GPNMB promotes proliferation of developing eosinophils.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation.

Roles of RUNX1 and PU.1 in CCR3 Transcription.

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.

Comparison of Proliferative Effect of Human Lactoferrin and Its Proteolytic Peptide on Normal and Transformed Epithelial Cells.

Human lactoferrin (hLF) is an iron-binding glycoprotein with a variety of functions. hLF undergoes proteolytic cleavage to smaller peptides in the stomach following ingestion. In the present study, we evaluated the effects of hLF and its proteolytic product, human lactoferrin peptide (hLFP), on the proliferation of two epithelial cells, HEK293 normal cells and KATO III gastric carcinoma cells, using an MTT assay and expression of proliferative nuclear cell antigen (PCNA), a notable proliferation marker. When the two epithelial cells were stimulated with hLF and hLFP in the presence of fetal bovine serum (FBS), hLFP stimulated proliferation of both cell types at lower concentrations than hLF by two orders of magnitude. The cancer cells exhibited proliferative responses to both hLF and hLFP at lower concentrations by 2∼3 orders of magnitude than the normal cells. Either hLF or hLFP alone did not support appreciable proliferation of these cell lines in the absence or low concentrations of FBS. Bovine serum albumin or its proteolytic product failed to promote cellular proliferation even in the presence of 10 % FBS, indicating the specificity of the proliferative activity of hLF and hLFP. These data highlight feasibility of hLF and its peptide for adjuvants for tissue culture medium.

Surface display expression of Bacillus licheniformis lipase in Escherichia coli using Lpp'OmpA chimera.

The lipase from Bacillus licheniformis ATCC14580 was displayed on the cell surface of Escherichia coli using Lpp'OmpA as the anchoring protein. The expressed Lpp'OmpA-lipase fusion protein has a molecular weight of approximately 35 kDa, which was confirmed by SDS-PAGE and western blot analysis. The Lpp'OmpA-lipase fusion protein was located on the cell surface, as determined by immunofluorescence confocal microscopy and flow cytometry. The enzyme activity of the surface-displayed lipase showed clear halo around the colony. The cell surface-displayed lipase showed the highest activity of 248.12 ± 9.42 U/g (lyophilized cell) at the optimal temperature of 37°C and pH 8.0. The enzyme exhibited the highest activity toward the substrate p-nitrophenyl caprylate (C8). These results suggest that E. coli, which displayed the lipase on its surface, could be used as a whole cell biocatalyst.

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum.

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris.

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ( 630 )) or a short-TIP1 fragment (ScTIP ( 120 )) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice.

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.