Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Tissue susceptibility factors in cadmium carcinogenesis

Recently, in two separate studies we have observed cadmium (Cd)-induction of prostatic tumors (PT) in rats. Cd (sc or im) at doses nontoxic to the testes markedly increased PT formation (2.5 μmol/kg, sc, 8 PT/29 exposed, 28%; 30 μmol/kg, im, 11/26, 42%; control 14/127, 11%). The administration of zinc (Zn; 1 mmol/kg, sc, at ?6, 0 and +18 h) to prevent testicular toxicity and tumors from Cd (30 μmol/kg, sc, 0 h) also resulted in an elevated incidence of PT (8/27, 30%). The nature of the metal-binding proteins in the prostate has not been defined, although metallothionein (MT), a low M_r Cd-binding protein that confers tolerance to Cd, is deficient in other target tissues of Cd carcinogenesis, such as the rat testes. Using a technique that extracts MT from liver, a low-M_r Cd-binding protein was extracted from both ventral (VP) and dorsal prostate (DP) and isolated by gel filtration. In contrast to the two forms of rat MT, reverse phase HPLC of VP and DP extract eluted 1 and 5 forms, respectively. The amino acid compositions of the VP and DP proteins were quite distinct from MT, with much less cys than MT and the presence of residues not found in MT (leu, tyr, phe). Thus Cd-induction of PT appears to be dependent on functional testes and, as is the case with Cd-induced testicular formation, appears to be associated with a deficiency of MT.     

An empirical analysis of the factors contributing to 20-year decrease in soil pH in an old-field plantation of loblolly pine

The pH of weak-acid solutions is controlled by acid concentration (HA + A^–), the degree of acid dissociation (A^–/HA), and the strength of the acids present (pK_a). We developed an empirical approach that allows the relative importance of each of these factors to be estimated for soils. This empirical model was applied to soils collected from an old-field plantation of loblolly pine (Pinus taeda L.) at 5 and 25 years of age. During this period, soil pH dropped by 0.3 to 0.8 units, and extractable calcium, magnesium and potassium declined by 20 to 80%. The empirical model indicates that the decline in pH resulted largely from the reduction in base saturation of the exchange complex. However, the average acid strength of the exchange complex decreased during the 20 years, preventing a greater decline of perhaps 0.1 to 0.2 units in the observed pH. The rate of decrease in the acid neutralizing capacity to pH 3.5 was about 1.3 kmol_c/ha annually, while the increase in base neutralizing capacity was about 2.7 and 1.6 kmol_c/ha annually to pH 5.5 and 8.2, respectively. Extractable alkali and alkaline earth cations declined by about 2.2 kmol_c/ha annually, matched by the rate of increase in aluminium. These changes demonstrated the dynamic nature of poorly buffered soils, and indicated that changes in soil acidity may be expected over a period of decades (especially following changes in land-use).     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

Summary The accumulation of interferon (IFN) -2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon -2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN -2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t _1/2 values for IFN -2 decreased with increasing incubation temperature. Pulse-chase studies using [³⁵S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN -2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN -2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.     

DNA loop domains in mammalian spermatozoa

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46±7 kb in sperm as compared with 16±11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.     

Bradyrhizobium japonicum Survival in and Soybean Inoculation with Fluid Gels

The utilization of gels, which are used for fluid drilling of seeds, as carriers of Bradyrhizobium japonicum for soybean (Glycine max (L.) Merr.) inoculation was studied. Gels of various chemical composition (magnesium silicate, potassium acrylate-acrylamide, grafted starch, and hydroxyethyl cellulose) were used, although the hydroxyethyl cellulose gels were more extensively investigated. Gel inocula were prepared by mixing gel powder with liquid cultures of B. japonicum (2% [wt/vol]). The population of B. japonicum USDA 110 did not change in each gel type during 8 days of incubation at 28°C. These fluid gels were prepared with late-exponential-growth-phase cells that were washed and suspended in physiological saline. Mid-exponential-growth-phase B. japonicum USDA 110, 123, and 138 grew in cellulose gels prepared with yeast extract-mannitol broth as well as or better than in yeast extract-mannitol broth alone for the first 10 days at 28°C. Populations in these cellulose gels after 35 days were as large as when the gels had originally been prepared, and survival occurred for at least 70 days. Soybeans grown in sand in the greenhouse had greater nodule numbers, nodule weights, and top weights with gel inoculants compared with a peat inoculant. In soil containing 10³ indigenous B. japonicum per g of soil, inoculation resulted in increased soybean nodule numbers, nodule weights, and top weights, but only nodule numbers were greater with gel than with peat inoculation. The gel-treated seeds carried 10² to 10³ more bacteria per seed (10⁷ to 10⁸) than did the peat-treated seeds.     

Relationships among Cell Size, Membrane Permeability, and Preservative Resistance in Yeast Species

The rate of uptake of propanoic acid and the cell dimensions were measured for 23 yeasts differing in their resistance to weak-acid-type preservatives. Relationships between reciprocal uptake rate, reciprocal permeability, cell volume, cell area, volume/area, and the MICs of benzoic acid and propanoic acid for the yeasts were tested by correlation analysis on pairs of parameters. The MIC of methylparaben, which is not a weak-acid-type preservative, was included. The most significant relationships found were between both reciprocal uptake rate and reciprocal permeability and the MICs of propanoic and benzoic acids Cell volume, area, and volume/area were each individually correlated with propanoic and benzoic acid MICs, but less strongly. In multiple regression analyses, inclusion of terms for volume, area, or volume/area did not markedly increase the significance. The MIC of methylparaben was unrelated to the uptake and permeability parameters, but did show a correlation with cell volume/area. Schizosaccharomyces pombe was anomalous in having very low permeability. Exclusion of these outlying data revealed particularly strong relationships (P < 0.001) between both reciprocal uptake rate and reciprocal permeability and the benzoic acid MIC. MICs for Zygosaccharomyces bailii isolates were substantially higher than for the other species, and therefore Z. baillii isolates had a large influence on the regressions. However, the relationships observed remained significant even after removal of the Z. bailii data. In showing a correlation between the rate at which propanoic acid enters yeast cells and the ability of the cells to tolerate this and other weak-acid-type preservatives, but not methylparaben, the results suggest that the resistance mechanism, in which preservative is continuously removed from the cell, is a common and major determinant of the preservative tolerance of yeast species.     

Protein engineering of disulfide bonds in subtilisin BPN'

Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also possible that the rate of irreversible inactivation depends upon the kinetics and not the thermodynamics of unfolding and so the entropically stabilizing effect expected from a disulfide bond may not apply.