Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.     

Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein

The bile salt-activated farnesoid X receptor (FXR; NR1H4) controls expression of several genes considered crucial in maintenance of bile salt homeostasis. We evaluated the physiological consequences of FXR deficiency on bile formation and on the kinetics of the enterohepatic circulation of cholate, the major bile salt species in mice. The pool size, fractional turnover rate, synthesis rate, and intestinal absorption of cholate were determined by stable isotope dilution and were related to expression of relevant transporters in the livers and intestines of FXR-deficient (Fxr-/-) mice. Fxr-/- mice showed only mildly elevated plasma bile salt concentrations associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA levels of the bile salt export pump were decreased. Cholate pool size and total bile salt pool size were increased by 67 and 39%, respectively, in Fxr-/- mice compared with wild-type mice. The cholate synthesis rate was increased by 85% in Fxr-/- mice, coinciding with a 2.5-fold increase in cholesterol 7alpha-hydroxylase (Cyp7a1) and unchanged sterol 12alpha-hydroxylase (Cyp8b1) expression in the liver. Despite a complete absence of ileal bile acid-binding protein mRNA and protein, the fractional turnover rate and cycling time of the cholate pool were not affected. The calculated amount of cholate reabsorbed from the intestine per day was approximately 2-fold higher in Fxr-/- mice than in wild-type mice. Thus, the absence of FXR in mice is associated with defective feedback inhibition of hepatic cholate synthesis, which leads to enlargement of the circulating cholate pool with an unaltered fractional turnover rate. The absence of ileal bile acid-binding protein does not negatively interfere with the enterohepatic circulation of cholate in mice.     

Crystal structure of octakis(2,3,6-tri-O-methyl)-gamma-cyclodextrin x 4.5 H2O: evidence for conformational flexibility of permethylated cyclodextrins

Octakis(2,3,6-tri-O-methyl)-gamma-CD (TRIMEG) cocrystallized at 18 degrees C with 4.5 water molecules in the orthorhombic space group P2(1)2(1)2(1), unit cell dimensions a = 10.7879(3), b = 29.0580(9), c = 32.2909(11) A. The TRIMEG macrocycle is in a 'round' form with all glucose units oriented syn, and one O-6-CH3 methoxy group points 'toward' the molecular cavity. The TRIMEG x 4.5 H2O molecules are stacked to form infinite cylinders with the central cavities aligned into channels filled for each TRIMEG by 4.5 water molecules distributed over 15 partially occupied sites. This structure differs from the two known structures of TRIMEG in which two diametrically opposed glucoses are oriented anti to yield an 'elliptical' form, and their O-6-CH3 groups are directed 'toward' the cavity and close it at this side to form a bowl-shaped molecule.     

Adaptation of the food-borne pathogen Bacillus cereus to carvacrol

Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations. A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol. Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells. Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C. The addition of carvacrol to cell suspensions of adapted B. cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol. During adaptation, changes in the fatty acid composition were observed. The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased. The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells. It could be concluded that B. cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.     

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.     

Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (?/?) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [¹³C]-linoleic acid absorption was determined by measuring [¹³C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [¹³C]-linoleic acid at 2 h after enteral administration was significantly lower in (?/?) compared to (+/+) mice (P≤0.05), net intestinal absorption of dietary linoleic acid or of [¹³C]-linoleic acid was similar in (+/+) and (?/?) mice. Molar percentage of linoleic acid and the triene:tetraene ratio were not different in whole plasma or in liver of (?/?) compared to (+/+) mice. Present data indicate that biliary phospholipids are involved in the rate of appearance in plasma of enterally administered linoleic acid, but are not required for net intestinal absorption or plasma status of linoleic acid.