Phospholipase C‐gamma (PLC‐gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC‐gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC‐gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild‐type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC‐gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy‐terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC‐gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy‐terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association.

We have tested one aspect of the allosteric dimerization model for the activation of EGF receptor (EGFR) by EGF: whether EGF binding favors dimerization of the receptor. For this to be true, EGF molecules must bind with higher affinity to dimeric receptors than to monomeric receptors. We have tested this directly in a defined system using the soluble, extracellular ligand binding domain of EGFR monomers (sEGFR) and sEGFR dimers stabilized by treatment with a covalent cross-linking agent. We describe real-time kinetic measurements of EGF binding to receptor monomers and dimers employing the method of total internal reflection (surface plasmon resonance). Our data show that sEGFR dimers bound EGF with 30-40-fold higher affinity [KD = (2-3) x 10(-8) M] than did sEGFR monomers. The enhanced binding affinity of sEGFR dimers resulted mainly from a reduced off-rate with k(off) = 0.001 s-1 for sEGFR dimers as compared to k(off) = 0.06 s-1 for sEGFR monomers. These measurements indicate that dimerization of sEGFR increases its affinity for EGF by prolonging the amount of time that EGF remains bound to the receptor. This provides evidence that EGF binding stabilizes receptor dimerization and provides further support for the allosteric dimerization model as a mechanism for ligand induced receptor activation.

The hormone gastrin stimulates acid secretion by releasing histamine from gastric enterochromaffin-like (ECL) cells and induces ECL cell proliferation in vivo. This study uses a > 90% pure ECL cell preparation in culture to compare gastrin effects on histamine release, histidine decarboxylase (HDC) activity, and DNA synthesis. Gastrin and the cholecystokinin octapeptide (CCK-8, nonsulfated) induced histamine release from ECL cells (24-96 h of primary culture) within 5 min of incubation [concentration eliciting 50% of maximal response (EC50), 4 and 2 x 10(-11) M, respectively]. The CCK-B antagonist L-365,260 inhibited this effect [concentration inhibiting 50% of maximal response (IC50), 2 x 10(-8) M], whereas the CCK-A antagonist L-364,718 (10(-8) M) and the tyrosine kinase inhibitor genistein (10(-4) M) had no effect. Histamine release was associated with a biphasic elevation of intracellular Ca2+. Gastrin stimulated HDC activity two- to threefold after 60 min of incubation (EC50, 10(-10) M). Gastrin also increased DNA synthesis in ECL cells, with an EC50 of 1.7 x 10(-12) M as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Positive nuclear immunostaining increased two- to threefold in up to 20% of ECL cells after 48-96 h of incubation. This effect was inhibited by L-365,260 (IC50, 5 x 10(-9) M) and by genistein (10(-4) M) but was not altered by L-364,718 (10(-8) M). The antisecretory drugs omeprazole, lansoprazole, and pantoprazole did not affect BrdU incorporation in isolated ECL cells. In conclusion, acute and chronic gastrin effects on the ECL cell are mediated via CCK-B receptors but differ in apparent receptor affinity and signal transduction pathways.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep beta-lactoglobulin (BLG) 5'-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5'-sequences demonstrated that 3 kb of 5'-sequences were sufficient to support high levels of expression of BLG, and that the longer 5'-sequences did not improve upon the levels of expression. As such, the 3 kb 5'-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml-1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 micrograms ml-1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.

Microbial transglutaminase (MTG) was stably solid-phase immobilized on glass microbeads by using a second-generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins, including antibody fragments, as well as whole antibodies through distinct glutamines and, unprecedentedly, also through lysines with various bifunctional substrates with defined stoichiometries. With this method, we generated dual, site-specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling-a strategy anticipated to design antibodies for imaging and simultaneous therapy. Furthermore, we provide evidence that immobilized MTG features higher siteselectivity than soluble MTG.

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The expression of the adenovirus (Ad) early coding region la (Ela) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earli Oncogenic transformation by adenoviruses is a function of both the Ela and Elb (mp 4.5-11.3) regions as determined by the analysis of virus-transformed cells (5, 6), DNA transfection (7-9), and the use of viral mutants (10)(11)(12)(13)(14). DNA transfection studies have shown that Ela can partially transform baby rat kidney (BRK) cells (15). These Ela-transformed cells are "immortalized," whereas their morphology and growth pattern resemble those of untransformed cells. In addition to the immortalization function, Ela also appears to have a transforming function, since immortalized rat embryo cells cannot be transformed by DNA constructs capable of expressing Elb without Ela (9).The Ela gene also has been shown to facilitate the transformation of BRK cells by cellular oncogenes. The human cellular oncogene Ha-ras isolated from a bladder carcinoma is unable to transform BRK cells. However, Ha-ras can be complemented by the Ad2 Ela gene to produce full transformation and tumorigenecity (16).The Ela gene encodes three overlapping mRNAs that have identical 5' and 3' termini (Fig. 1). They differ in the size of the intron removed during splicing. Each uses a common 3'-splice site junction (acceptor) but differs in the location of the 5'-splice site junction (donor). The 13S and 12S mRNAs are expressed during the early stage of the productive cycle (21, 22) and in transformed cells (23,24). The 9S mRNA does not accumulate to significant levels except during the late stage of the productive cycle (25) and is not essential for viral growth during productive infection (26). The 9S mRNA has not been detected in any of the Ad-transformed cells examined (24,26). These studies indicate that the proteins encoded by the 13S mRNA and/or the 12S mRNA play an essential role in cell transformation. Viral mutants (27, 28) and plasmid constructs (29) have demonstrated the requirement of the 289-amino acid tumor antigen coded by the 13S mRNA for transformation.To investigate the role of the Ela 243-amino acid tumor antigen coded by the 12S mRNA in cell transformation and the productive cycle, we constructed an Ad2 Ela deletion mutant that specifically and precisely lacks the Ela 13S mRNA intervening sequence (IVS). It recently has been shown that the splicing of Ela mRNAs is nonsequentialthat each mRNA is produced from an unspliced nuclear precursor and not from an intermediate (i.e., 13S mRNA does not mature into 12S mRNA or 9S mRNA) (30). Therefore, our deletion mutant, which lacks the 13S IVS, cannot produce functional 12S mRNA. Our results demonstrate that the 243-amino acid tumor antigen is required for full transformation of rat embryo cells. MATERIALS AND METHODSConstr...