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External stimuli act in concert with intracellular signals to regulate a cell's genetic program, activating genes important in granulocytic lineage commitment, proliferation, and maturation. Signal transducer and activator of transcription 3 (STAT3), a transcription factor, has been implicated in mediating granulocytic differentiation. We have examined the role of STAT3 as a physiologic mediator of granulocytic kinetics. Distinct isoformsthe long form STAT3␣, the truncated forms STAT3␤ and STAT3␥, and a putative novel form STAT3␦-were expressed and activated in a maturation stage-specific manner. With the progression of differentiation, the ratio of isoforms shifted from predominantly STAT3␣ to STAT3␤. The kinetics of STAT3␥, generated through proteolytic cleavage of STAT3␣, coincided with but were inverse to those of STAT3␣. STAT3␦ was expressed at low levels and decreased with differentiation but was preferentially phosphorylated during an intermediate IntroductionHematopoietic lineage determination by transcriptional collaboration with cytokines is well established. 1 In particular, members of the STAT (signal transducer and activator of transcription) transcription factor family mediate many cytokine-induced responses in hematopoietic cells, including proliferation, differentiation, and survival. 2 Upon ligand binding, STATs are recruited to activated cell-surface receptors and become phosphorylated on a specific residue either directly or through association with a Janus kinase. Subsequently, the phosphorylated STATs homodimerize or heterodimerize and translocate to the nucleus, where they regulate transcription by binding to specific DNA promoter elements. 2,3 The STAT family consists of 7 members, most of which are ubiquitously expressed. However, individual STAT proteins may be differentially activated depending on the cell type or tissue. 2,[4][5][6] During myeloid differentiation, various isoforms of STAT3 and STAT5 are activated in a cell type-and maturation state-dependent manner. While STAT5 appears to be important for proliferative responses to interleukin-3 (IL-3), granulocyte-macrophage colonystimulating factor (GM-CSF), and G-CSF, 7,8 STAT3 is activated by G-CSF and appears to be the major STAT protein driving G-CSFmediated granulocytic differentiation. 9-16 Expression of dominant negative STAT3 9 or G-CSF receptor (G-CSFR) mutants lacking regions in the membrane distal domain, which contains recruitment sites for STAT3, [16][17][18][19][20] blocks granulocytic differentiation, demonstrating a requirement for STAT3 activation in growth arrest and morphologic granulocytic differentiation.Three distinct isoforms of STAT3, all derived from a single gene, have been identified 11,21 : STAT3␣ (p92), the full-length isoform expressed in most cells; STAT3␤ (p83), an alternatively spliced RNA form of STAT3␣ in which 55 amino acids of the C-terminal transactivation domain are replaced by 7 unique residues 22 ; and STAT3␥ (p72), also a C-terminal truncated form of STAT3␣ but derived posttranslationally th...

Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species. On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields. Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation. Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation. Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.

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