From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced. ANABAENA has nine DNA MTases. Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M. AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.

Objective— To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice. Methods and Results— SR-BI deficient (−/−) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI −/− × CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI −/− mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI −/− × CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet ( P <0.003). Conclusions— Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

The scavenger receptor BI (SR-BI) is highly expressed in hepatocytes, where it mediates the uptake of lipoprotein cholesterol, promotes the secretion of cholesterol into bile, and protects against atherosclerosis. Despite a strong correlation between the hepatic expression of SR-BI and biliary cholesterol secretion, little is known about SR-BI trafficking in response to changes in sterol availability. Using a well characterized polarized hepatocyte cell model, WIF-B, we determine that in cholesterol-depleted cells, SR-BI is extensively located on the basolateral surface, where it can access circulating lipoproteins. However, in response to cholesterol loading, SR-BI undergoes a slow transcytosis to the apical bile canaliculus independently of lipoprotein binding and new protein synthesis. In cholesterolreplete WIF-B cells, SR-BI that resides on the canalicular membrane is dynamically associated with defined microdomains and does not rapidly recycle to and from the subapical or basolateral regions. Taken together, these data demonstrate that hepatic SR-BI transcytosis is regulated by cholesterol and suggest that SR-BI has a stationary function on the bile canaliculus. High density lipoproteins (HDL)2 have a functional role in the protection against cardiovascular disease. HDL mediates both cholesterol removal from lipid-laden macrophages and delivery to the liver for subsequent biliary secretion in a process termed "macrophage reverse cholesterol transport." The cholesterol removal is mediated, in part, by the well established HDL receptor, scavenger receptor class BI (SR-BI) (1) (reviewed in Ref. 2).SR-BI is a two-transmembrane domain cell surface glycoprotein with short intracellular N-and C-terminal domains (3). The receptor is ubiquitously expressed at low levels and is highly expressed in steroidogenic, intestinal, and hepatic tissues (1). SR-BI binds a large array of ligands, including HDL and native or modified low density lipoproteins (LDL).Hepatic SR-BI protects against atherosclerosis by promoting the final stages of macrophage reverse cholesterol transport (5). In contrast to the holo-particle uptake of the LDL receptor pathway, SR-BI mediates cholesterol, cholesteryl ester, and phospholipid uptake via a selective pathway whereby lipids are transferred down their concentration gradient through a hydrophobic channel into the membrane (6). Importantly, if the concentration gradient is reversed, SR-BI can also perform cholesterol efflux (7).Although lipid delivery to and from lipoprotein particles occurs mainly on the plasma membrane, both SR-BI and HDL have been shown to internalize into and recycle from endocytic compartments (8 -10). SR-BI also localizes on the apical domain in gall bladder epithelial cells (11), testicular Sertoli cells (12), isolated primary mouse hepatocytes (13), primary mouse hepatocyte couplets (8), and hepatic tissues sections (14) and has been shown to undergo regulated transcytotic movement in polarized Madin-Darby canine kidney cells (15).Our laboratory has recently demonstra...

Previously, we have reported that thalidomide (Thd) can enhance neutrophil function in female B6C3F1 mice. The present study was intended to evaluate the mechanisms underlying the enhanced neutrophil responses following Thd treatment intraperitoneally (100 mg/kg) for 14 or 28 days. Treatment with Thd increased the numbers of neutrophils in the spleen, peripheral blood, bone marrow, peritoneal cavity and lung of female B6C3F1 mice when compared to the vehicle control mice. Thd treatment for 14 days increased the percentages and the number of neutrophils in the spleen in the first eight hours (peaking at 2 h) after the last Thd treatment, and it returned to the baseline after 24 h. However, Thd treatment for 28 days increased the percentages and number of neutrophils in the spleen even at the 24-h time point after the last Thd treatment. These neutrophils were demonstrated to be functional by the myeloperoxidase activity assay. Further studies have ruled out the possibility of an increased bone marrow granulopoiesis following Thd treatment. Flow cytometric analysis of the surface expression of adhesion molecules suggested that Thd treatment for either 14 or 28 days decreased the surface expression of either CD18 or CD44 by bone marrow neutrophils. On the other hand, the surface expression of both CD18 and CD44 by splenic neutrophils was increased following Thd treatment for 28 days but not for 14 days. No effect was produced for other cell surface molecules such as CD62L and CD11a. It was possible that decreased surface expressions of CD18 and CD44 facilitated neutrophils' release from the bone marrow; increased surface expressions of CD44 and CD18 by splenic neutrophils after 28 days of Thd treatment increased their ability to remain in the periphery. Taken together, Thd treatment increased neutrophils in female B6C3F1 mice, at least partially, through differentially modulating the surface expression of CD18 and CD44 by the neutrophils in the bone marrow and spleen.

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