Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase.Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phanid prokaryotic expression vector and screened with a monoclonal , was ligated to the EcoRP-Sal I fragment of the expression vector pCEV-lacZ to generate the plasmid pCEV-bek (Fig. 1A). pCEV-lacZ is a pUC-based plasmid expression vector that produces a 3-gal fusion protein under the control of the lac promoter (see below).Construction of ApCEV-IacZ Prokaryotic Expression Vector. The vector designed for prokaryotic cDNA expression cloning was similar to Agtll but incorporated several additional features. cDNAs can be inserted by the automatic directional cloning method (16), allowing construction of unidirectional libraries with high efficiency. Use of unidirectional libraries to screen negative signals excludes antisense cDNA clones, which might inhibit the expression of cDNAs in host cells (like the bek kinase used in these experiments). It also increases the possibility of cDNA expression from the promoter in the vector. Since ApCEV-lacZ is a phagemid vector, the plasmid can be rescued by a simple procedure (17).To construct ApCEV-lacZ, the lacZ structural gene (encoding a-gal) of pMC1871 (Pharmacia) was cloned into pUCSVneo4ARI, a precursor plasmid of pCEV27 (17). Multiple cloning sites similar to those in ApCEV27 were created by inserting oligonucleotides into the EcoRI site of the IacZ gene. The resulting plasmid, pCEV-lacZ, was linearized at the Not I site and, after multiple excision sites were attached (17), cloned into ApCEV27 Not I arms to create ApCEV-lacZ.Library Construction and Screening. An oligo(dT)-primed M426 human fibroblast cDNA library was constructed in ApCEV-lacZ. To screen the library, bacteria (Escherichia coli Y1091; Clontech) containing the pCEV-bek plasmid were infected with phage (1.2 x 104 plaque-forming units per 150-mm plate) and grown for 4 hr at 3TC on agar plates containing ampicillin (50 pug/ml). The plates were overlaid with nitrocellulose filters impregnated with 10 mM IPTG and were incubated for an additional 4 hr at 37cC, as described (18). The filters were removed, washed with phosphatebuffered saline containing 0.05% Tween 20 (PBST), and then blocked in PBST containing 3% dry milk for 1 hr at room temperature. Filters were probed with mouse monoclonal anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY) overnight at room temperature, rinsed four times with PBST, incubated with 1251-labeled protein A for 30 min,

To determine the biologic activity of interleukin-6 (IL-6) on megakaryocytopoiesis and thrombocytopoiesis in vivo, the cytokine was administered intraperitoneally to mice every 12 hours at varying doses for five days or for varying time intervals, based on the kinetic analysis of IL-6 serum levels indicating the peak of 40 minutes following injection, with no detection at 150 minutes. A dose-response experiment showed that IL-6 increased platelet counts in a dose- dependent fashion at a plateau stimulation level of 5 micrograms. Administration of 5 micrograms of IL-6 reproducibly elevated platelet counts at five days by approximately 50% to 60% of increase. Moreover, a striking increase in megakaryocytic size in response to IL-6 was elicited by the treatment, but no change in megakaryocyte numbers; whereas IL-6 administration did not expand CFU-MK numbers. The in vivo studies in this manner had negligible effects on other hematologic parameters, with the minor exception of monocyte levels. These data show that IL-6 acts on maturational stages in megakaryocytopoiesis and promotes platelet production in vivo in mice, suggesting that IL-6 functions as thrombopoietin.

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In selected cases, injection of 40 units of BTX-A into the turbinates, as a single agent, may help the symptomatic control of AR for up to 8 weeks.

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