BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis.

The RAS/RAF signaling pathway is an important mediator of tumor cell proliferation and angiogenesis. The novel bi-aryl urea BAY 43-9006 is a potent inhibitor of Raf-1, a member of the RAF/MEK/ERK signaling pathway. Additional characterization showed that BAY 43-9006 suppresses both wild-type and V599E mutant BRAF activity in vitro.

Single-chain urokinase alone or complexed to its receptor in tetracycline-induced pleuritis in rabbits.

Intrapleural loculation can increase morbidity in hemothoraces or parapneumonic effusions. Intrapleural fibrin precedes visceral-parietal pleural adhesions. We speculated that single-chain urokinase plasminogen activator alone or bound to its receptor could prevent these adhesions by their relative resistance to local inhibition by plasminogen activator inhibitors.

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes.

Novel interactions between urokinase and its receptor.

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm).

Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity.

Bryodin 1 (BD1) is a type I ribosome-inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBD1) has been produced and crystallized. To gain insight into the relationship of rBD1 structure and function, we investigated the role of sequences in a region (residues 128-156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site.

Expression and characterization of bryodin 1 and a bryodin 1-based single-chain immunotoxin from tobacco cell culture.

Bryodin 1 (BD1) is a potent ribosome-inactivating protein (RIP) isolated from the plant Bryonia dioica. It is relatively nontoxic in rodents (LD50 > 40 mg/kg) and represents a potential improvement over other RIPs and bacterial toxins that have been used in immunotoxins. Recombinant BD1, expressed in Escherichia coli, localizes to insoluble inclusion bodies necessitating denaturation and refolding steps to generate active protein.

Construction, expression, and characterization of BD1-G28-5 sFv, a single-chain anti-CD40 immunotoxin containing the ribosome-inactivating protein bryodin 1.

The major limitation to the use of immunotoxins in the clinic is the toxicity associated with the toxin moiety. BD1-G28-5 single-chain Fv (sFv) is a single-chain immunotoxin targeted to human CD40 and consists of bryodin 1 (BD1), a plant ribosome-inactivating protein that is 20-30-fold less toxic in animals than commonly used toxins, fused to the sFv region of the anti-CD40 monoclonal antibody G28-5. This immunotoxin was expressed in Escherichia coli and purified from refolded inclusion bodies.

Molecular, biological, and preliminary structural analysis of recombinant bryodin 1, a ribosome-inactivating protein from the plant Bryonia dioica.

Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequence encoding BD1 was isolated from B. dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues.

Characterization of ribosome-inactivating proteins isolated from Bryonia dioica and their utility as carcinoma-reactive immunoconjugates.

Two ribosome-inactivating proteins (RIPs) were isolated and characterized from the roots of Bryonia dioica. One of these was a novel 27-kDa protein termed bryodin 2 (BD2), while the second was a previously reported RIP, referred to here as bryodin 1 (BD1). The amino-terminal sequence obtained for BD2 was similar, but distinct from BD1, ricin A chain, trichosanthin, and momorcharin.

In vivo activities of acidic fibroblast growth factor-Pseudomonas exotoxin fusion proteins.

Fibroblast growth factor receptors are highly expressed in a variety of cancer cells and activated vasculature. Using chimeric toxins targeted to cell-surface a FGF receptors, we have demonstrated specific cytotoxic activity to these cell types. These molecules, aFGF-PE40 and aFGF-PE40 KDEL, are fusion proteins containing acidic FGF and either a 40- or a 66-kDa binding defective form of Pseudomonas exotoxin, respectively.

Basic fibroblast growth factor-Pseudomonas exotoxin chimeric proteins; comparison with acidic fibroblast growth factor-Pseudomonas exotoxin.

We have constructed growth factor-toxin chimeric molecules composed of basic fibroblast growth factor (bFGF) and two different binding mutant forms of Pseudomonas exotoxin termed bFGF-PE40 and bFGF-PE4E KDEL. The chimeric molecules were expressed in Escherichia coli and localized to both inclusion bodies and the spheroplast cytoplasm. The bFGF-toxin fusion protein that was isolated and purified from inclusion bodies was 3-fold more active in inhibiting protein synthesis than that purified from spheroplast cytoplasm.

BR96 sFv-PE40, a potent single-chain immunotoxin that selectively kills carcinoma cells.

We have constructed a single-chain immunotoxin composed of the carcinoma-reactive antibody BR96 and a truncated form of Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, was expressed in Escherichia coli and localized to the inclusion bodies. We purified and identified two species of BR96 sFv-PE40, monomers and aggregates.

Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin.

We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography.

Homodimeric forms of bombesin act as potent antagonists of bombesin on Swiss 3T3 cells.

Two Lys3-bombesin dimers were prepared by crosslinking epsilon-amino groups Lys3-bombesin with noncleavable (glutaraldehyde) and cleavable [dimethyl-3,3'-dithiobispropionimidate (DTBP)] crosslinkers. The dimers were purified by HPLC ion-exchange chromatography and were shown to have retained immunoreactivity with an anti-bombesin monoclonal antibody directed against the C-terminal binding region of bombesin. The glutaraldehyde cross-linked bombesin dimer specifically inhibited binding of 125I-GRP to its receptor on Swiss 3T3 cells.

Antibody-forming cells in the contralateral and ipsilateral lymph nodes draining the locally immunized supramammary/suprainguinal region.

The Jerne hemolytic plaque assay was used to compare the number of antibody forming cells in the ipsilateral supramammary/suprainguinal lymph node which drains the udder, its counterpart area in males, of dairy goats inoculated with the antigen, sheep red blood cells, and in the contralateral lymph node which drains the corresponding non-inoculated area. Parenteral immunization was shown to have suppressing effects upon the local immune responses to the subsequently applied antigens. Three monthly intramammary inoculations of the antigen induced significant numbers of indirect plaque-forming cells (i.

Lymphoid organ weights and organ:body weight ratios of growing beagles.

Although dogs, especially beagles, are used extensively in biological and clinical investigations, the literature dealing with normal biological measurements of their lymphoid organs is scanty. This study was undertaken to provide the information on the weight of lymphoid organs of beagles. The thymus, spleen, and prescapular, popliteal, and mesenteric lymph nodes of 95 normal beagle dogs, from one day to 11 months of age, were weighed and compared with body weights.

Establishment of asparaginase- and guinea pig complement-resistant subline of murine lymphoblastic leukemia L5178y cells.

Asparaginase-resistant subline L5178Y/Asp of murine lymphoblastic leukemia L5178Y cells was established from resistant clones isolated from soft agar cultures treated with Escherichia coli asparaginase. The L5178Y/Asp cells were found to be over 1000-fold more resistant to E. coli asparaginase, co-resistant to inhibitory effect of guinea pig serum (complement), more tumorigenic for syngeneic DBA/2 mice, and to grow much better (higher plating efficiency) in soft agar medium.