A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment.

To investigate the molecular basis and physiological regulation of exceptionally high levels of aromatase (P450arom) activity in the brain of teleost fish, a 2927 bp P450arom cDNA encoding a 510 amino acid protein was isolated from a goldfish brain cDNA library. The brain-derived cDNA had 53% and 61-62% sequence identity when compared with human placental and fish ovarian P450arom forms, respectively, and higher homologies in conserved functional domains. Goldfish brain poly(A) RNA was translatable in vitro to a 56 kDa P450arom immunoprecipitation product. Northern blot analysis using the brain cDNA revealed a major 3.0 kb transcript of high abundance in brain (FB, forebrain > M/HB, mid/hindbrain), but no signal in ovary, testis or liver. P450arom mRNA varied seasonally in brain, with a peak at the onset of gonadal regrowth (February) that preceded the annual rise in enzyme levels and was 4-fold (FB) or 50-fold (M/HB) higher than during reproductive inactivity (July-December). Known markers of neurogenesis and estrogen action in brain (28S rRNA, beta-actin and beta-tubulin transcripts) each had unique seasonal patterns which differed from P450arom mRNA. In vivo steroid treatment showed that estrogen and aromatizable androgen increase FB and M/HB levels of P450arom mRNA 8- and 4-fold, respectively. P450arom mRNA in pituitary and retina had a different regulation. Southern analysis provided no evidence for multiple genes encoding the brain derived cDNA or for brain-specific gene amplification. Results imply that high accumulated levels of P450arom mRNA are the major determinant of high measured enzyme activity in goldfish brain, and that physiological regulation of mRNA expression in the natural environment is mediated by aromatization of androgen to estrogen.

Interaction of the v-Rel oncoprotein with NF-kappaB and IkappaB proteins: heterodimers of a transformation-defective v-Rel mutant and NF-2 are functional in vitro and in vivo.

The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of the Rel/NF-kappa B family of transcription factors. The mechanism by which v-Rel malignantly transforms chicken spleen cells is not precisely known. To gain a better understanding of functions needed for transformation by v-Rel, we have now characterized the activities of mutant v-Rel proteins that are defective for specific protein-protein interactions. Mutant v-delta NLS, which has a deletion of the primary v-Rel nuclear localizing sequence, does not interact efficiently with I kappa B-alpha but still transforms chicken spleen cells approximately as well as wild-type v-Rel, indicating that interaction with I kappa B-alpha is not essential for the v-Rel transforming function. A second v-Rel mutant, v-SPW, has been shown to be defective for the formation of homodimers, DNA binding, and transformation. However, we now find that v-SPW can form functional DNA-binding heterodimers in vitro and in vivo with the cellular protein NF-kappa B p-52. Most strikingly, coexpression of v-SPW and p52 from a retroviral vector can induce the malignant transformation of chicken spleen cells, whereas expression of either protein alone cannot. Our results are most consistent with a model wherein Rel homodimers or heterodimers must bind DNA and alter gene expression in order to transform lymphoid cells.