In the studies reported herein, we show that two complementary experimental models: inbred strains of mice (i.e. C57BL/6 and C3H/HeJ), and a differentiated line of rat hepatoma cells (i.e. L35 cells), require the activation of cytokines by monocyte/macrophages to display bile acid negative feedback repression of cholesterol 7␣-hydroxylase (CYP7A1). Feeding a bile acid-containing atherogenic diet for 3 weeks to C57BL/6 mice led to a 70% reduction in the expression of hepatic CYP7A1 mRNA, whereas no reduction was observed in C3H/HeJ mice. The strain-specific response to repression of CYP7A1 paralleled the activation of hepatic cytokine expression. Studies using cultured THP-1 monocyte/ macrophages showed that the hydrophobic bile acid chenodeoxycholate, a well established potent repressor of CYP7A1, induced the expression of mRNAs encoding interleukin 1 (IL-1) and tumor necrosis factor ␣ (TNF␣). In contrast, the hydrophilic bile acid ursodeoxycholate, which does not repress CYP7A1, did not induce cytokine mRNA expression by THP-1 cells. Chenodeoxycholate activation of cytokines by THP-1 cells was blocked by the peroxisome proliferator-activated receptor ␥ agonist rosiglitazone. The expression of cytokines (e.g. IL-1 and TNF␣) by THP-1 cells paralleled with the ability of these cells to produce conditioned medium that when added to rat L35 hepatoma cells, repressed CYP7A1. Moreover, rosiglitazone, which blocks cytokine activation by macrophages, also blocked the repression of CYP7A1 normally exhibited by C57BL/6 mice fed the bile acid-containing atherogenic diet. The combined data indicate that the activation of cytokines may mediate CYP7A1 repression caused by feeding mice an atherogenic diet containing bile acids.Bile acids, the major metabolites produced from cholesterol, are amphipathic steroid detergents necessary for the digestion and absorption of fat soluble nutrients from the intestine (1-3). The conversion of cholesterol to bile acids is regulated by the expression of cholesterol 7␣-hydroxylase (CYP7A1), 1 a cytochrome P450 enzyme unique to the liver parenchymal cell (4 -6). Bile acid synthesis exhibits negative feedback regulation (7,8) by decreasing the enzymatic activity of CYP7A1 (9). It is generally accepted that bile acids can inhibit the transcription of the CYP7A1 gene (1-3).Many different experimental models have been used to examine bile acid negative feedback regulation of CYP7A1 and some have yielded conflicting results. Bile acid negative feedback repression of CYP7A1 has been experimentally demonstrated by infusing bile acids into the intestine of bile fistulae rats (10) and hamsters (11). The ability of different bile acids to repress CYP7A1 expression correlates with the hydrophobic index of the infused bile acid; CDCA is a potent repressor, whereas UDCA is not (12). The finding that infusing taurocholate into the portal vein of bile fistulae mice was unable to repress CYP7A1 led to the conclusion that a factor produced within the enterohepatic circulation may be required to repress CYP7A1...

Abstract-C57BL/6J mice are susceptible to atherosclerosis when fed a diet consisting of fat, cholesterol, and taurocholate.The susceptibility to diet-induced atherosclerosis is linked to a reduction in plasma high density lipoprotein (HDL). Diet-induced reduction of plasma HDL shows a physiological and a genetic correlation with repression of cholesterol-7-␣-hydroxylase, the liver-specific enzyme that regulates the conversion of cholesterol into bile acids. To examine the hypothesis that the repression of cholesterol-7-␣-hydroxylase is responsible for initiating the metabolic alterations leading to the formation of atherosclerosis and gallstones, we determined whether constitutive transgenic expression of cholesterol-7-␣-hydroxylase in C57BL/6J mice would confer resistance to these 2 common human diseases. When fed the atherogenic diet, nontransgenic littermates, but not cholesterol-7-␣-hydroxylase transgenic mice, accumulated cholesterol and cholesterol esters in their livers and plasma. Although the atherogenic diet caused a marked decrease in plasma HDL cholesterol in nontransgenic mice, HDL levels in transgenic mice remained relatively unchanged. Moreover, the ability of cholesterol-7-␣-hydroxylase transgenic mice to maintain cholesterol and lipoprotein homeostasis completely prevented the formation of atherosclerosis and gallstones. These data establish the integral role that cholesterol-7-␣-hydroxylase has in maintaining hepatic cholesterol homeostasis and, thus, in the susceptibility to the formation of gallstones and atherosclerosis.

The finding that expression of a cholesterol 7␣-hydroxylase (CYP7A1) transgene in cultured rat hepatoma cells caused a coordinate increase in lipogenesis and secretion of apoB-containing lipoproteins led to the hypothesis that hepatic production of apoB-containing lipoproteins may be linked to the expression of CYP7A1 (Wang, S.-L., Du, E., Martin, T. D., and Davis, R. A. (1997) J. Biol. Chem. 272, 19351-19358). To examine this hypothesis in vivo, a transgene encoding CYP7A1 driven by the constitutive liver-specific enhancer of the human apoE gene was expressed in C56BL/6 mice. The expression of CYP7A1 mRNA (20-fold), protein (ϳ10-fold), and enzyme activity (5-fold) was markedly increased in transgenic mice compared with non-transgenic littermates. The bile acid pool of CYP7A1 transgenic mice was doubled mainly due to increased hydrophobic dihydroxy bile acids. In CYP7A1 transgenic mice, livers contained ϳ3-fold more sterol response element-binding protein-2 mRNA. Hepatic expression of mRNAs encoding lipogenic enzymes (i.e. fatty-acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, squalene synthase, farnesylpyrophosphate synthase, 3-hydroxy-3-methylglutarylCoA reductase, and low density lipoprotein receptor) as well as microsomal triglyceride transfer protein were elevated ϳ3-5-fold in transgenic mice. CYP7A1 transgenic mice also displayed a >2-fold increase in hepatic production and secretion of triglyceride-rich apoB-containing lipoproteins. Despite the increased hepatic secretion of apoB-containing lipoproteins in CYP7A1 mice, plasma levels of triglycerides and cholesterol were not significantly increased. These data suggest that the 5-fold increased expression of the low density lipoprotein receptor displayed by the livers of CYP7A1 transgenic mice was sufficient to compensate for the 2-fold increase production of apoB-containing lipoproteins. These findings emphasize the important homeostatic role that CYP7A1 plays in balancing the anabolic lipoprotein assembly/secretion pathway with the cholesterol catabolic bile acid synthetic pathway.Hepatic lipoprotein secretion requires apoB having a size that is sufficiently large to allow the formation of a lipoprotein particle containing a neutral lipid core, the availability of lipids (i.e. phospholipids, triglycerides, cholesterol, and cholesterol esters), and the intraluminal chaperone/lipid transfer protein microsomal triglyceride transfer protein (MTP) 1 (reviewed in Refs. 1-6). The assembly of apoB-containing lipoproteins is abrogated when these essential requirements are not satisfied, resulting in rapid degradation of apoB within the hepatocyte (7). The most characterized pathway responsible for the rapid, co-translational degradation of incompletely translocated apoB is via a ubiquitin-dependent proteasome process (8 -11). Several additional pathways that may contribute to the intracellular degradation of apoB have been described (12)(13)(14)(15).Cholesterol 7␣-hydroxylase (CYP7A1) is a liver-specific enzyme that regulates the production of bil...

Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7‐like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan‐associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7‐like phage‐encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7‐like phages may be smaller than previously hypothesized.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

No abstract

Cholesterol-7 ␣ -hydroxylase (CYP7A1) regulates the pathway through which cholesterol is converted into bile acids. The unique detergent properties of bile acids are essential for the digestion and intestinal absorption of hydrophobic nutrients. Bile acids have potent toxic properties (e.g., membrane disruption) and there are a plethora of mechanisms to limit their accumulation in blood and tissues. The discovery of farnesoid X receptor (FXR), the nuclear receptor activated specifically by bile acids, has opened new insights into these mechanisms. Bile acid activation of FXR has been shown to repress the expression of CYP7A1 via increasing the expression of small heterodimer partner (SHP), a non-DNA binding protein. The increased abundance of SHP causes it to associate with liver receptor homolog (LRH)-1, an obligate factor required for transcription of CYP7A1. Recent studies show there is an "FXR/ SHP-independent" mechanism that also represses CYP7A1 expression. This "FXR/SHP-independent" pathway involves the interaction of bile acids with liver macrophages (i.e., Kupffer cells), which induces the expression, and secretion of cytokines. These inflammatory cytokines, which include tumor necrosis factor ␣ and interleukin-1 ␤ , act upon liver parenchymal cells causing a rapid repression of the CYP7A1 gene. -

Two enzymes of mammalian cellular mevalonate biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, have been shown to be regulated by exogenous sterols. It has been demonstrated that these enzymes are regulated, at least in part, by transcriptional control of their synthesis. We have previously described a somatic cell mutant (CR1) of the CHO-K1 cell line that is defective in regulation of the activity of these enzymes in response to exogenous sterols. In this report, we demonstrate that this mutant is defective in regulation of the mRNA levels for HMG-CoA reductase and HMG-CoA synthase by 25-hydroxycholesterol and mevinolin. In the case of HMG-CoA reductase, this loss of apparent transcriptional control is not accompanied by a comparable loss in regulation of synthesis of this enzyme. This observation is consistent with prior studies suggesting that HMG-CoA reductase can be regulated translationally. We also show that CR1 cells exhibit a constitutively rapid rate of degradation of HMG-CoA reductase.