ABSTRACT
Cyclooxygenase 1b (COX-1b) is a splice variant of COX-1, containing a retained intron 1 within the signal peptide sequence. COX-1b mRNA is found in many species, but the existence of a functionally active protein, which is possibly related to different species-dependent lengths of intron 1, is controversially discussed. The human intron 1 comprises 94 bp, and the resulting frameshift at the intron 1-exon 2 junction creates a premature stop codon. Nevertheless, full-length human COX-1b protein expression, including translated intron 1 and the signal peptide, has been reported and was explained by a frameshift repair. In this study, the fate of COX-1b mRNA in a human overexpression system is analyzed. Independent of the hypothetical frameshift repair mechanism, the splicing of the COX-1b intron 1, resulting in COX-1 mRNA and removal of the signal peptide during protein maturation, with subsequent generation of a COX-1 protein is demonstrated.
Subject(s)
Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandin-Endoperoxide Synthases/genetics , Protein Biosynthesis , Amino Acid Sequence , Base Sequence , Cyclooxygenase 1/chemistry , Cyclooxygenase 1/metabolism , DNA, Complementary/genetics , Exons/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Introns/genetics , Liver/enzymology , Mass Spectrometry , Molecular Sequence Data , Prostaglandin-Endoperoxide Synthases/chemistry , Protein Sorting Signals , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Stomach/enzymologySubject(s)
Catechin/analogs & derivatives , Thrombin/biosynthesis , Thromboplastin/antagonists & inhibitors , Blood Platelets/drug effects , Blood Platelets/metabolism , Catechin/chemistry , Catechin/pharmacology , Humans , In Vitro Techniques , Molecular Structure , Tea/chemistry , Thromboplastin/pharmacologyABSTRACT
Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre-incubation with EGCG concentration-dependently inhibited thrombin-induced aggregation and phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP-76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase-dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3' position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2' position (epigallocatechin) did not.