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.     

Molecular organization of great millet (Sorghum vulgare) DNA

Approximately 52% of the nuclear genome of great millet(Sorghum vulgare) consists of repetitive DNA which can be grouped into very fast, fast and slow components. The reiteration frequencies of the fast and slow reassociating components are {dy7000} and 92 respectively. Approximately 90% of the genome consists of repeated sequences interspersed amongst themselves and with single copy sequences. The interspersed repeat sequences are of three sizesviz. > 1·5 kilobase pairs, 0·5–1·0 kilobase pairs and 0·15–0·30 kilobase pairs while the size of the single copy sequences is 3·0 kilobase pairs. Hence the genome organization of great millet is essentially of a mixed type NCL communication No. 3527.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Subcellular Localization, Partial Purification, and Characterization of a Dynorphin Processing Endopeptidase from Bovine Pituitary

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.     

Neue,abgewandelte pseudoguajanolide aus Psilostrophe villosa

Investigation of the North American species Psilostrophe villosa afforded, in addition to known compounds, five new sesquiterpene lactones, which all turn out to be modified pseudoguaianolides, most of them closely related to hymenolide. However, one of the lactones is a new type of nor-pseudoguaianolide. Furthermore a new monoacetylated borneol-β-D-glucoside is present. The chemotaxonomic situation is discussed briefly.     

Development and Validation of Real-Time PCR for Rapid Detection of Mecistocirrus digitatus

Hematophagous activity of Mecistocirrus digitatus, which causes substantial blood and weight loss in large ruminants, is an emerging challenge due to the economic loss it brings to the livestock industry. Infected animals are treated with anthelmintic drugs, based on the identification of helminth species and the severity of infection; however, traditional methods such as microscopic identification and the counting of eggs for diagnosis and determination of level of infection are laborious, cumbersome and unreliable. To facilitate the detection of this parasite, a SYBR green-based real-time PCR was standardized and validated for the detection of M. digitatus infection in cattle and buffaloes. Oligonucleotides were designed to amplify partial Internal Transcribed Spacer (ITS)-1 sequence of M. digitatus. The specificity of the primers was confirmed by non-amplification of DNA extracted from other commonly occurring gastrointestinal nematodes in ruminants. Plasmids were ligated with partial ITS-1 sequence of M. digitatus, serially diluted (hundred fold) and used as standards in the real-time PCR assay. The quantification cycle (Cq) values were plotted against the standard DNA concentration to produce a standard curve. The assay was sensitive enough to detect one plasmid containing the M. digitatus DNA. Clinical application of this assay was validated by testing the DNA extracted from the faeces of naturally infected cattle (n = 40) and buffaloes (n = 25). The results were compared with our standard curve to calculate the quantity of M. digitatus in each faecal sample. The Cq value of the assay depicted a strong linear relationship with faecal DNA content, with a regression coefficient of 0.984 and efficiency of 99%. This assay has noteworthy advantages over the conventional methods of diagnosis because it is more specific, sensitive and reliable.     

AT2R agonist NP‐6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm

Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP‐6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP‐6A4–induced AT2R activation could mitigate AngII‐induced abdominal aortic aneurism (AAA) using AngII‐treated Apoe^?/? mice. Male Apoe^?/? mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre‐treated subcutaneously with NP‐6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP‐6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP‐6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII‐induced increases in monocyte chemotactic protein‐1, osteopontin and proteolytic activity of the aorta. However, NP‐6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.