A Continuous Fluorescence Assay of Renin Activity

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate K_m 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability.     

Ectomycorrhizal diversity on the roots of Pitch pine (Pinus rigida Mill.) saplings as influenced by remediation and soil metal content

From 1913 to 1980, two zinc smelters in Palmerton, Pennsylvania, emitted large quantities of atmospheric pollutants nearly eliminating forests along a ridge above the town. In 2008, a remediation treatment was applied to the land above one of the smelters that included the planting of several locally adapted plant species. It also included mineral fertilization and mycorrhizal inoculation. One of the species, the Pitch pine (Pinus rigida, Mill.), is a native tree that is both tolerant of metalliferous soils and obligatorily ectomycorrhizal. This report summarizes the results of two observational studies conducted 5 years after the remediation treatment. The first study's objective was to compare ectomycorrhizal communities on treated Pitch pine saplings, with communities on naturally regenerating saplings in an adjacent non-remediated area. The second study's objective was to determine if the composition of the fungal communities on root tips of naturally regenerating Pitch pine saplings differed with distance from the smelters. Fungal community compositions were determined using internal transcribed spacer rRNA sequences. Comparisons of sequences from the remediated and non-remediated sites revealed that communities at the remediated sites had lower taxonomic diversity and were dominated by members of a genus in the remediation inoculant. The results of the smelter-proximity study indicated that although fungal diversity did not differ markedly with distance from the smelters, the relative abundances of some taxa were greater on saplings growing directly above the smelters, where the soils contained highest concentrations of zinc and cadmium.     

Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5′ promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5′ up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Prostaglandin H Synthetase-Mediated Metabolism of Dopamine: Implication for Parkinson's Disease

Abstract: Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and post-mortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E₂ (PGE₂, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE₂ synthesis. Dopamine stimulated PHS synthesis of PGE₂. [³H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H₂O₂-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H₂O₂-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. ³²P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/µg DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.     

Prostaglandin F2 alpha increases responsiveness of pulmonary airways in dogs

We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on the responsiveness of pulmonary airways in dogs. Airway responsiveness was assessed by determining the bronchoconstrictor response to increasing concentrations of acetylcholine aerosol delivered to the airways. In each of five dogs, we determined responsiveness during treatment with physiologic saline, histamine, or PGF2 alpha aerosols. The doses of histamine and PGF2 alpha were determined by establishing the largest dose of each which could be given to the dog without causing bronchoconstriction (subthreshold doses). We found that airway responsiveness was not significantly different during histamine treatment than after saline, however, responsiveness increased during treatment with PGF2 alpha. In addition, the hyperresponsiveness induced by PGF2 alpha was prevented by pretreatment with the ganglion blocking drug hexamethonium (5 mg/kg given intravenously). The results show that PGF2 alpha specifically increases the responsiveness of pulmonary airways in doses that do not cause bronchoconstriction, and suggest that the hyperresponsiveness involves a neural mechanism such as increased responsiveness of airway sensory nerves.     

Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections

Estrogen has been shown to affect ventromedial hypothalamic (VMH) nerve cell nucleoli in ovariectomized rats, by causing an increase in the number of electron-dense aggregates associated with nucleoli. In order to characterize these nucleolus-associated structures and other nuclear components, we examined the ultrastructure of ventromedial hypothalamic nucleoli and nuclei revealed by enzyme digestions (pepsin, RNase and DNase) in resinless thin sections. Digestion by pepsin did not cause obvious alterations in the morphology of the nucleolus or its related structures. Pepsin treatment followed by RNase, however, reduced the density of the nucleolus, while that of the nucleolus-associated structure and other related structures remained unchanged. Conversely pepsin treatment followed by DNase, reduced the density of nucleolus-associated and other chromatin structures, but had no effect on the density of the nucleolus. Pepsin treatment followed by RNase and then DNase treatment, reduced the density of the nucleolus and nucleolus-associated structures. A residual nucleolus and nucleolus-associated structure remained after this treatment. Stereo viewing of resinless sections shows that the nucleolus, its associated structures, and other related structures, are associated with fine filaments that may comprise the nuclear matrix. The nucleolus-associated structure containing DNA may direct RNA synthesis at an increased rate in estrogen-treated hypothalamic cells.