Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies.

Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.     

Monoclonal antibodies to the hydrogenase ofThiocapsa roseopersicina

Monoclonal antibodies were produced to electrophoretically pure hydrogenase fromThiocapsa roseopersicina. Protein immunoelectroblotting was used to identify the hydrogenase-specific antibodies. Among the 18 monoclonal antibodies selected by enzyme immunoassay, three were found to react with highly immunogenic trace contaminating proteins. One cell line produced antibody that inhibitied hydrogenase activity. This was the first specific inhibitor of the hydrogenase function. The results suggest that monoclonal antibodies could provide valuable new informations about the enzyme structure as well.     

Initiation of early breeding in a population of Microtus townsendii (Rodentia) with the secondary plant compound 6-MBOA

Summary Feeding the secondary plant compound 6-Methoxybenzoxazolinone (6-MBOA) during winter to a free living population of Microtus townsendii accelerated breeding in females. Both the recruitment of young and sexual maturation of these young were advanced by four weeks in comparison with a control population. Overwintered females supplied with 6-MBOA matured at a lower body weight than control females. But there was no such weightat-maturity difference between the grids for males. During April and May 72% of all juveniles captured came from the experimental population. Winter reproduction and early recruitment of young stimulated by 6-MBOA could have important population consequences for these voles.     

Thymic epithelium in vitro. IV. Regulation of growth and mediator production by epidermal growth factor

The regulation of thymic epithelial cell function has been examined using pure cultures of morphologically distinct thymic epithelial cells and the ubiquitous hormone epidermal growth factor (EGF). Small thymic epithelial cells, TECS, had receptors for EGF with high affinity, Kd = 1.2 X 10(-9) M, and exhibited increased DNA synthesis and increased RNA synthesis upon stimulation with EGF. In addition, incubation of TECS monolayers with EGF resulted in enhanced production of prostaglandin E2. In contrast, large thymic epithelial cells, TECL, did not express receptors for EGF and demonstrated no biological response to the hormone. These results suggest the possibility that intrathymic regulation of lymphoid cells may occur via the action of "nonimmunologic" mediators on thymic epithelial cells. They further suggest the more general possibility that immunologic and nonimmunologic hormonal systems may be linked via intersecting cellular pathways.     

Enzymatic activity and filament assembly of Acanthamoeba myosin II are regulated by adjacent domains at the end of the tail

Polyclonal antibodies raised against a synthetic peptide consisting of the last 19 amino acids at the end of the coiled-coil region of the heavy chains inhibited the actin-activated Mg2+-ATPase activity of myosin II and its ability to form filaments. Antibodies against a synthetic peptide corresponding to the 21 adjacent amino acids at the beginning of the non-helical tailpiece, which include the three regulatory phosphorylatable serines, had no effect on either activity.     

Statistical discrimination of fractal and Markov models of single-channel gating.

A statistical comparison is presented of Markov and fractal models of ion channel gating. The analysis is based on single-channel data from two types of ion channels: open times from a 90 pS Ca-activated K channel from GH3 pituitary cells, and closed times from a nonselective channel from rabbit corneal endothelium (Liebovitch et al., 1987a). Maximum likelihood methods were used to fit the data. For both data sets the best Markov model had three exponential components. The best Markov model had a higher likelihood than the fractal model, and the Asymptotic Information Criterion favored the Markov model for each data set. A more detailed analysis, using the Monte Carlo methods described in Horn (1987), showed that the Markov model was not significantly better than the fractal model for the corneal endothelium channels. The inability to discriminate the models definitively in this case was shown to be due in part to the small size of the data set.     

Protein that induces cell differentiation causes nicks in double-stranded DNA

The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double-stranded DNA. We now report that purified myeloid cell differentiation-inducing protein causes single strand breaks (nicks) in double-stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.