Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.     

Invariant chain trimers are sequestered in the rough endoplasmic reticulum in the absence of association with HLA class II antigens

HLA class II antigens are heterodimeric cell surface glycoproteins that interact with antigenic peptides to form complexes recognizable by CD4-positive T cells. During their biosynthesis, class II antigens are retained in a post-Golgi compartment in association with the invariant chain, which dissociates before class II cell surface expression. To address whether the invariant chain mediates this post-Golgi retention, its transport and assembly were examined in cells that do not express HLA class II antigens. Pulse-chase analysis and endoglycosidase digestions showed that very little invariant chain proceeded as far as the trans-Golgi in class II-negative cell lines. Immunofluorescence studies suggested that in these cells the invariant chain is sequestered in the RER. Gel filtration and cross-linking data showed that RER-localized invariant chain is present as trimers or aggregated trimers. Multimerization is mediated by lumenal interactions; a proteolytic fragment of the invariant chain corresponding to the lumenal domain remained trimeric as determined by cross-linking analysis. Similar transport and structural characteristics were observed for a pool of excess invariant chain in class II-positive cells, suggesting that an excess of invariant chain in the ER may be important for class II antigen function. These results have important implications for the transport of cellular proteins in general and for the role of the invariant chain in class II antigen biosynthesis.     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

Osteoclast biology in the osteopetrotic (op) rat

Osteopetrosis is a metabolic bone disease characterized by reduced bone resorption. From experimental studies of various osteopetrotic mutations has emerged the hypothesis that each is unique with respect to mechanisms whereby osteoclast development and/or function are reduced. The osteopetrotic (op) mutation in the rat was discovered in Fatty/ORL stock over a decade ago. The paucity of data about osteoclast biology in this mutation prompted this study of cytological, cytochemical, and ultrastructural features of osteoclasts. In op rats, osteoclasts are significantly reduced in number, but are larger and more vacuolated than in normal littermates. Mutant osteoclasts can form ruffled borders and clear zones, but their ability to fragment and excavate bone surfaces is greatly impaired. Cytoplasmic vacuoles in op osteoclasts are randomly distributed and greatly enlarged, and they stain weakly for two cytochemical characteristics of osteoclasts, tartrate-resistant acid phosphatase and acid ATPase. These findings suggest that an abnormality in the lysosomal/vacuolar system, an important component of the resorptive mechanism, may be involved in the interception of osteoclast function in this mutation.     

Purification of L-[3H]nicotine eliminates low affinity binding

Some studies of L-[3H]nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicates that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-[3H]nicotine eliminates the low affinity site.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.     

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.