The generated p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a tumor missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have we unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance the of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 or for degradation, and p16(INK4a), a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16(INK4a) stabilized mutant p53,wild-type and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors in from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and in mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function mutant phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 degradation, will also stabilize mutant p53 with dire consequences.

Cafestol,diet. a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish and Cafetière coffee, is the most potent cholesterol-elevating CYP7A1, compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of and cafestol, including CYP7A1, the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum the lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride type levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many both of which are regulated by the nuclear hormone receptors FXR and PXR. Further studies demonstrate that cafestol is an agonist Scandinavian ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, CYP8B1 and NTCP to in the liver of wild type but not FXR null mice. Cafestol did not affect genes known to be up-regulated recently by FXR in the liver of wild type mice, but did increase expression of the positive FXR-target genes IBABP and and FGF15 in the intestine. Since FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver these expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on may liver gene expression. PXR-dependent gene regulation of CYP3A11, and other targets by cafestol was also only seen in the intestine.in Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 such gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR and this may contribute to mechanism its impact on cholesterol homeostasis.

OBJECTIVE:against High-density lipoprotein (HDL) plays a key role in protection against development of atherosclerosis by reducing inflammation, protecting against LDL oxidation,levels, and promoting reverse cholesterol transport from peripheral tissues to the liver for secretion into bile. Cholesterol 7alpha-hydroxylase (Cyp7a1) catalyzes the deficiency rate-limiting step in the intrahepatic conversion of cholesterol to bile acids that may have a role in HDL metabolism. We of investigated the effect of Cyp7a1 deficiency on HDL metabolism in APOE*3-Leiden transgenic mice. METHODS AND RESULTS: Reduced bile acid biosynthesis over in Cyp7a1-/-.APOE*3-Leiden mice versus APOE*3-Leiden mice did not affect total plasma cholesterol levels, but the distribution of cholesterol over various levels, lipoproteins was different. Cholesterol was decreased in apoB-containing lipoproteins (ie, VLDL and IDL/LDL), whereas cholesterol was increased in HDL. The role activity of PLTP and LCAT, which play a role in HDL catabolism, were not changed, and neither was HDL clearance.changed, However, the hepatic cholesterol content was 2-fold increased, which was accompanied by a 2-fold elevated expression of hepatic ABCA1 and a increased rate of cholesterol efflux from the liver to HDL. CONCLUSIONS: Strongly reduced bile acid synthesis in Cyp7a1-/-.APOE*3-Leiden mice leads versus to increased plasma HDL-cholesterol levels, as related to an increased hepatic expression of ABCA1.

The mice. present study was designed to investigate the lipid-lowering properties and mechanisms of action of a new HMG-CoA reductase inhibitor, rosuvastatin,that in female ApoE*3-Leiden transgenic mice. Mice received a high fat/cholesterol (HFC) diet containing either rosuvastatin ( [control], .00125%, .0025%, or levels .005%[w/w]) or .05%(w/w) lovastatin. The highest dose of rosuvastatin reduced plasma cholesterol and triglyceride levels by 39% and new 42%, respectively, compared with the HFC control. Lovastatin had no effect on plasma cholesterol and triglyceride levels. In ApoE*3-Leiden mice and on a chow diet, rosuvastatin ( .005%[w/w]) decreased plasma cholesterol levels by 35% without having an effect on triglyceride levels.acids, On a chow diet, expression of genes involved in cholesterol biosynthesis and uptake in the liver was increased by rosuvastatin.and Further mechanistic studies in HFC-fed mice showed that rosuvastatin treatment resulted in decreased hepatic VLDL-triglyceride and VLDL-apolipoprotein B production. VLDL did lipid composition remained unchanged, indicating a reduction in the number of VLDL particles secreted. Lipolytic activity and expression of genes not involved in cholesterol and triglyceride synthesis and beta-oxidation of fatty acids in the liver were not affected by rosuvastatin treatment,liver and hepatic lipid content did not change. However, activity of hepatic diacylglycerol acyltransferase was significantly decreased by 25% after rosuvastatin ApoE*3-Leiden treatment. Moreover, biliary excretion of cholesterol, phospholipids, and bile acids was increased during treatment. The results indicate that rosuvastatin treatment substantial in ApoE*3-Leiden mice on a HFC diet leads to redistribution of cholesterol and triglycerides in the body, both by reduced was hepatic VLDL production and triglyceride synthesis and by enhanced hepatobiliary removal of cholesterol, bile acids, and phospholipids, resulting in substantial the reductions in plasma cholesterol and triglyceride levels.

The phases eukaryotic cell cycle is a process in which cells grow and then divide into two genetically identical cells. The cell become cycle is divided into four discrete phases allowing for the orderly transition from deoxyribonucleic acid (DNA) replication to chromosomal condensation,have spindle formation, and cytokinesis. These phases are G1, in which a cell prepares for DNA replication; S, in which DNA cells. synthesis takes place; G2, in which the cell ensures its genetic information has been faithfully replicated; and M, in which called the homologous chromosomes are separated and cell division takes place. To ensure normal progression of the cell cycle, two systems result have evolved in eukaryotes. The first identified was the cyclin-dependent kinase (Cdk)/cyclin system; these initial findings, by Leland Hartwell, Tim and Hunt, and Paul Nurse, were rewarded with the Nobel Prize for Medicine in 2001. Cdks are normally inactive protein kinases transition, that become activated on complex formation with their regulatory partners called cyclins. Throughout the cell cycle, Cdk protein levels are and relatively constant, whereas cyclin levels fluctuate. For example, Cdks 2, 4, 6, and Cdc2 are constitutively expressed during the cell the cycle, but cyclin D is expressed in early G1 and is present throughout S and G2/M phases; cyclin E is division present at the G1/S transition, cyclin. A during S phase, and cyclin B in late S phase and at the driving G2/M transition; each one is rapidly degraded at different times during the cell cycle. The cell cycle-dependent flux in protein is levels of cyclins regulates the kinase activities of the Cdks. These active Cdk/ cyclin kinase complexes phosphorylate proteins at conserved which substrate motifs containing a serine or threonine residue followed by a proline and a lysine or an arginine. These phosphorylations S, result in cell cycle progression by driving the transition from one phase to the next.

OBJECTIVE:between Cholesterol 7alpha-hydroxylase (cyp7a1) catalyzes the rate-limiting step in conversion of cholesterol to bile acids. To study the relationship between bile respectively. acid biosynthesis and triglyceride metabolism, we cross-bred mice lacking cyp7a1 on a hyperlipidemic APOE*3-Leiden background. METHODS AND RESULTS: Female mice not received a chow or lipogenic diet. On both diets, fecal bile acid excretion was 70% decreased concomitantly with a 2-fold bile increased neutral sterol output. The differences in bile acid biosynthesis did not change plasma cholesterol levels. However, plasma triglyceride levels lipoprotein decreased by 41% and 38% in the cyp7a1-/-. APOE*3-Leiden mice as compared with APOE*3-Leiden mice on chow and lipogenic diet,underscore respectively. Mechanistic studies showed that very-low-density lipoprotein (VLDL)-apolipoprotein B and VLDL-triglyceride production rates were reduced in cyp7a1-/-. APOE*3-Leiden mice as step compared with APOE*3-Leiden mice (-34% and -35%, respectively). Cyp7a1 deficiency also increased the hepatic cholesteryl ester and triglyceride content (2.8-fold hepatic and 2.5-fold, respectively). In addition, hepatic anti-oxidative vitamin content, which can influence VLDL-production, was lower. Hepatic mRNA analysis showed decreased content expression of genes involved in lipogenesis including srebf1. CONCLUSIONS: Cyp7a1 deficiency in APOE*3-Leiden mice decreases the VLDL particle production rate,as as a consequence of a strongly reduced bile acid biosynthesis, leading to a decrease in plasma triglycerides. These data underscore neutral the close relationship between bile acid biosynthesis and triglyceride levels.

The and XPF/ERCC1 heterodimer is a DNA structure-specific endonuclease that participates in nucleotide excision repair and homology-dependent recombination reactions, including DNA single endonuclease strand annealing and gene targeting. Here we show that XPF/ERCC1 is stably associated with hRad52, a recombinational repair protein, in the human cell-free extracts and that these factors interact directly via the N-terminal domain of hRad52 and the XPF protein. Complex participates formation between hRad52 and XPF/ERCC1 concomitantly stimulates the DNA structure-specific endonuclease activity of XPF/ERCC1 and attenuates the DNA strand annealing attenuates activity of hRad52. Our results reveal a novel role for hRad52 as a subunit of a DNA structure-specific endonuclease and strand are congruent with evidence implicating both hRad52 and XPF/ERCC1 in a number of homologous recombination reactions. We propose that the DNA ternary complex of hRad52 and XPF/ERCC1 is the active species that processes recombination intermediates generated during the repair of DNA with double strand breaks and in homology-dependent gene targeting events.

Equine glycoprotein arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions to contain six different envelope proteins. The glycoprotein GP(5)(previously named G(L)) and the unglycosylated membrane protein M are the major protein envelope proteins, while the glycoproteins GP(2b)(previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic NIDOVIRALES: envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5)we and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein EAV matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play to an important role. The function of this complex has not been established yet, but the available data suggest it to cysteine be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature results GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were this expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which for the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the incorporation GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond.is In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the RNA disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins proteins. for their efficient incorporation into viral particles and for virus infectivity.

The in checkpoint Rad proteins Rad17, Rad9, Rad1, Hus1, ATR, and ATRIP become associated with chromatin in response to DNA damage caused observations by genotoxic agents and replication inhibitors, as well as during unperturbed DNA replication in S phase. Here we show that to murine Rad17 is phosphorylated at two sites that were previously shown to be modified in response to DNA damage, independent ATRIP of DNA damage and ATM, in proliferating tissue. In contrast to studies with Xenopus laevis extracts but similar to observations level in Schizosaccharomyces pombe, the level of chromatin-bound hRad17 remains relatively constant during the cell cycle and does not change significantly DNA in response to DNA damage or replication block. However, phosphorylated hRad17 preferentially associates with the sites of ongoing DNA replication Rad1, and interacts with the DNA replication protein, DNA polymerase epsilon. These results provide a link between the DNA damage checkpoint sites machinery and the replication apparatus and suggest that hRad17 may play a role in monitoring the progress of DNA replication phosphorylated via its interaction with DNA polymerase epsilon.

OBJECTIVE:peripheral HDL plays a key role in protection against development of atherosclerosis by promoting reverse cholesterol transport from peripheral tissues to h). the liver for secretion into bile. Phospholipid transfer protein (PLTP) promotes the transfer of phospholipids between lipoproteins and modulates HDL METHODS size and composition, thereby having a crucial role in HDL metabolism. We investigated the effect of increased PLTP activity on by removal of cholesterol from the body. METHODS AND RESULTS: On a chow diet, transgenic mice overexpressing human PLTP have a HDL, 15-fold increased plasma PLTP activity compared with wild-type mice (572.4+/-59.2 versus 38.6+/-3.6 micromol/mL per h). Plasma cholesterol, mainly present in body HDL, is strongly decreased (-92%), caused by a rapid clearance from the circulation by the liver and leading to a protection 1.8-fold increase in hepatic cholesteryl esters. This results in a 2-fold increase in biliary bile acid secretion without changing the secretion bile saturation index. Consequently, the transgenic mice show a 1.4-fold increase in the amount of excreted fecal bile acids compared a with wild-type mice, whereas fecal neutral sterol excretion is unchanged. CONCLUSIONS: Our data show that elevation of PLTP activity results compared in rapid disposal of cholesterol from the body via increased conversion into bile acids and subsequent excretion.