Jae B. Park

Clovamide-Type Phenylpropenoic Acid Amides, N -Coumaroyldopamine and N -Caffeoyldopamine, Inhibit Platelet-Leukocyte Interactions via Suppressing P-Selectin Expression

N-Coumaroyldopamine and N-caffeoyldopamine are clovamide-type phenylpropenoic acid amides found in Theobroma cacao. In this article, N-coumaroyldopamine and N-caffeoyldopamine were investigated to determine their effects on P-selectin expression and platelet-leukocyte interactions in vitro and in vivo models. At the concentration of 0.05 μM, they were able to inhibit P-selectin expression on the platelets by 33 (P < 0.011) and 30% (P < 0.012), respectively. The inhibition was partially blocked by β2-adrenoceptor antagonists, suggesting that β2 receptors are probably engaged in the inhibition. N-Caffeoyldopamine and N-coumaroyldopamine could also suppress platelet-leukocyte interactions in blood samples by 36 (P < 0.013) and 32% (P < 0.011), respectively, at the same concentration (0.05 μM). In an animal study, mice administrated orally with N-caffeoyldopamine (50 and 100 μg/35 g of body weight) also showed great reduction in the P-selectin expression and platelet-leukocyte interactions by 31 to 45% (P < 0.011) and 34 to 43% (P < 0.014), respectively. These data suggest that the clovamide-type phenylpropenoic acid amides are able to suppress platelet-leukocyte interactions via inhibiting P-selectin expression.

Cloning, Sequencing, and Characterization of Alternatively Spliced Glutaredoxin 1 cDNA and Its Genomic Gene CHROMOSOMAL LOCALIZATION, mRNA STABILITY, AND ORIGIN OF PSEUDOGENES

Alternatively spliced human glutaredoxin (Grx1as) cDNA was isolated from a neutrophil cDNA library, using a 32P-labeled human glutaredoxin (Grx1) cDNA probe under non-stringent conditions. The sequence of Grx1as cDNA indicated that the open reading frame of the gene was identical to the open reading frame of the previously reported first human glutaredoxin (Grx1) cDNA, but the 3′-untranslated region of Grx1as was not homologous to Grx1 cDNA. Northern blot and RT-PCR analyses showed Grx1as mRNA was expressed in normal human neutrophils and transformed cells including U937, HL-60, THP, and Jurkat cells. Cloning and sequencing of the genomic gene corresponding to Grx1as cDNA showed that two different glutaredoxin cDNAs (Grx1as and Grx1) were generated from the same genomic gene via alternative splicing. Origination of Grx1as and Grx1 from the same gene was confirmed by chromosomal localization of the Grx1as gene to chromosome 5q13, the same location where the Grx1 gene was localized previously. During screening of the Grx1as genomic gene, two additional glutaredoxin pseudogenes were also isolated. Surprisingly, these pseudogenes contained 3′-untranslated regions that were nearly identical to the 3′-untranslated regions of Grx1as, not Grx1, cDNA. Because 3′-untranslated regions may be important in stabilizing mRNAs, the effect of the two 3′-untranslated regions of Grx1 and Grx1as on mRNA stability was investigated using luciferase reporter vectors with the 3′-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vector containing the 3′-untranslated region of Grx1as cDNA compared with the 3′-untranslated region of Grx1 cDNA. These data indicate that Grx1as cDNA is an alternatively spliced human Grx1 cDNA and that the Grx1as 3′-untranslated region may have a role in stabilizing mRNA.