Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

Separation and localization of the four cysteine-949 residues in human alpha 2-macroglobulin using fluorescence energy transfer.

By use of the intermediate form (I-form) [Gettins, Crews, & Cunningham (1989) Biochemistry 28, 5613-5618], alpha 2-macroglobulin can be specifically labeled with fluorescent probes in a manner that allows the determination of the topology of the four thiol ester derived Cys949 residues within this large tetrameric protease inhibitor. Freshly prepared I-form alpha 2-macroglobulin was reacted with 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonate (1,5-I-AEDANS) to produce alpha 2-macroglobulin specifically and stoichiometrically labeled with 1,5-AEDANS (donor) at the two Cys949 SH groups in the first protease interaction site. Upon subsequent reaction of this labeled species with chymotrypsin, the remaining two bait regions and thiol ester linkages were opened, generating two free SH groups on the two Cys949 residues in the second protease interaction site. These SH groups were specifically and stiochiometrically labeled with 5-(iodoacetamido)fluorescein (acceptor). Fluorescence energy transfer from donor to acceptor results in 82% loss of AEDANS fluorescence intensity. By use of an R0(2/3) value of 43.5 A, calculated from the spectral parameters of this system, an R(2/3) separation between donor and acceptor of 33.9 A was calculated. From fluorescence anisotropy measurements of both donor and acceptor attached to alpha 2-macroglobulin, upper and lower limits on the separation of 43.4 and 26.1 A, respectively, were calculated. These separations, small in the context of the alpha 2-macroglobulin tetramer, which has approximate dimensions of 190 x 90 x 90 A, severely restrict the possible locations of the four Cys949 residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.     

Crab shell-crushing predation and gastropod architectural defense

The shell-breaking behavior of the crabs Ozius verreauxii Saussure 1853 and Eriphia squamata, Stimpson 1859 from the Bay of Panama is described. The master claws of both these crabs are well designed for breaking shells. Small shells, relative to the size of a crab predator, are crushed by progressively breaking off larger segments of a shell's apex, while larger shells are peeled by inserting a large dactyl molar into the aperture of a shell and progressively chipping away the lip of the shell.

Heavy gastropod shells are shown to be less vulnerable to crab predators than lighter shells, and narrow shell apertures and axial shell sculpture are demonstrated to be architectural features that deter crab predation. The incidence of architectural features which deter crab predation appears to be higher for smaller gastropod species than for larger gastropods which are too large for most crab predators. Large fish predators prey upon both gastropods and shell-crushing crabs. To avoid fish predators, both these prey groups seek refuge under rocks when covered by the tide. Fish predation thus appears to enforce a close sympatry between smaller gastropods and their crab predators.     

Partial characterization of a Trypanosoma cruzi-released decomplementing factor

Trypanosoma cruzi releases a factor (SCAF) when grown in vitro which decomplements normal mouse, human, and guinea pig sera. The production and potency of SCAF was dependent on the density of cultured parasites, parasite viability and proliferative capacity, and duration of culture. The in vitro interaction between SCAF and serum complement (C') occurred rapidly and was complete within 30 min of mixing. The administration of SCAF to normal mice resulted in up to 50% reduction in hemolytic C' activity, whereas SCAF had no effect on the C' levels in mice infected wit T. cruzi for more than 10 days. The active moiety of SCAF was shown to be a nonproteinaceous substance(s) with a molecular weight of approximately 23,000 daltons.     

Validation of a primary production model of the desert shrub Larrea tridentata using soil-moisture augmentation experiments

Summary In previous papers we have described and verified a primary production model of the desert shrub Larrea tridentata. Here we address the validation phase of the evaluation of this model. Two versions of the model which differ in the priority scheme used for allocating carbon to reproductive or vegetative organs were compared on the basis of their usefulness and reliability over a range of soil-moisture conditions. Over an entire growing season when soil-moisture conditions were near normal both versions of the model were adequate predictors of total above-ground vegetative growth and one was an adequate predictor of reproductive growth as well. A more detailed analysis revealed that the versions varied in the range of soil-moisture conditions over which they were adequate and that neither was adequate when soil-moisture had remained high for extended periods. The validation process has revealed some likely areas for model improvement to increase adequacy.     

Trypanosoma cruzi-induced suppressor substance III. Activation of suppressor cells

Serum from mice infected with Trypanosoma cruzi (SSS) is known to interact with normal spleen cells to induce an immunosuppressed condition and activate splenic suppressor cells. The induction of immunosuppression by SSS was shown to be independent of, and precede the activation of, suppressor cells. Suppressor-cell activation, however, was demonstrable only after the induction of immunosuppression. Furthermore, mice that were given two aliquots of SSS at different intervals of time, exhibited suppression of humoral responses of similar duration and magnitude, regardless of the SSS transfer regimen, whereas both the length and degree of suppressor-cell activity was critically dependent on the interval of time between SSS transfers. SSS interacted with spleen cells via a trypsin-sensitive membrane site which was regenerable within a 4- to 5-hr period, yet the suppressive effects of SSS on spleen cells following interaction was resistant to treatment with trypsin. The interaction between SSS and spleen cells during brief adsorption protocols leads to immunosuppression only because extensive washing of SSS-treated spleen cells did not reverse the immunosuppression process, but did prevent the development of detectable suppressor cells. The phenomenon of suppressor-cell activation was further distinguished from immunosuppression in that supernates from culture of spleen cells derived from SSS-treated mice or T. cruzi-infected contained a factor that activated suppressor cells, but did not directly induce a state of suppression in the responding cell population.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

The synthesis and cytotoxic activity of 1,3,4-tri-O-acetyl-2,6-dideoxy-L-arabino- and -L-lyxo-hexopyranose

Methyl 2,6-dideoxy-α-L-arabino-hexopyranoside (6) was prepared from L-rhamnose in five steps. Hydrolysis of6 with 50% aqueous acetic acid gave 2,6-dideoxy-L-arabino-hexopyranose. Treatment of 3,4-di-O-acetyl-L-rhamnal with acetic acid in the presence of acetic anhydride and 2% sulfuric acid afforded 1,2,3-tri-O-acetyl-2,6-dideoxy-L-arabino-hexopyranose in 65% yield. Selective benzoylation and subsequent mesylation of 6 afforded methyl 3-O-benzoyl-2,6-dideoxy-4-O-mesyl-α-L-arabino-hexopyranoside, which was treated with sodium benzoate and sodium azide in hexamethylphosphoric triamide to give the corresponding 3,4-dibenzoyl 9 and 4-azido 11 analogs. Hydrogenation and N-acetylation of 11 afforded the 4-acetamido derivative 12. Deprotection of 9 and 12 gave 2,6-dideoxy-L-lyxo-hexopyranose and 4-acetamido-2,4,6-trideoxy-L-lyxo-hexopyranose, which were characterized as their peracetates. The free and corresponding peracetylated derivatives were assayed for their ability to inhibit the growth of P388 leukemia cells in culture. Although the free sugars did not inhibit the replication of these tumor cells under the conditions employed, their peracetylated derivatives demonstrated significant activity.