Recombinant human interferon-fl2 was produced in Escherichia coli by direct expression of cDNA encoding the mature protein sequence. At concentrations that stimulate DNA synthesis and growth in B-cell hybridomas and plasmacytomas, the cytokine was found to exert a strong inhibition on the growth of a number of carcinoma and leukemia/lymphoma cell lines. This antigrowth effect was observed in clonogenic assays and by measurements of cell number and thymidine incorporation in growing cultures. The effect was blocked by antibodies to a synthetic peptide from the N terminus of the molecule. Normal diploid fibroblasts were inhibited at concentrations higher than those needed for breast carcinoma cells.Cell growth appears regulated by the dual action of growth factors promoting competence or progression through the cell cycle and of growth-inhibitory signals provided by cell contacts or secreted proteins, which limit proliferation (1-3). Extracellular growth inhibitors allow homeostatic networks of regulation-for example, when a counteracting growth inhibitor is secreted in response to a growth factor. Thus, it has been proposed that some interferons (IFNs) may function as autocrine growth inhibitors limiting cell proliferation after growth and differentiation stimuli and preventing neoplastic growth (3-6). IFNs of the a, 83, and y subclasses are characterized by growth-inhibitory activity on a wide spectrum of cells (3, 7), but other groups of cytokines, such as type ( transforming growth factors, have similar functions (2). We are studying the cytokine IFN-(32, originally identified and cloned as an IFN-,3-like antiviral activity from human fibroblasts (8)(9)(10)(11)(12).* This protein is encoded by genes on human chromosome 7, which, as is the case for IFN-y, are not closely related to the IFN-a and -(81 gene cluster (10, 11).More recently, the same protein was identified as a differentiation factor for B lymphocytes that promotes their ability to secrete immunoglobulins (BSF-2 activity) (13) and as a growth stimulatory factor for plasmacytomas and hybridomas (HPGF activity) (14,15). The same cytokine was also shown to promote the synthesis of acute-phase reactants, fibrinogen, antiproteases, and complement proteins in hepatocytes (hepatocyte stimulatory factor activity) (16)(17)(18) and to have a multilineage hematopoietic function (19,20). The protein is induced in fibroblasts, monocytes, and other cells by viruses or double-stranded RNA, by bacterial products as lipopolysaccharides (LPS), as well as by growth factors and inflammatory cytokines, including platelet-derived growth factor, interleukin 1, and tumor necrosis factor (6-10, 14, 18). These numerous inducers and activities make it difficult at present to define which is the main activity or function of IFN-(32/BSF-2/hepatocyte stimulatory factor (21); it is also not clear that the multiple forms of the protein secreted from mammalian cells all have identical functions (18,20). We show here that unmodified recombinant Escherichia coliproduced IFN-f...
Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
A model for studying the efficiency of photodynamic action with a photosensitizer placed exclusively on the bacterial cell wall has been used. Bacteriochlorophyllide molecules, conjugated to rabbit immunoglobulin G (IgG), were synthesized. The conjugated pigment bacteriochlorophyll (Bchl)-IgG bound with high specificity to protein-A residues naturally exposed on the cell wall of the bacterium Staphylococcus aureus Cowan I. In bacterial suspensions the phototoxicity of the targeted conjugates (0.5-2.5 pigment per IgG molecule) was dose dependent (LD50 = 1.7 microM) in the presence of light (lambda > 550 nm) and inhibited by native IgG but not by ovalbumin, suggesting selective interaction with protein-A on the bacterial cell wall. No dark toxicity was noticed even with the highest conjugate concentration tested. In contrast, the photocytotoxicity of bacteriochlorophyll-serine (Bchl-Ser, LD50 = 0.07 microM) used as a nontargeted control was not inhibited by IgG. In spite of its lower apparent potency, Bchl-IgG was found to be 30 times more efficacious than Bchl-Ser: At LD50, only 66,000 Bchl-IgG molecules were bound per bacterium compared to 1,900,000 molecules of Bchl-Ser. The higher efficacy of Bchl-IgG is explained by its exclusive position on the bacterial cell wall. Consequently, photogeneration of oxidative species is confined to the cell wall and its vicinity, a seemingly highly susceptible domain for photodynamic action. In considering the design of cell-specific sensitizers for bacterial and cancer therapies, it would be beneficial to identify the more discretely sensitive subcellular domains as targets.
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