Biomedical subjects

J W Bulock

Publications and source records attributed to J W Bulock.

4 recordsLinked to original sources

PubMed1991

An LRE (leucine-arginine-glutamate)-dependent mechanism for adhesion of neurons to S-laminin.

S-laminin is a homolog of laminin that is concentrated in the synaptic cleft of the neuromuscular junction. We previously showed that the tripeptide LRE is a crucial determinant for binding of ciliary motoneurons to recombinant s-laminin. Here, we describe a neuroblastoma-spinal neuron hybrid cell line, NSC-34, that binds to an LRE-containing s-laminin fragment and to a synthetic LRE-protein conjugate. NSC-34 cells exhibit several properties of motoneurons; other cell lines tested were not motoneuron-like and did not display LRE-dependent adhesion. We therefore used NSC-34 cells to characterize the LRE-dependent adhesion mechanism. Inhibition studies with a series of 20 tripeptide LRE analogs showed that the cells exhibit a high degree of selectivity for LRE, and suggested that ligand binding requires a combination of electrostatic and hydrophobic interactions. The effects of cations on LRE-dependent adhesion are unlike those of previously described adhesion molecules including the integrins, a family of receptors for extracellular matrix proteins, including laminin. Specifically, adhesion to LRE does not require divalent cations and is inhibited by Ca2+ (but not by Mg2+) in the physiological range. In contrast, adhesion of NSC-34 cells to laminin is LRE- and Ca2+ independent but Mg2+ dependent, and appears to be mediated by integrins. Additionally, experiments using mixed substrates demonstrated that LRE-protein conjugates inhibit neurite outgrowth promoted by laminin. Finally, we show that, under ionic conditions that minimize integrin-dependent adhesion, NSC-34 cells bind to s-laminin-rich basal laminae in tissue sections in an LRE-dependent manner. Together, these results suggest that LRE comprises a motoneuron-selective adhesion site that is accessible in native basal laminae and that acts to inhibit neurite outgrowth.

Amino Acid Sequence↗

PubMed1990

Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells. A comparative study.

A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.

Carcinoma, Hepatocellular↗

PubMed1990

Multiple peptide synthesis using a single support (MPS3).

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.

Amino Acid Sequence↗

PubMed1989

Primary sequence of a motor neuron-selective adhesive site in the synaptic basal lamina protein S-laminin.

S-laminin, a novel homolog of laminin, is concentrated in a subset of basal laminae including the basal lamina that passes between motor nerve terminals and muscle fibers at the neuromuscular junction. Here we used recombinant fragments to localize a neuronal attachment site to the C-terminal 10% of s-laminin. We then used synthetic peptides spanning the active fragment to identify the primary sequence of the adhesive site as Leu-Arg-Glu (LRE): neurons attach to an immobilized LRE-containing peptide, and soluble LRE blocks attachment of neurons to the s-laminin fragment. Whereas ciliary ganglion neurons (which normally innervate muscle fibers) adhered well both to laminin and to an s-laminin fragment, sensory and central neurons and several neuronal cell lines all adhered well to laminin but poorly to the s-laminin fragment. Together, these results define a motor neuron-selective attachment site on s-laminin.

Amino Acid Sequence↗