Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.     

Water utilization of tropical hardwood hammocks of the Lower Florida Keys

Summary Predawn water potential of representative plant species, together with stable isotope composition of stem water and potential water sources were investigated in four low-elevation tropical hardwood hammocks in the Lower Florida Keys, during a one year period. Hammock species had the lowest water potentials when soil water content was low and/or soil salinity was high, but differences in groundwater salinity had no effect on the water potential. Comparison of D/H ratio of plant stem water with soil and ground water corroborates the conclusion that they are primarily utilizing soil water and not groundwater. Thus, tropical hardwood hammocks are buffered from saline groundwater, and are able to thrive in areas where groundwater salinity is as high as 25. The effect of sea level rise on these forests may depend more on changes in the frequency of tidal inundation of the soil surface than on changes in groundwater salinity.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Soil restoration under pasture after lignite mining: Management effects on soil biochemical properties and their relationships with herbage yields

The recovery of soil biochemical properties under grazed, grass-clover pasture, after simulated lignite mining, was studied over a 5-year period in a mesic Typic Dystrochrept soil at Waimumu, Southland, New Zealand. The restoration procedures involved four replacement treatments, after A, B, and C horizon materials had been separately removed, from all except the control, and stockpiled for 2–3 weeks. In each replacement treatment, the effects of ripping to 1.8 m depth, mole drainage, and the use of fertilizer nitrogen were also investigated.Replacement treatment markedly influenced the recovery of herbage production and soil organic C and total N contents, N mineralization, microbial biomass (as indicated by mineral-N flush) and invertase and sulphatase activities. The effectiveness of replacement treatments decreased in the order: 1. control (no stripping or replacement). 2 A, B, and C horizon materials replaced in the same order. 3. A, B, and C horizon materials each mixed with an equal amount of siltstone overburden and replaced in order, 4. A and B horizon materials mixed before replacing over C horizon material.Ripping increased herbage production, net N mineralization, and to some extent microbial biomass. Drainage had little, if any, effect.Fertilizer N also stimulated herbage production, but depressed clover growth. Over 2.5 years, it had little detectable effect on the soil properties.Increases in soil invertase and, to a lesser extent, sulphatase activity during the trial were closely related to changes in herbage production. Microbial biomass increased more rapidly than did soil organic C in the early stages of the trial.Rates of net N mineralization strongly suggest that N availability would have limited pasture growth, especially in the treatments with mixed soil materials.     

Hairpin formation in the self-complementary dodecamer d-GGTACGCGTACC and derivatives containing GA and IA mispairs

The dodecamer d-GGTACGCGTACC and four derivatives with GA and IA mispairs in the 6,7 and 5,8 positions have been examined in dilute solution and 0.01-0.1 M sodium chloride. Concentration dependence of Tm, gel electrophoresis, and equilibrium centrifugation indicate that these self-complementary oligomers can form hairpins under the present conditions. Thermal transitions measured in the ultraviolet primarily represent melting of hairpin to coil [cf. Scheffler et al. (1968, 1970)]. The Tm values show little or no depression for 6,7 substitution but rather large depression for 5,8 replacement. We interpret the results to indicate that the 6,7 sequences have two-base loops and five base pair stems and that the 5,8 sequences have four-base loops and four base pair stems. A concurrent theoretical modeling study [Raghunathan et al. (1991) Biochemistry (following paper in this issue)] provides support for this interpretation.     

Thermodynamics of antiparallel hairpin-double helix equilibria in DNA oligonucleotides from equilibrium ultracentrifugation

Five highly palindromic DNA dodecamers, four of which may form G-A or I-A purine-purine mispairs at either the 5.8 or 6.7 positions, have been studied at sedimentation equilibrium in the analytical ultracentrifuge. Each DNA oligonucleotide forms an equilibrium mixture of ordered antiparallel hairpin and double-stranded helical structures in solutions of 0.1 or 0.5 M NaCl between 5 and 40 degrees C. The dimeric duplex is favored by conditions of high salt and low temperature. The monomer-dimer equilibrium constants vary from 5 x 10(6) to 5 x 10(3) and are unique for each DNA dodecamer. Analysis of the temperature dependence of the equilibrium constants shows that the double helix to hairpin conversion is driven by a positive entropy change and is associated with an endothermic enthalpy change. The mispair substitutions at the 5.8 positions and the IA(6.7) mispair have the greatest tendency toward hairpin formation and exhibit significantly larger entropy changes than the nonmispaired dGGTACGCGTACC parent sequence and the thermodynamically similar GA(6.7) DNA. The consequences of such hairpin-double helix equilibria must be considered in the interpretation of other kinds of experiments carried out on oligonucleotides at different concentrations.     

Nerve growth factor receptors are preaggregated and immobile on responsive cells.

It has been hypothesized that signal transduction occurs by ligand-induced receptor clustering and immobilization. For many peptide receptors, cross-linking by anti-receptor antibodies is sufficient for receptor activation. This is not, however, the case for nerve growth factor receptor (NGFR). Using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP), we have analyzed the distribution and diffusibility of NGFR on a series of cell lines. We have found the following: (1) Cells expressing high-affinity responsive NGFR's display clustered NGFR's even in the absence of ligand. In contrast, NGFR's in nonresponsive cell lines are diffusely distributed. (2) Receptors on responsive cell lines are largely nondiffusing while most receptors on nonresponsive cell lines are relatively free to diffuse. (3) NGF does not greatly alter the distribution or diffusion properties of the NGFR on either nonresponsive or responsive cell lines. Thus, NGFR is preclustered and immobile on responsive cells, which suggests that immobilization of NGFR prior to ligand binding is required for signal transduction.