ABSTRACT
A fibrinolytic enzyme, designated as brevinase, was purified from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Brevinase cleaved both the Aalpha- and Bbeta-chains of fibrinogen but did not affect the gamma-chain. It showed beta-fibrinogenase activity devoid of fibrinogen clotting and caseinolytic activity. The fibrinolytic activity was completely inhibited by PMSF, DFP, Pefabloc, and DTT, indicating brevinase is a serine protease requiring disulfide bridge(s) for its activity. It kept 80% of the initial activity after heating at 100 degrees C for 3 min, showed an equal maximum activity in the pH range from 5.5 to 8.5, and was inactivated by Zn(2+). Brevinase consists of two polypeptide chains of 16.5 and 17 kDa linked by disulfide bridge(s). The N-terminal amino acid sequences of 16.5 and 17 kDa chains showed homology to the N-terminal and the internal (central region) amino acid sequences of single-chain fibrinolytic enzymes in snake venom, respectively.
Subject(s)
Crotalid Venoms/enzymology , Fibrinogen/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Cations, Divalent/pharmacology , Disulfides/chemistry , Disulfides/metabolism , Dithiothreitol/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Stability , Hot Temperature , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Sequence Analysis , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/isolation & purification , Zinc/pharmacologyABSTRACT
The mechanism for apoptosis induced by aburatubolactam C was investigated in human Jurkat T cells. When the cells were treated with 3 micrograms/ml of aburatubolactam C, apoptotic DNA fragmentation was first detectable in 3 hr and then increased time-dependently in accordance with upregulation in the protein level of Fas ligand (FasL). Both the DNA fragmentation and upregulation of FasL expression reached a maximal level in 7-8 hr, at which time a significant increase in the tyrosine phosphorylation of multiple cellular proteins was detected, suggesting that the enhanced tyrosine phosphorylation of cellular proteins may result from activation of Fas-mediated death signaling. However, these aburatubolactam C-induced cellular changes and accompanied apoptosis were completely blocked in the presence of genistein, a known protein tyrosine kinase inhibitor. These results indicate that upregulation of FasL expression dictated by protein tyrosine kinase activation and subsequent mediation of Fas death signaling account for aburatubolactam C-induced apoptosis in Jurkat T cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Lactams , Membrane Glycoproteins/metabolism , Pyrroles/pharmacology , DNA Fragmentation/drug effects , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fas Ligand Protein , Genistein/pharmacology , Humans , Jurkat Cells , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Tyrosine/metabolism , Up-Regulation/drug effectsABSTRACT
By using radiolabeled murine cyclin D3 cDNA as a probe, two cyclin D3 genomic clones, MCD3P-117 and MCD3P-327, were isolated from a murine genomic library constructed with murine liver DNA. Physical mapping and DNA sequence analysis revealed that these clones contain approximately 1.5 kb uninterrupted linear sequence similar to murine cyclin D3 cDNA, indicating that the 1.5 kb sequence is a processed pseudogene for cyclin D3. When the nucleotide sequence of the cyclin D3 pseudogene was compared with that of cyclin D3 cDNA at the nucleotide level, the pseudogene contained 229 bp of 5'- and 371 bp of 3'-untranslated regions, and a recognizable complete coding region that is 90% identical to murine cyclin D3. This sequence is bounded by the repeat sequence (GC/AGCTCTCC), which is common to many processed pseudogenes. However, multiple genetic lesions, including substitution, deletion and/or insertion events that result in modification of the reading frame were found in the pseudogene sequence. The pseudogene appeared to accumulate 67 random point mutations in the functional coding region composed of 879 nucleotide positions. It is thus estimated that the cyclin D3 pseudogene arose approximately 11 million years (Myr) ago. These data provide the first characterization of murine cyclin D3 pseudogene and insight into its evolutionary age.
Subject(s)
Cyclins/genetics , Pseudogenes , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cyclin D3 , DNA Primers/genetics , DNA, Complementary , Evolution, Molecular , Liver/metabolism , Mice , Molecular Sequence Data , Point Mutation , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Time FactorsABSTRACT
Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of Cushing's syndrome in infants, children and young adults. It is characterized by non-adrenocorticotropic hormone-dependent hypersecretion of cortisol by multiple, pigmented nodules of hyperplastic adrenocortical cells. Biochemically, PPNAD is characterized by elevated levels of plasma and urinary cortisol that are not suppressed by high doses of dexamethasone (8mg/d for 2 days). Pathologically, the adrenal glands contain multiple dark brown or black nodules and the intervening cortical tissue is atrophic. Recognition of this diagnosis, although rare, is important, as bilateral adrenalectomy is the treatment of choice. We experienced a case of Cushing's syndrome due to primary pigmented nodular adrenocortical disease and report it with reviews of the literature.
