31 research outputs found
Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces
We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4,400; FD-10, MW 9,500; FD-40, MW 40,500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1 % of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient Papp for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher Papp values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the Papp of small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compounds absorption from the peritoneal cavity, judging from absorption clearance CLa calculated by utilizing the peritoneal organ surface area
Absorption of phenolsulfonphthalein as a model across the mesenteric surface in rats to determine the drug absorption route after intraperitoneal administration
The purpose of this study is to clarify absorption characteristics of a drug across the mesenteric surface which occupies a large area of absorption in the peritoneal cavity in order to determine the drug absorption route after intraperitoneal administration. Absorption of phenolsulfonphthalein (PSP) as a model after application to the mesenteric surface was investigated in rats, by employing a cylindrical diffusion cell attached to the mesentery with or without blood vessels. PSP was absorbed from the rat mesenteric surface, followed by its appearance in the plasma and bile, regardless of blood vessel existence. The absorption ratios of PSP in 6 h were calculated to be 92.1 % and 83.6 % from the mesenteric surface with and without blood vessels, respectively. We then employed an experimental system by sticking a polyethylene cap (PE cap) on the surface of the other side to exclude the influence of absorption of the drug from the other organ surfaces that penetrated across the mesentery. The PE cap-sticking decreased the appearance of PSP in the plasma from the mesenteric surface with blood vessels and eliminated the PSP absorption completely from the mesenteric surface without blood vessels. Accordingly, blood vessels on the mesenteric surface actually play an important role in drug absorption, but the contribution of the mesenteric surface to drug absorption from the peritoneal cavity is unlikely to be significant due to there being a small effective area of blood vessels
Absorption characteristics of model compounds with different molecular weights from the serosal caecal surface in rats
The purpose of this study is to clarify the absorption characteristics of drugs across the serosal cecal surface membrane occupying a large absorption area in the peritoneal cavity in rats. Absorptions of phenolsulphonphthalein (PSP) and fluorescein isothiocyanate-dextrans (FDs) as model drugs after application to the rat serosal cecal surface were investigated in rats, employing a cylindrical diffusion cell. PSP was absorbed from the rat serosal cecal surface, followed by appearance in the plasma and bile. The time course of the remaining PSP amount in the diffusion cell obeyed first-order kinetics, and its rate constant Ka was calculated to be 8.01 x 10-3 min-1. No significant difference was seen in the absorption ratio of PSP which was approximately 90 % in 6 h among three doses (0.3, 0.5 and 1 mg), suggesting a linearity of absorption. Moreover, the absorption ratios of FDs from the rat serosal cecal surface at 3 h decreased with an increase in the molecular weight (24.7% for FD-4, 12.8% for FD-10 and 3.4% for FD-40)
Binding of 14-3-3β but not 14-3-3σ controls the cytoplasmic localization of CDC25B: Binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B
The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3β, 14-3-3ε and 14-3-3σ, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3β bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3σ bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3β drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3σ and CDC25B did not affect the subeellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3β, even when other putative 14-3-3 binding sites were mutated. 14-3-3ε resembled 14-3-3β with regard to its binding to CDC25B and the control of CDC25B subcellujar localization. The results of the present study indicite that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3σ has effects on CDC25B other than the control of its subcellular localization
Delivery advantage to the unilateral kidney by direct drug application to the kidney surface in rats and pharmacokinetic verification based on a physiological model
The objective of this study was to evaluate the drug delivery advantage to the unilateral kidney by direct drug application to the rat kidney surface based on a physiological pharmacokinetic model. Under anesthesia, a cylindrical diffusion cell (i.d. 6 mm, area 0.28 cm(2)) was attached to the right kidney surface in rats. Phenolsulfonphthalein (PSP), an organic anion chosen as a model compound, was added into the diffusion cell. The free PSP concentration in the right (applied) kidney after application to the right kidney surface at a dose of 1 mg was significantly higher than that of the left (non-applied) kidney until 60 min after application. Similarly, the urinary excretion rate of free PSP from the applied kidney was much faster than that from the non-applied kidney, with a 2.6 times larger excreted amount in 240 min. These results imply the possibility that a considerable drug delivery advantage to the unilateral kidney could be obtained after direct absorption from the kidney surface. This tendency was also observed at the other application doses of 0.3 and 1.5 mg. On the other hand, fluorescein isothiocyanate dextran (Mw 4400, FD-4) was equally excreted into the urine from each kidney and the renal concentrations in the applied and non-applied kidneys were almost the same, possibly due to the involvement of passive transport for the absorbed FD-4, i.e. glomerular filtration. The computer simulations of free PSP concentrations in the plasma and each kidney based on a physiological model after kidney surface application were consistent with the respective experimental data. Moreover, the delivery advantage of kidney surface application of PSP was verified by its comparison with other routes such as i.v. and i.a. administrations
Studies on the Apg12-Apg5・Apg16 complex essential for autophagy
In eukaryotic cells, the majority of intracellular bulk degradation occurs in the vacuole/lysosome, an acidic compartment that contains various hydrolytic enzymes. Autophagy is the main pathway to deliver cytoplasmic components to the vacuole in yeast, or to the lysosome in mammalian cells, for degradation. In this process, portion of cytoplasm are sequestered within the double-membrane structure termed autophagosome, which subsequently fuses with the vacuole/lysosome. The sequestered components are, then, degraded by the vacuolar or lysosomal hydrolases for reuse. The best-known role of autophagy is a cellular survival response to starvation, but it also plays important roles in developmental process and cell differentiation. Taking advantage of yeast genetics, autophagy defective mutants (apg) were isolated and so far 15 APG genes have been cloned. All of them seem to be involved in the step of autophagosome formation. During the characterization of Apg proteins, a ubiquitin-like conjugation system, the Apgl2 system, was found to be essential for autophagy. In this system, Apg12 is covalently bound to Apg5, which is catalyzed by Apg7 and Apgl0. Mammalian homologues of Apgl2 and Apg5 have been identified and undergo a similar covalent linkage, indicating that this conjugation system is conserved in mammalian cells. Studies using mammalian cells further revealed that the Apg12-Apg5 conjugate localized to pre-autopagosomal membrane and is required for elongation of the membrane to form a complete spherical autophagosome. But the exact molecular role of the Apg12-Apg5 conjugate is still unknown. Recently, Apgl6 was found to interact with the Apg12-Apg5 conjugate in yeast. Apg16 is a 150-amino acid protein that contains a carboxyl-terminal coiled-coil motif. Since Apg16 is the only molecule identified to interact with the Apg12-Apg5 conjugate and required for function of the conjugate, further characterization of Apg16 would provide valuable insights into the molecular role of the Apg12-Apg5 conjugate. In this study, she found that Apg12-Apg5 conjugate formed a ~350-kDa complex with Apg16 in the cytosol of yeast cells, irrespective of autophagy induction. As Apg16 formed homo-oligomer through the coiled-coil region and cross-linked Apg12-Apg5, she generated an in vivo system that allows us to control the oligomerization state of Apg16. With this system, she demonstrated concretely that formation of the ~350-kDa complex depends on oligomerization of Apg16 and formation of this complex is required for autophagy in yeast. Although the Apg12-Apg5 conjugation system is highly conserved among eukaryotes, there is no significant homologue of Apg16. In mammalian cells, the Apg12-Apg5 conjugate forms a ~800-kDa complex, much larger than the yeast Apg12-Apg5・Apg16 complex. Purification of this ~800-kDa complex identified a novel Apg5-interacting protein containing seven WD repeats. She demonstrated that this newly WD repeat protein, named Apg16L, is a functional counterpart of yeast Apg16. Mouse Apg16L interacts with Apg5 at its amino-terminal region and forms a homo-oligomer through its coiled-coil region to form the ~800-kDa complex as yeast Apg16. Apg16L associates with pre-autophagosomal membrane with the Apg12-Apg5 conjugate, suggesting that the ~80O-kDa complex functions in autophagosome formations in mammalian cells. Since multiple homologues of Apg16L exist in higher eukaryotes, the autophagic machinery is well conserved through evolution. On the other hand, WD domain, which is thought to be a platform for protein-protein interaction, is not found in yeast Apg16. As the WD domain of Apg16L is not required for interaction with Apg5 and homo-oligomerization, the ~800-kDa complex is expected to interact with other proteins. Identification of such proteins would be helpful to understand the molecular mechanism of the Apg12-Apg5 conjugate in autophagosome formation