidogyne arenaria race 2, M. incognita race 1, M. incognita race 3, and M. javanica) measuring gall index, root necrosis, and egg mass number. Root necrosis affected the gall index-egg mass relationship. At lower root necrosis values, a stronger relationship existed between gall index and egg mass number than at higher root necrosis values. Root tissue was destroyed by root necrosis, and normal root-knot nematode reproduction would not occur, even though root galling was still observed. The races of M. incognita tested had a greater effect in predisposing C. sativus and C. metuliferus to root necrosis than did M. arenaria race 2 or M. javanica. This study showed that root necrosis had an adverse affect on the relationship between gall index and egg mass number in cucumber.

Resistance to Root-knot Nematodes in Cucumber and Horned Cucumber.

Two experiments were conducted in the greenhouse. In one experiment, cucumber (Cucumis sativus) and horned cucumber (C. metuliferus) cultigens were evaluated for resistance to four root-knot nematode species (Meloidogyne arenaria, M. hapla, M. incognita, and M. javanica), and, in a second experiment, a standard (12-week) test was compared with a rapid (6-week) test. In the first experiment, horned cucumber cultigens varied in response to the Meloidogyne species. 'Sumter' cucumber was more susceptible than the horned cucumber to Meloidogyne incognita, M. javanica, and M. arenaria. All cultigens were more resistant to M. hapla than to the other root-knot nematode species. In the second experiment, best results were obtained when the test was run for 12 weeks rather than 6 weeks after planting (or 10 and 4 weeks after inoculation, respectively). All cultigens were more resistant to M. arenaria than to either M. incognita or M. javanica.

Uptake and metabolism of [3H]choline mustard by cholinergic nerve terminals from rat brain.

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.

Disposition of preformed mineral in matrix vesicles. Internal localization and association with alkaline phosphatase.

Studies were made on the disposition of mineral ions in matrix vesicles (MV) and their relationship to alkaline phosphatase by treatment of MV-enriched microsomes (MVEM) with graded levels of Ca2+-chelating agents to complex accessible ions, fractionation of MVEM on hypertonic sucrose gradients at two different pH values (7.5 and 8.0) to evaluate for the presence of calcium phosphate mineral, and passage of MVEM through cation-exchange columns to determine the accessibility of the Ca2+. The effect of removal of Ca2+ and Pi on subsequent ability of MVEM to induce mineral formation from synthetic cartilage lymph was also determined. Passage through cation-exchange columns revealed that MV Ca2+ was not freely exchangeable, but coeluted in the void volume with alkaline phosphatase. However, upon incubation in synthetic cartilage lymph, progressively more Ca2+ was retained by the column. These findings indicate that, initially, the majority of Ca2+ in MVEM is internal and not readily exchangeable, but as Ca2+ accumulates, progressively more becomes external. The mineral in MV is labile and readily susceptible to loss; treatment with graded levels of EGTA removed major portions of the original Ca2+ and Pi. 45Ca uptake by these mineral-depleted MV was markedly reduced, even in the presence of alkaline phosphatase substrates. Sucrose gradient fractionation of MVEM caused extensive loss of Pi, but not Ca2+, from the low-density alkaline phosphatase-rich fractions. This reveals that Ca2+ and Pi are not initially coupled together: Pi is largely soluble, whereas Ca2+ must be tightly bound. In the high-density vesicles, large amounts of both Ca2+ and Pi are present. The slightly enhanced recovery at higher pH suggests the presence of a solid mineral phase. During mineralization by MV, Ca2+ became externalized, and concomitantly alkaline phosphatase activity declined. This suggests that a direct association exists between the enzyme and the developing mineral.