Hormones regulate glucose homeostasis, in part, by controlling the expression of gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK). Insulin and glucocorticoids reciprocally regulate PEPCK expression primarily at the level of gene transcription. We demonstrate here that glucocorticoids promote, whereas insulin disrupts, the association of CREB-binding protein (CBP) and RNA polymerase II with the hepatic PEPCK gene promoter in vivo. We also show that accessory factors, such as CCAAT/enhancerbinding protein ␤ (C/EBP␤), can recruit CBP to drive transcription. Insulin increases protein levels of liverenriched transcriptional inhibitory protein (LIP), an inhibitory form of C/EBP␤, in a phosphatidylinositol 3-kinase-dependent manner. LIP concomitantly replaces liver-enriched transcriptional activator protein on the PEPCK gene promoter, which can abrogate the recruitment of CBP and polymerase II, culminating in the repression of PEPCK expression and the attenuation of hepatocellular glucose production.

The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed p⌬CREC/EBP, binds C/EBP␣ and ␤ but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved cotransfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (p⌬CREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBP␣ are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBP␤ provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBP␤ needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBP␤ fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBP␤ contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.

Our series demonstrates that blood loss during RRP continues to decrease. The respectable blood loss and low transfusion rates in this series were due to refinements in surgical technique rather than to perioperative modifications. To our knowledge the identification of BMI as a predictor of blood loss and transfusion is novel. These data serve as a benchmark for future comparisons and argue for continued refinements in techniques to decrease blood loss, particularly in overweight and obese patients.

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ς1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ς1 cleavage upon conversion of virions to ISVPs. We examined the ς1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ς1 cleavage. The ς1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ς1 proteins of the remaining strains. Thr249 occupies the d position of a heptad repeat motif predicted to stabilize ς1 oligomers through α-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ς1 known as the neck. Substitution of Thr249 with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ς1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ς1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ς1 after Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ς1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ς1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

Laparoscopic nephrectomy of the left renal remnant and ligation of the left distal ureter.