Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif

Scavenger receptor, class B, type I (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester without the uptake and degradation of the particle. In transfected cells SR-BI recognizes HDL, low density lipoprotein (LDL) and modified LDL, protein-free lipid vesicles containing anionic phospholipids, and recombinant lipoproteins containing apolipoprotein (apo) A-I, apoA-II, apoE, or apoCIII. The molecular basis for the recognition of such diverse ligands by SR-BI is unknown. We have used direct binding analysis and chemical cross-linking to examine the interaction of murine (m) SR-BI with apoA-I, the major protein of HDL. The results show that apoA-I in apoA-I/palmitoyl-oleoylphosphatidylcholine discs, HDL(3), or in a lipid-free state binds to mSR-BI with high affinity (K(d) congruent with 5-8 microgram/ml). ApoA-I in each of these forms was efficiently cross-linked to cell surface mSR-BI, indicating that direct protein-protein contacts are the predominant feature that drives the interaction between HDL and mSR-BI. When complexed with dimyristoylphosphatidylcholine, the N-terminal and C-terminal CNBr fragments of apoA-I each bound to SR-BI in a saturable, high affinity manner, and each cross-linked efficiently to mSR-BI. Thus, mSR-BI recognizes multiple sites in apoA-I. A model class A amphipathic alpha-helix, 37pA, also showed high affinity binding and cross-linking to mSR-BI. These studies identify the amphipathic alpha-helix as a recognition motif for SR-BI and lead to the hypothesis that mSR-BI interacts with HDL via the amphipathic alpha-helical repeat units of apoA-I. This hypothesis explains the interaction of SR-BI with a wide variety of apolipoproteins via a specific secondary structure, the class A amphipathic alpha-helix, that is a common structural motif in the apolipoproteins of HDL, as well as LDL.     

Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3).

Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL-2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells.     

Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.     

Receptor interaction between eastern equine encephalitis virus and chicken embryo fibroblasts.

The attachment of eastern equine encephalitis virus to chicken embryo fibroblasts was studied at 0 degrees C. The binding specifically responsible for initiating infection was studied in the initial experiments by employing plaque-forming ability as the measured response. Results from these initial studies were closely paralleled in studies of binding of radiolabeled virus under the same conditions. Binding that had occurred at the pH optimum, pH 6.5, could be reversed only at higher pH. The observed pH dependence of virus attachment suggested the interaction of at least two ionizable species in the initial binding of virus to cell, and that one to three attachments must occur between virus and cell prior to infection.     

Limb regeneration and survival of prolactin treated hypophysectomized adult newts

Hypophysectomized adult newts exhibited 98% survival and limb regeneration at 23 days post-hypophysectomy when injected intraperitoneally every other day with prolactin (0.015 U/newt) and kept continuously in aquaria with 1 × 10^?7 concentration of thyroxine. Thyroxine alone was no more effective than saline injections. Prolactin (1.2 U/newt every other day) alone increased survival and limb regeneration, but less effectively than did the prolactin-thyroxine combination.     

Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas.

BACKGROUND: A lack of standardized assays and consensus of cell definition has lead to a wide variation in the reported range of circulating endothelial cells (CECs). METHODS: An automated rare cell analysis system was used to enumerate nucleated, CD146+/CD105+/CD45- CECs in 4 mL of blood. RESULTS: Recoveries of spiked HUVECs were linear over a range of 0-1,241 cells (R2>or=0.99) with recoveries of >or=70% at each spike level. Correlation coefficient values for interoperator variability and duplicate sample variation were (R2=0.99 and 0.90), respectively. Correlation of CEC counts between tubes 1-2 and 2-3 drawn from the same subject in sequence differed (R2=0.48 and 0.63, respectively). The normal CEC reference range established in 249 healthy donors was 1-20 CECs/mL blood. CEC counts were significantly higher in the 206 metastatic carcinoma patients (P<0.0001). CONCLUSION: CECs can be accurately and reproducibly enumerated in blood and are elevated in metastatic carcinomas compared with healthy donors. Phlebotomy procedures can affect endothelial cell counts.