Determination of human body burden baseline date of platinum through autopsy tissue analysis.
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Results of analysis for platinum in 97 autopsy sets are presented. Analysis was performed by a specially developed emission spectrochemical method. Almost half of the individuals studied were found to have detectable platinum in one or more tissue samples. Platinum was found to be deposited in 13 of 21 tissue types investigated. Surprisingly high values were observed in subcutaneous fat, previously not considered to be a target site for platinum deposition. These data will serve as a human tissue platinum burden baseline in EPA's Catalyst Research Program. (+info)
Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes.
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Dysfunction of the pancreatic beta cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the beta cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the beta cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic beta cell and suggest that defects in insulin signaling at the level of the beta cell may contribute to the observed alterations in insulin secretion in type 2 diabetes. (+info)
Freeze-fracture replication of organized tissue without cryoprotection.
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Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas. (+info)
Further studies on the mechanism of adrenaline-induced lipolysis in lipid micelles.
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Lipase [EC 3.1.1.3] depleted lipid micelles, in which lipolysis was not elicited by adrenaline, were prepared from lipid micelles. When these lipase-depleted lipid micelles incubated with adipose tissue extract containing lipase activity, adrenaline-induced lipolysis was restored to almost the same level as that of native lipid micelles. Adrenaline-induced lipolysis was not restored when the lipase-depleted lipid micelles were homogenized or sonicated. Various tissue extracts from kidney, lung, liver, and pancreas, and post-heparin plasma, which contained lipase activity, restored adrenaline-induced lipolysis in lipase-depleted lipid micelles. (+info)
Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH.
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Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH approximately 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents. (+info)
His ... Asp catalytic dyad of ribonuclease A: histidine pKa values in the wild-type, D121N, and D121A enzymes.
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Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue. (+info)
Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT).
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Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates. (+info)
Cloning and characterization of a secreted frizzled-related protein that is expressed by the retinal pigment epithelium.
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The Wnt/frizzled cell signaling pathway has been implicated in the determination of polarity in a number of systems, including the Drosophila retina. The vertebrate retina develops from an undifferentiated neuroepithelium into an organized and laminated structure that demonstrates a high degree of polarity at both the tissue and cellular levels. In the process of searching for molecules that are preferentially expressed by the vertebrate retinal pigment epithelium (RPE), we identified secreted frizzled-related protein 5 (SFRP5), a member of the SFRP family that appears to act by modulating Wnt signal transduction. SFRP5 is highly expressed by RPE cells, and is also expressed in the pancreas. Within the retina, the related molecule SFRP2 is expressed specifically by cells of the inner nuclear layer. Thus, photoreceptors are likely to be bathed by two opposing gradients of SFRP molecules. Consistent with SFRP5 's postulated role in modulating Wnt signaling in the retina, it inhibits the ability of Xwnt-8 mRNA to induce axis duplication in Xenopus embryos. The human SFRP5 gene consists of three coding exons and it maps to chromosome 10q24.1; human SFRP2 maps to 4q31.3. Based on the biology and complementary expression patterns of SFRP2 and SFRP5, we suggest that they may be involved in determining the polarity of photoreceptor, and perhaps other, cells in the retina. (+info)