Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.     

General acid/base catalysis in the active site of Escherichia coli thioredoxin

Enzymic catalysts of thiol:disulfide oxidoreduction contain two cysteine residues in their active sites. Another common residue is an aspartate (or glutamate), the role of which has been unclear. Escherichia coli thioredoxin (Trx) is the best characterized thiol:disulfide oxidoreductase, and in Trx these three active-site residues are Cys32, Cys35, and Asp26. Structural analyses had indicated that the carboxylate of Asp26 is positioned properly for the deprotonation of the thiol of Cys35, which would facilitate its attack on Cys32 in enzyme-substrate mixed disulfides. Here, Asp26 of Trx was replaced with isologous asparagine and leucine residues. D26N Trx and D26L Trx are reduced and oxidized more slowly than is wild-type Trx during catalysis by E.coli thioredoxin reductase. Stopped-flow spectroscopy demonstrated that the cleavage of the mixed disulfide between Trx and a substrate is slower in the D26N and D26L enzymes. Buffers increase the rate of mixed disulfide cleavage in these variants but not in wild-type Trx. These results indicate that Asp26 serves as an acid/base in the oxidation/reduction reactions catalyzed by Trx. Specifically, Asp26 protonates (during substrate oxidation) or deprotonates (during substrate reduction) the thiol of Cys35. A similar role is likely filled by the analogous aspartate (or glutamate) residue in protein disulfide isomerase, DsbA, and other thiol:disulfide oxidoreductases. Moreover, these results provide the first evidence for general acid/base catalysis in a thiol:disulfide interchange reaction.     

Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells

The seasonal variation in the nutrient composition of Enhydra fluctuans and Marsilea quadrifolia, two edible semi-aquatic plants, was studied in order to promote their consumption as green leafy vegetables. Both plants had a high crude protein content throughout all harvesting seasons. Enhydra fluctuans had a low ash content and was a good source of beta-carotene (3.7 to 4.2 mg/100 g on a fresh weight basis). Marsilea quadrifolia exhibited wide fluctuations between seasons and was not very promising in nutrient composition when compared to other commonly used green leafy vegetables.     

A least mean square error synthesis with pattern constraints atmultiple frequencies

The pattern synthesis techinque described allows the power pattern to be optimized in a very useful sense over the desired operating band of the antenna array. This is an improvement over previous techniques involving constraints on such artificial parameters as source norm and superdirectivity ratio, which do not, in fact, ensure a useful pattern bandwidth. The procedure described provides minimum mean square error over an arbitrary number of discrete frequencies