IntroductionAn enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques.

Tissue needs for retinoids are believed to be satisfied through the delivery in the circulation of retinol by its specific plasma transport protein, retinol-binding protein (RBP), which circulates as a 1-to-1 protein complex with transthyretin (TTR). The binding of RBP to TTR is thought to prevent filtration of retinol-RBP in the kidney and to play a role in secretion of RBP from hepatocytes. Recently a strain of mice (TTR-) that totally lacks immunoreactive TTR was produced by targeted mutagenesis. We have explored the effects of TTR deficiency on retinol and RBP metabolism in this mutant strain. In pooled plasma from the TTR- mice retinol levels averaged 6% of those of wild type animals. Similarly, plasma RBP in the TTR- mice was found to be 5% of wild type levels. Hepatic retinol and retinyl ester levels were similar for mutant and wild type mice, suggesting that the mutation affects neither the uptake nor storage of dietary retinol. Levels of retinol and retinyl esters in testis, kidney, spleen, and eye cups from TTR- mice were normal. Plasma all-trans-retinoic acid levels for the TTR- mice were 2.3-fold higher than those of wild type (425 versus 190 ng/dl). Kidney RBP levels were similar for the mutant and wild type mice and we were unable to detect intact RBP in urine from TTR- mice. Hepatic RBP levels in the TTR- mice were 60% higher than those of wild type mice (39.8 versus 25.0 micrograms of RBP/g of tissue). These data may suggest that there is a partial blockage in RBP secretion from TTR- hepatocytes that leads to lessened plasma levels of retinol-RBP.

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Monoclonal antibody (mAb) 5C3 directed against human p140 TrkA is a structural and functional mimic of nerve growth factor (NGF) and an artificial receptor agonist. mAb 5C3 binds in the NGF-docking site and, like NGF, it promotes TrkA internalization, TrkA and phosphatidylinositol-3 kinase tyrosine phosphorylation, and increased transformation of TrkA-expressing fibroblasts. More important, mAb 5C3 protects human TrkA-expressing cells from apoptotic death in serum-free media. Interestingly, agonistic activity is observed with monomeric F(ab) 5C3 fragments. mAb 5C3 (Kd approximately 2 nM) was used to study features of ligand binding by TrkA and the distribution of TrkA protein in normal human brain.

The conformation and internal dynamics of a bioactive cyclic peptide, N-acetyl-YCTDEKQCY, derived from the C-D loop of ␤-nerve growth factor (␤-NGF) were analyzed by solution NMR spectroscopy. NMR experimental data were used to calculate an ensemble of peptide structures. All of the structures had a ␤-turn at residues Asp 4 -Gln 7 but could be divided into two families according the presence or absence of a hydrogen bond at Gln 7 . Comparison of the calculated structures with the corresponding C-D loops from the x-ray structures of the NGF revealed striking similarity. The orientation of Glu 5 , Lys 6 , and Gln 7 side chains in the NGF mimetic was very similar to the C-D loop of NGF. These residues are known to participate in interactions with the TrkA receptor. Relaxation measurements of the peptidomimetic ␣-carbons at 13 C natural abundance and calculated dynamic parameters suggest that the loop region of peptide is well structured but that residues Thr 3 , Asp 4 , Glu 5 , and Lys 6 undergo slow conformational exchange. These results suggest that conformational similarity and possibly peptide dynamics are responsible for the bioactivity of the peptide. Nerve growth factor (NGF)1 is a member of the neurotrophin family of polypeptide growth factors. NGF promotes the growth and survival of sympathetic, trigeminal, dorsal root ganglia neurons and cholinergic neurons of the basal forebrain. Two cell surface neurotrophin receptors have been characterized: the low affinity p75 receptor (K D ϳ10 Ϫ9 M), which is common to all members of the neurotrophin family (1-3), and the intermediate affinity receptors (K D ϳ10 Ϫ11 M), TrkA, TrkB, and TrkC, which afford the binding specificity of the different neurotrophins (4 -7). TrkA is selective for NGF and has tyrosine kinase enzymatic activity that mediates most of the downstream signaling for this growth factor. Highest affinity binding of NGF (K D ϳ10 Ϫ12 M) occurs in cells coexpressing TrkA and p75 (4, 8).NGF, like other neurotrophins, is a dimer of two identical polypeptides of 118 residues (9). X-ray structures of NGF (10, 11), a brain-derived neurotrophic factor-neurotrophin-3 heterodimer (12), and the 7 S NGF complex (13) have been determined. The neurotrophin protomers have the same overall topology of seven ␤-strands connected by three disulfide bridges arranged in a cysteine knot motif. Comparison of the primary sequence of different neurotrophins shows clusters of high homology (Ͼ50% identity) and high diversity. The hypervariable domains occur in the exposed ␤-loops (residues 29 -35, 43-48, and 92-98), in an exposed reverse turn (residues 59 -66), in a solvent-exposed ␤-strand (residues 79 -89), and at both N and C termini. By analogy with findings from other growth factors, the hypervariable domains are believed to be responsible for Trk receptor selectivity