The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
The Ras target AF-6 has been shown to serve as one of the peripheral components of cell–cell adhesions, and is thought to participate in cell–cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of ∼220 kD (p220) to investigate the function of AF-6 at cell–cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell–cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.
Liver is composed of various kinds of cells, including hepatic parenchymal cells (hepatocytes) and nonparenchymal cells, and separation of these cells is essential for cellular therapies and pharmacological and metabolic studies. Here, we present microfluidic devices for purely hydrodynamic and size-dependent separation of liver cells, which utilize hydrodynamic filtration. By continuously introducing cell suspension into a microchannel with multiple side-branch channels, cells smaller than a specific size are removed from the mainstream, while large cells are focused onto a sidewall in the microchannel and then separated into two or three groups. Two types of PDMS-glass hybrid microdevices were fabricated, and rat liver cells were successfully separated. Also, cell size, morphology, viability and several cell functions were analyzed, and the separation performances of the microfluidic devices were compared to that of a conventional centrifugal technique. The results showed that the presented microfluidic devices are low-cost and suitable for clinical use, and capable of highly functional separation with relatively high-speed processing.
Abstract-Postprandial hyperlipidemia (PH) is recognized as a significant risk factor for cardiovascular disease. The present study, involving rats with streptozotocin (STZ)-induced diabetes, was performed to establish a PH model and to examine the relation between small intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and serum lipid levels in the postprandial state. The small intestinal ACAT activities in normal rats during the experimental period were 4 to 5 pmol/mg protein per minute. In contrast, in the diabetic rats, the ACAT activities were 2 to 3 times higher than activities seen in normal rats from 7 to 21 days after the STZ injection in the absence of a high fat diet and hyperplasia in the gut. In an oral fat-loading test that used diabetic rats that had been injected with STZ (60 mg/kg) intravenously 14 days previously, the postloading changes in the serum concentrations of total cholesterol (TC) and triglyceride (TG) were significantly greater in the diabetic rats than in normal rats. Single oral administration of (1s,2s F-1394, 3 to 30 mg/kg), a potent ACAT inhibitor, suppressed the post-fat-loading elevation of serum TC levels in the diabetic rats in a dose-dependent manner without affecting serum glucose levels. Furthermore, the small intestinal ACAT activity, serum TG levels, and lymphatic absorption of TC and TG in the rats that were administered F-1394 (30 mg/kg) were reduced by Ϸ90%, 70%, 30%, and 15%, respectively. This is the first evidence that elevated ACAT activity in the gut, unlike hyperplasia and hyperphagia, induces PH in rats. Our results strongly suggest that F-1394 may be a potential treatment for PH in humans. C oronary heart disease (CHD) resulting from premature atherosclerosis is a major cause of death in insulindependent and non-insulin-dependent diabetic patients. 1,2 Abnormalities in the metabolism of plasma lipoproteins and lipids in the postprandial state have been reported in diabetic patients, 3-9 and now evidence is being accumulated indicating that postprandial increases in triglyceride (TG)-rich lipoprotein, 10,11 which is known as one of the atherogenic remnant lipoproteins, 12,13 TG, 14,15 and cholesterol 16 remarkably contribute to the occurrence of CHD in such patients.A number of studies have reported that rats with streptozotocin (STZ)-induced diabetes (STZ-diabetic rats) shows severe hyperlipidemia after exogenous fat loading. [17][18][19] It has been suggested that the severe hyperlipidemia occurring in STZ-diabetic rats fed a high fat diet could be attributable to a marked increase in fat absorption via the gut, which in turn could be due to an abnormal increase in small intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) activity. 20 Indeed, repeated administration of an ACAT inhibitor, such as 21 CL-277082, 22,23 or FR 145237, 24 markedly decreases both the serum total cholesterol (TC) level and enhanced small intestinal ACAT activity in STZ-diabetic rats fed a high fat diet. However, previous investigators using diabetic mo...
The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and H) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and H resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosomelike structures. Peaks I and H were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosomal structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.
These results imply the usefulness of hepatocyte sheets in ectopic transplantation, as well as the need of trophic factors for hepatocyte survival.
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