It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A-and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.
Uremic toxins have been suggested to promote progression of chronic renal failure by damaging tubular cells. Previous in vitro studies have indicated that some uremic toxins induce oxidative stress and activate NF-ĸB to upregulate plasminogen activator inhibitor-1 in tubular cells. These mechanisms may promote tubulointerstitial fibrosis. The present study examined whether uremic toxins induce glomerular and tubulointerstitial damage in vivo. Two uremic toxins, hippuric acid (HA) or indoleacetic acid (IAA), were tested in two independent experiments (HA-treated rats vs. non-HA-treated controls, IAA-treated rats vs. non-IAA-treated controls). The uremic toxins were administered to subtotally nephrectomized rats. Renal functions were measured periodically and glomerular sclerosis and interstitial fibrosis were examined at the end of the experimental period (18 and 24 weeks, respectively, after subtotal nephrectomy for HA and IAA treatments). Glomerular filtration rate (inulin clearance) at the end of the study period was significantly lower in uremic toxin-treated rats than in control rats (HA-treated rats: 0.090 ± 0.004 ml/min/100 g body weight vs. non-HA-treated controls: 0.125 ± 0.013, IAA-treated rats: 0.068 ± 0.006 versus non-IAA-treated controls: 0.100 ± 0.013; both p < 0.05). Beta-N-acetyl-glucoseamidase excretion was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.55 ± 0.05 U/day vs. control: 0.39 ± 0.04 at week 18, IAA-treated: 0.35 ± 0.02 vs. control: 0.26 ± 0.07 at week 16; both p < 0.05). Glomerular sclerosis index was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.85 ± 0.16 versus control: 0.48 ± 0.10, IAA-treated: 1.13 ± 0.25 vs. control: 0.57 ± 0.10; both p < 0.05). Significant enlargement of interstitial fibrosis was observed in indoleacetic acid-treated rats. These results indicate that overload of uremic toxins accelerates the loss of kidney function, glomerular sclerosis and tubulointerstitial injury in a rat model of chronic renal failure. The present study suggests the potential benefit of early intervention to remove various uremic toxins in delaying the onset of end-stage renal failure in patients with progressive renal disease.
Several common modes of crystal growth provide particularly simple and elegant examples of spontaneous pattern formation not only in nature but also under artificial circumstances. We have already reported that well-organized ZnO whiskers are epitaxially grown using a chemical vapor deposition technique [Satoh et al..: Jpn. J. of Appl. Phys. 38 (1999) L586]. One aim of this study is to determine the optimum growth conditions for obtaining the structure containing homogeneous whiskers grown with a relatively high growth rate. A substrate temperature of 550°C and a vaporizing temperature of 125°C are the most appropriate for obtaining homogeneous whiskers. Whiskers are highly oriented in the a-and c-axes directions of the hexagonal structure. The growth rate reached a maximum value as high as 7.5 nm/s.
1 5-Hydroxytryptamine (5-HT; 1 nM ± 100 mM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC 50 ; 68 ± 84 nM) was more sensitive than the longitudinal muscle (EC 50 ; 1.3 ± 1.44 mM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the eects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations. 2 Pretreatment with tetrodotoxin (1 mM), propranolol (1 mM), atropine (1 mM), guanethidine (10 mM) or L-NAME (100 mM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells. 3 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT45-HT45-MeOT48-OH-DPAT. On the other hand, oxymethazoline, a-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no eect on spontaneous contractions, even at 10 ± 100 mM. 4 Inhibition by 5-HT was not decreased by either pindolol (1 mM), ketanserin (1 mM), tropisetron (10 mM), MDL72222 (1 mM) or GR113808 (10 mM), but was antagonized by the following compounds in a competitive manner (with pA 2 values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86). 5 Ro 20-1724 (20 mM) and rolipram (10 mM) signi®cantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 mM) nor dipyridamole (10 mM) altered the response of 5-HT. 6 5-HT (1 nM ± 1 mM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle. 7 From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT 7 receptor, mediates the inhibitory eect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT 7 receptors.
ZnO whiskers were epitaxially grown by a chemical-vapor deposition technique employed at atmospheric pressure. Highly oriented ZnO whiskers grew at a substrate temperature of 550°C on (0001)α-Al2O3 substrates with a growth rate of 3.7 nm/s. X-ray diffractometry revealed that the epitaxial relationship between the whiskers and the substrate was determined as ZnO[1010](0001)//Al2O3[1210](0001) or ZnO[1210](0001)//Al2O3[1010](0001). In addition, the full-width at half maximum value of the (0002) reflection was as low as 0.43°. Images obtained using a scanning electron microscope were analyzed and it was found that the whisker tip likely has a radius of curvature of approximately 20 nm. The typical number density of the whiskers has reached 1.3×105mm-2.
The mineralization and biodegradation of cerebrospinal fluid shunting systems were studied using material from 25 shunts that had been implanted for between 6 days and 10 years. New unused materials were also examined for comparison. Surface changes in six systems could be observed under an operating microscope. Substantial quantities of a white deposit had adhered to the tubing in four of the shunts. These changes were most advanced in the galeal penetrative portion of the shunts and are believed to have been caused by mechanical stress. Scanning electron microscopic analysis revealed surface wrinkles, microscopic holes, and tiny particles, suggesting deterioration of the material itself. An energy-dispersive analysis using x-rays demonstrated that the surface deposits were due to mineralization of calcium phosphate and that the tiny particle growth was aluminum. These changes may be a consequence of the degradation of silicone rubber. A discriminant analysis of the mineralization was carried out; thus, the age of the host and the duration of system implantation could be correlated with the incidence of mineralization (p less than 0.1). A measurement of the physical properties showed progressive change with a remarkable deterioration in systems implanted for more than 5 years.
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