Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Effects of chemical composition on nitrogen mineralization from green manures of seven tropical leguminous trees

Green manures from seven tropical leguminous trees were incubated with soil to determine the rates and controls of net nitrogen release. Fresh green manure (leaves and succulent twigs) was mixed with moist soil and incubated in polyethylene bags. Net N mineralization from green manures was estimated by the accumulation of extractable ammonium and nitrate minus the accumulation in soil alone. Patterns of N mineralization were complex, differed among species, and at 12 weeks ranged from 10 to 65 percent of original green-manure N. Cumulative net N mineralization was negatively correlated with initial soluble polyphenol content in the early phases of decomposition (1 through 8 weeks) and with initial lignin content in later phases (4 through 12 weeks). Neither initial percent N nor lignin: N ratio were strongly correlated with N mineralization. The best chemical index of N release was the initial polyphenol: N ratio. This study confirms previous findings that N mineralization from tropical legumes is controlled more by soluble polyphenols than by lignin or N content.     

Soil acidification as caused by the nitrogen uptake pattern of Scots pine (Pinus sylvestris)

Three-year-old Scots pine (Pinus sylvestris) trees were grown on a sandy forest soil in pots, with the objective to determine their NH₄/NO₃ uptake ratio and proton efflux. N was supplied in three NH₄-N/NO₃-N ratios, 3:1, 1:1 and 1:3, either as ¹⁵NH₄+¹⁴NO₃ or as ¹⁴NH₄+¹⁵NO₃. Total N and ¹⁵N acquisition of different plant parts were measured. Averaged over the whole tree, the NH₄/NO₃ uptake ratios throughout the growing season were found to be 4.2, 2.5, and 1.5 for the three application ratios, respectively. The excess cation-over-anion uptake value (C_a-A_a) appeared to be linearly related to the natural logarithm of the NH₄/NO₃ uptake ratio. Further, this uptake ratio was related to the NH₄/NO₃ ratio of the soil solution. From these relationship it was estimated that Scots pine exhibits an acidifying uptake pattern as long as the contribution of nitrate to the N nutrition is lower than 70%. Under field circumstances root uptake may cause soil acidification in the topsoil, containing the largest part of the root system, and soil alkalization in deeper soil layers.     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.     

Root turnover as determinant of the cycling of C,N, and P in a dry heathland ecosystem

Root production and turnover were studied using sequential core sampling and observations in permanent minirhizotrons in the field in three dry heathland stands dominated by the evergreen dwarfshrub Calluna vulgaris and the grasses Deschampsia flexuosa and Molinia caerulea, respectively. Root biomass production, estimated by core sampling, amounted to 160 (Calluna), 180 (Deschampsia) and 1380 (Molinia) g m^-2 yr^-1, respectively. Root biomass turnover rate in Calluna (0.64 yr^-1) was lower compared with the grasses (Deschampsia: 0.96 yr^-1; Molinia 1.68yr^-1)). Root length turnover rate was 0.75–0.77 yr^-1 (Deschampsia) and 1.17–1.49 yr^-1 (Molinia), respectively. No resorption of N and P from senescing roots was observed in either species. Input of organic N into the soil due to root turnover, estimated using the core sampling data, amounted to 1.8 g N m^-2 yr^-1(^Calluna), 1.7 g N m^-2 yr^-1 (Deschampsia) and 19.7 g N m^-2 yr^-1 (Molinia), respectively. The organic P input was 0.05, 0.07 and 0.55 g P M^-2 yr^-1, respectively. Using the minirhizotron turnover estimates these values were20–22% (Deschampsia) and 11–30% (Molinia) lower.When the biomass turnover data were used, it appeared that in the Molinia stand root turnover contributed 67% to total litter production, 87% to total litter nitrogen loss and 84% to total litter phosphorus loss. For Calluna and Deschampsia these percentages were about three and two times lower, respectively.This study shows that (1) Root turnover is a key factor in ecosystem C, N, and P cycling; and that (2) The relative importance of root turnover differs between species.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

OPTIMUM N:P RATIOS AND COEXISTENCE OF PLANKTONIC ALGAE1

The optimum atomic ratio of N to P, the ratio at which one nutrient limitation changes over to the other, was determined in seven species of freshwater planktonic algae. The ratio varied over a wide range among species; the average for these species was 17. If the cellular nutrient ratios in marine species are comparable with those in freshwater organisms, Redfield's ratio of 15 is remarkably close to the average. Cellular N:P ratios varied over a 24-h period under a light:dark cycle. The variation of the optimum ratio between species and diel change in cellular N:P ratios within a species could play an important role in population dynamics by enhancing the probability of coexistence of species.